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https://openalex.org/W2949100880	http://pure-oai.bham.ac.uk/ws/files/74388354/ALICE_collaboration_Energy_dependence_of_exclusive_J_photoproduction_off_protons_in_ultra_peripheral_European_Physical_Journal_C_2019.pdf	English	null	Energy dependence of exclusive $$\mathrm {J}/\psi $$ photoproduction off protons in ultra-peripheral p–Pb collisions at $$\sqrt{s_{\mathrm {\scriptscriptstyle NN}}} = 5.02$$ TeV	European physical journal. C, Particles and fields	2,019	cc-by	17,724	Link to publication on Research at Birmingham portal Publisher Rights Statement: Publisher Rights Statement: Acharya, S., Acosta, F.T., Adam, J. et al. Eur. Phys. J. C (2019) 79: 402. https://doi.org/10.1140/epjc/s10052-019-6816-2 Energy dependence of exclusive J/ψ photoproduction off protons in ultra-peripheral p–Pb collisions at √sNN = 5.02 TeV ALICE Collaboration; Acharya, S.; Andrews, H. A.; Evans, D.; Graham, Kay; Jevons, Oliver; Jones Peter; Jusko Anton; Krivda Marian; Kvapil Jakub; Lietava Roman; Villalobos Baillie Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): ALICE Collaboration, Acharya, S, Andrews, HA, Evans, D, Graham, K, Jevons, O, Jones, P, Jusko, A, Krivda, M, Kvapil, J, Lietava, R, Villalobos Baillie, O, Willsher, E & Zardoshti, N 2019, 'Energy dependence of exclusive J/ψ photoproduction off protons in ultra-peripheral p–Pb collisions at √sNN = 5.02 TeV', European Physical Journal C, vol. 79, no. 5, 402. https://doi.org/10.1140/epjc/s10052-019-6816-2 Link to publication on Research at Birmingham portal General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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Energy dependence of exclusive J/ψ photoproduction off protons in ultra-peripheral p–Pb collisions at √sNN = 5.02 TeV ALICE Collaboration⋆ CERN, 1211 Geneva 23, Switzerland Received: 25 September 2018 / Accepted: 30 March 2019 / Published online: 12 May 2019 © CERN for the beneﬁt of the ALICE collaboration 2019 ALICE Collaboration⋆ CERN, 1211 Geneva 23, Switzerland ALICE Collaboration⋆ CERN, 1211 Geneva 23, Switzerland Received: 25 September 2018 / Accepted: 30 March 2019 / Published online: 12 May 2019 © CERN for the beneﬁt of the ALICE collaboration 2019 Received: 25 September 2018 / Accepted: 30 March 2019 / Published online: 12 May 2019 © CERN for the beneﬁt of the ALICE collaboration 2019 Abstract The ALICE Collaboration has measured the energy dependence of exclusive photoproduction of J/ψ vec- tor mesons off proton targets in ultra–peripheral p–Pb colli- sions at a centre-of-mass energy per nucleon pair √sNN = 5.02 TeV. The e+e−and μ+μ−decay channels are used to measure the cross section as a function of the rapidity of the J/ψ in the range −2.5 < y < 2.7, correspond- ing to an energy in the γ p centre-of-mass in the interval 40 < Wγ p < 550 GeV.Themeasurements,whichareconsis- tent with a power law dependence of the exclusive J/ψ pho- toproduction cross section, are compared to previous results from HERA and the LHC and to several theoretical models. They are found to be compatible with previous measure- ments. saturation effects, because the cross section for this process depends, at leading order in pQCD, on the square of the gluon density in the target [8]. The mass of the charm quark provides a scale large enough to allow calculations within pQCD. A reduction in the growth rate of the cross section for this process as the centre-of-mass energy of the photon-target system increases would signal the onset of gluon saturation effects. This process has been extensively studied in ep interac- tions at the HERA collider. Both ZEUS and H1 measured the energy dependence of exclusive J/ψ photoproduction off protons at γ p centre-of-mass energies, Wγ p, from 20 to 305 GeV [9–11]. H1 has also measured exclusive ψ(2S) photo- production off protons with Wγ p between 40 and 150 GeV [12]. The exclusive photoproduction of charmonium can also be studied at hadron colliders, where one of the incoming hadrons is the source of the photons and the other the target. Energy dependence of exclusive J/ψ photoproduction off protons in ultra-peripheral p–Pb collisions at √sNN = 5.02 TeV CDF measured exclusive J/ψ production at mid-rapidities in p¯p collisions at the Tevatron [13], while LHCb reported measurements of exclusive J/ψ and ψ(2S) production at for- ward rapidities in pp collisions at the LHC [14]. In addition, ϒ production has been studied, both at HERA [15–17] and in pp collisions at the LHC [18]. In all cases, the experimental signature is the production of a vector meson in a collision in which there is no other hadronic activity, apart from the possible emission of a few very forward neutrons. ⋆e-mail: alice-publications@cern.ch Take down policy hil h i i Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions wh uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access ely and investigate. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we the work immediately and investigate. Download date: 24. Oct. 2024 Eur. Phys. J. C (2019) 79:402 https://doi.org/10.1140/epjc/s10052-019-6816-2 Regular Article - Experimental Physics 1 Introduction One of the most interesting aspects of perturbative Quantum Chromodynamics (pQCD) is the evolution of the structure of hadrons in terms of quarks and gluons towards the high- energy limit [1]. Very precise measurements at HERA [2,3] have shown that the gluon structure function in protons rises rapidly at small values of the Bjorken x variable, which cor- responds to the high-energy limit of QCD. This rise, inter- preted as the growth of the probability density function for gluons, has to be damped at some high energy to satisfy uni- tarity constraints [4]. Although gluon saturation [5] is the most straightforward mechanism to slow down the growth of the cross section, no compelling evidence for this effect has been found so far. Gluon saturation would have impor- tant implications in small-x physics and in the early stages of ultra-relativistic heavy-ion collisions at RHIC and LHC; con- sequently, ﬁnding evidence for gluon saturation has become a central task for present experiments and for future projects [6,7]. In hadron colliders, as the incoming particles can each be both source or target, there are two possible centre-of-mass energies for the photon-target system. This ambiguity can be resolved if the contribution of one beam acting as the source is much stronger than the other. This is the case for p–Pb col- lisions, because photon emission grows with the square of the electric charge and thus emission by the Pb ion is strongly enhanced with respect to that from the proton. ALICE pub- lished the ﬁrst measurement of exclusive photoproduction of J/ψ, at forward and backward rapidities, in p–Pb collisions at the LHC [19]. Theexclusivephotoproductionofcharmoniumoffprotons (γ p →J/ψp) is a very clean probe with which to search for 123 123 402 Page 2 of 18 402 Page 2 of 18 Eur. Phys. J. C (2019) 79 :402 2.1 The ALICE central barrel In this Letter we present the measurements of exclusive J/ψ photoproduction in collisions of protons with Pb nuclei at a centre-of-mass energy per nucleon pair √sNN = 5.02 TeV. The measurements are performed in the semi-backward (−2.5 < y < −1.2), mid (\|y\| < 0.8) and semi-forward (1.2 < y < 2.7) rapidity intervals, where the rapidity y of the J/ψ is measured in the laboratory frame with respect to the direction of the proton beam. 1 Introduction The value of Wγ p is determined by the rapidity of the J/ψ according to W 2 γ p = 2EpMJ/ψ exp(−y), where MJ/ψ is the J/ψ mass and Ep = 4 TeV is the energy of the proton beam. Thus, the measurements presented here span the Wγ p range from 40 to 550 GeV. Together with the measurements presented in [19] (for the rapidity ranges −3.6 < y < −2.6 and 2.5 < y < 4), ALICE data map the Wγ p dependence of the cross section for this process from about 20 to 700 GeV, which, using the standard deﬁnition x = M2 J/ψ/W 2 γ p, cor- responds to three orders of magnitude, from ∼2 × 10−2 to ∼2 × 10−5. The innermost component of the central barrel is the Inner Tracking System (ITS) [22]. The ITS contains six cylindrical layers of silicon detectors, with the innermost layer at a radius of 3.9 cm with respect to the beam axis and the outermost layer at 43 cm. The two rings closest to the beam form the Silicon Pixel Detector (SPD) and cover the pseudorapidity ranges \|η\| < 2 and \|η\| < 1.4, for the inner and outer layers respectively. It is a ﬁne granularity detector, having about 107 pixels. In addition to functioning as a tracking device, the SPD signals can be used to build triggers. The other four layers, two employing silicon drift chambers and two silicon microstrips, are used in this analysis exclusively for tracking. The Time Projection Chamber (TPC), surrounding the ITS, is a large cylindrical detector used for tracking and for particle identiﬁcation [23]. Its active volume extends from 85 to 247 cm in the radial direction and has a total length of 500 cm longitudinally, centred on the interaction point. A 100 kV central electrode separates its two drift volumes, providing an electric ﬁeld for electron drift. The two end-plates are instru- mented with Multi-Wire-Proportional-Chambers (MWPCs) with 560,000 readout pads in all, allowing high precision track measurements in the transverse plane. The z coordi- nate is given by the drift time in the TPC electric ﬁeld. The TPC acceptance covers the pseudorapidity range \|η\| < 0.9 for tracks fully traversing it. Ionization measurements made along track clusters are used for particle identiﬁcation. 2 Experimental set-up The ALICE detector and its performance are described in detail in [20,21]. It consists of two main sections: a central barrel placed in a large solenoid magnet (B = 0.5 T), cov- ering the pseudorapidity region \|η\| < 0.9, and a muon spec- trometer covering the range −4.0 < η < −2.5. In addition, the analyses presented here make use of two other detector systems, V0 and ZDC, which are placed near the beam pipe and cover large pseudorapidities. Beyond the TPC lies the Time-of-Flight detector (TOF) [24]. It is a large cylindrical barrel of Multigap Resistive Plate Chambers (MRPCs) giving very high precision timing for tracks traversing it. Its pseudorapidity coverage matches that of the TPC. It is also capable of delivering signals for triggering purposes [25]. Because the proton and Pb beams had different energies, 4 TeV and 1.58 TeV per nucleon respectively, the nucleon- nucleon centre-of-mass was shifted, with respect to the lab- oratory frame, by yNN = 0.465 in the direction of the proton beam. Collisions were performed in two conﬁgura- tions, denoted by p–Pb and Pb–p below, by reversing the directions of the LHC beams. According to the naming con- vention the ﬁrst named particle (e.g. ‘p’ in p–Pb) is the one where the beam travels from the interaction region towards the muon spectrometer. Note that the convention in ALICE is that the pseudorapidity is negative in the direction of the muon spectrometer. In contrast, the laboratory rapidity is measured with respect to the direction of the proton beam and changes sign accordingly for the p–Pb and Pb–p conﬁg- urations. In the semi-backward and semi-forward analyses, the negative rapidity points come from the Pb–p data, and the positive rapidity points from the p–Pb. 2.3 The V0 and the ZDC systems Two sets of hadronic Zero Degree Calorimeters (ZDC) are located at 112.5 m on either side of the interaction point. Each station consists of two detectors, one to detect protons and the other neutrons. The neutron ZDCs detect neutrons emitted in the very forward region (\|η\| > 8.7). The calorimeters detect the Cherenkov light produced in quartz ﬁbers by the hadronic showers. Each detector is read out by ﬁve photomultipliers. Their timing and energy resolution allow one to select events with neutrons produced at very forward rapidity, as could be the case in events with proton dissociation. The integrated luminosity Lint was corrected for the prob- ability that the exclusivity requirements could be spoilt by multiple interactions in the same bunch crossing. This pile- up correction is on average 5%. The total luminosity of the sample used for the mid-rapidity analysis is 2.1 nb−1 (4.8 nb−1) in the p–Pb (Pb–p) period, while for the semi-forward (semi-backward) analysis it is 3.1 nb−1 (3.7 nb−1) in the p– Pb (Pb–p) period. Note that in the mid-rapidity case, the p–Pb and Pb–p data samples have been analysed together, taking into account the inversion of the rapidity sign, to increase the size of the central rapidity data sample. The V0 counters [26] consist of two arrays of 32 scintilla- tor cells each, covering the range 2.8 < η < 5.1 (V0A on the side opposite to the muon spectrometer) and –3.7 < η < – 1.7 (V0C, on the same side as the muon spectrometer) and positioned respectively at z = 340 cm and z = −90 cm from the interaction point. The raw signals are used in the trigger, but are reﬁned ofﬂine for use in the ﬁnal data selections. For example, these detectors provide timing information with a resolution better than 1 ns, which can be used to distinguish two different timing windows, one corresponding to the pas- sage of particles coming from collisions, and the other to particles coming from upstream interactions with residual gas molecules, termed “beam-gas” interactions. 2.2 The ALICE muon spectrometer The muon spectrometer consists of a 3 Tm dipole mag- net, coupled with a tracking and a triggering system. A ten interaction-length conical front absorber, made of carbon, concrete and steel, is placed between the interaction point (IP) and the muon spectrometer tracking system, between 0.9 and 5 m from the IP, to ﬁlter out primary hadrons. Muon tracking is performed by means of ﬁve tracking stations, each one made of two planes of Cathode Pad Chambers. A 7.2 interaction-length iron wall is placed after the tracking sta- tions. It is followed by the muon trigger system, based on two stations equipped with Resistive Plate Chambers. The trigger system can provide single-muon and dimuon triggers with a programmable pT threshold. The pT threshold is an approximate value owing to the coarse grain of the trigger spatial information. In addition, there is a conical absorber made of tungsten, lead and steel, that surrounds the beam The ALICE apparatus consisted of 18 detector systems at the time the data used in this study were taken. The detectors relevant for the analysis are described in more detail below. 123 123 Eur. Phys. J. C (2019) 79 :402 402 Page 3 of 18 402 Page 3 of 18 402 Three triggers were used for the semi-backward and semi- forward analyses. The ﬁrst was active during the p–Pb data taking period. It required a muon candidate in the muon spec- trometer trigger system with a transverse momentum above 0.5 GeV/c (as assessed in the trigger system electronics), at most 4 cells with signal in the V0C and no activity in the V0A. The SPD requirement was to have at least 1 pixel hit in either the inner or the outer layer and less than 7 pixels ﬁred in the outer layer. Two different triggers were active during the Pb–p period. For the ﬁrst one, in addition to the previ- ous conditions, it was also required that at least one cell of the V0C had a signal. The second one had the same require- ments as the ﬁrst, but in addition vetoed signals from V0A compatible with beam-gas timing. These tighter conditions were required owing to an increased background trigger rate. pipe at small angles (θ < 2◦) and shields the spectrometer from secondary particles produced in interactions of primary particles in the beam pipe. 3 Data samples 3.1 Event and track selection for the mid-rapidity analysis 3.3 Selection of background samples 3.2 Event and track selection for the semi-backward and semi-forward analyses Several background samples, one for each analysis, were selected. The events in these samples satisfy the condi- tions listed in the previous sections, and in addition there is required to be a signal, compatible with the presence of a neutron, in the ZDC placed in the proton direction. Both like-sign and opposite-sign dilepton candidates are accepted. Several background samples, one for each analysis, were selected. The events in these samples satisfy the condi- tions listed in the previous sections, and in addition there is required to be a signal, compatible with the presence of a neutron, in the ZDC placed in the proton direction. Both like-sign and opposite-sign dilepton candidates are accepted. The events used in the semi-backward and semi-forward analyses fulﬁlled the following criteria. 1. They ﬁred the corresponding trigger. 1. They ﬁred the corresponding trigger. These samples are highly efﬁcient to tag dissociative pro- cesses (where the target proton breaks up) and other hadronic collisions and were used to build templates to subtract the remaining background in the real data samples as explained below. As the trigger suppresses events with higher pT dilep- tons (pT ≥0.5 GeV/c), due to the condition on azimuthal separation, the number of events in this pT range is limited. These samples are highly efﬁcient to tag dissociative pro- cesses (where the target proton breaks up) and other hadronic collisions and were used to build templates to subtract the remaining background in the real data samples as explained below. As the trigger suppresses events with higher pT dilep- tons (pT ≥0.5 GeV/c), due to the condition on azimuthal separation, the number of events in this pT range is limited. Therefore, each background spectrum was used to gener- ate a high statistics sample, which was smoothed to pro- duce the ﬁnal template. This is referred to as “non-exclusive background” in the analysis of the pT spectrum. The selec- tions and smoothing algorithms were varied to estimate the systematic uncertainties, and the background pT distribution was veriﬁed independently by selecting hadronic activity in V0C instead of the ZDC. 2. There was exactly one track reconstructed with the TPC and the ITS. 3. 2.4 Triggers The pseudorapidity ranges are different in p–Pb and Pb–p owing to the additional V0C trigger requirement (see Sect. 2.4). To build dilepton candidates the tracks were separated into electrons and muons using the energy loss measured by the TPC, such that: 1. the quadratic sum of the normalised deviation from the corresponding Bethe-Bloch expectation for the positive and negative track had to be less than 16; 6. The two tracks had to have opposite electric charge. 6. The two tracks had to have opposite electric charge. 7. The rapidity, measured in the laboratory frame, of the dimuon candidate, formed by the 4-vector sum of the two tracks with the muon-mass hypothesis, had to be in the range 1.2 < y < 2.7 for the p–Pb and −2.5 < y < −1.2 for the Pb–p sample, respectively. 2. the rapidity, measured in the laboratory frame, of the dilepton candidate, formed by the 4-vector sum of the two tracks with the corresponding mass hypothesis, had to be in the range \|y\| < 0.8. 8. In addition, the events had to have no signal in V0A, at most one cell with a signal in V0C, (in both cases as determined by the ofﬂine reconstruction), no signal in either of the neutron ZDCs and at most one tracklet in the SPD layers. In addition, the events had to 3. havenosignalinV0asdeterminedbytheofﬂinedecision, and 4. no signal in either of the neutron ZDCs. 3.3 Selection of background samples This track fulﬁlled similar requirements to those for the central analysis tracks, with two modiﬁcations: it had to be compatible with a muon (within 4σ) according to the energy-loss measurement, and the acceptance was enlarged to \|η\| < 1.1. 4. Furthermore,thedistanceofclosestapproachofthistrack to the nominal interaction point had to be less than 15 cm in the direction along the beam-line. 5. The event also had to have exactly one track in the muon spectrometer such that: (a) the track is matched to a trigger track above the pT threshold; (b) the radial coordinate of the track at the end of the front absorber (Rabs) was required to be in the range 17.5 cm < Rabs < 89.5 cm; 2.4 Triggers The events used in the mid-rapidity analysis fulﬁlled the fol- lowing criteria. The events used in the mid-rapidity analysis fulﬁlled the fol- lowing criteria. Two different signatures have been used in the analyses described below. In one case, the mid-rapidity analysis, the J/ψ candidate decays into a pair of leptons (either e+e− or μ+μ−), both of which are detected with the central bar- rel detectors. The second case, the semi-forward (or semi- backward) analysis, corresponds to a J/ψ candidate decay- ing into a pair of muons where one of them is measured with the muon spectrometer and the other with the central barrel detectors. 1. They ﬁred the corresponding trigger. 2. TherewereexactlytwotracksreconstructedwiththeTPC and the ITS. 3. The primary vertex had at least two tracks deﬁning it. 4. Each track had at least 50 TPC space points out of a maximum of 159, a TPC-χ2/dof < 4, at least one SPD point and pseudorapidity \|η\| < 0.9. The trigger for the mid-rapidity analysis required at least two, and at most six, ﬁred pad-OR in the TOF detector [24], such that at least two of the signals came from modules sep- arated by more than 150 degrees in azimuth, since the J/ψ decay products are predominantly produced back-to-back in azimuth. The trigger also required no signal in V0A and no signal in V0C, to ensure that there is very little extra hadronic activity in the event, and at most six ﬁred pixel chips in the outer layer of the SPD. In addition there were required to be two inner-outer pairs of these pixels having a back-to-back topology. 5. The distance of closest approach of each track to the (nominal) primary vertex (DCA) was less than 2 cm in the longitudinal direction and less than 0.0182+0.035/p1.01 T cm, with pT in GeV/c, in the transverse direction [21]. 6. Neither track was associated with a kink candidate. 7. The coordinate of the primary vertex of the interaction along the beam-line direction was within 15 cm of the nominal interaction point. 8. The two tracks had opposite electric charge. 12 3 402 Page 4 of 18 Eur. Phys. J. C (2019) 79 :402 to calculate the distance of closest approach. The pseudorapidity ranges are different in p–Pb and Pb–p owing to the additional V0C trigger requirement (see Sect. 2.4). to calculate the distance of closest approach. 3.4 Monte Carlo samples The STARLIGHT Monte Carlo generator [27,28] was used to generate large samples for the following processes: exclu- sive J/ψ photoproduction off proton (γ p →J/ψp) and Pb (γ Pb →J/ψPb) targets, exclusive photoproduction of ψ(2S) decaying into J/ψ + X, and continuum dilep- ton production γ γ →l+l−. The physics models used by STARLIGHT are described in [28–30]. All generated events were passed through a full simulation of the detector based on GEANT 3 transport code [31] and subjected to the same analysischainasrealdata.TheMonteCarlotookintoaccount (c) it had a pseudorapidity in the range −4.0 < η < −2.5 (−3.7 < η < −2.5) in the p–Pb (Pb–p) period and fulﬁlled the so called p×DCA requirement. (c) it had a pseudorapidity in the range −4.0 < η < −2.5 (−3.7 < η < −2.5) in the p–Pb (Pb–p) period and fulﬁlled the so called p×DCA requirement. The p×DCA requirement uses the difference in the distributions for signal and beam-induced back- ground in the (p, DCA) plane, where p is the track momentum and DCA is the distance between the interaction vertex and the track extrapolated to the vertex transverse plane. If in a given event no inter- action vertex was found, the point (0, 0, 0) was used 12 3 Page 5 of 18 402 Eur. Phys. J. C (2019) 79 :402 402 the changing status of the detectors during data-taking, and their residual misalignment. the changing status of the detectors during data-taking, and their residual misalignment. sive and the dissociative dilepton production, while the tem- plate describes only the exclusive component. The number of candidates for coherent J/ψ production off lead has been estimated using STARLIGHT. The STARLIGHT predictions were rescaled so as to be compatible, within the uncertain- ties, with ALICE measurements in Pb–Pb collisions [34,35]. The normalisation of the exclusive J/ψ and non-exclusive background templates have been left free. Note that the non-exclusive background is lower at higher energies of the photon-target system, as has already been observed at HERA [10,11], and also by ALICE [19]. In all cases the polarization of the photoproduced char- monium was taken as transverse, as expected for photopro- duction of vector mesons. For the ψ(2S) sample three cases were studied, corresponding to an assumption of longitudi- nal, transverse and no polarisation of the J/ψ produced by the decay of the ψ(2S). 4.1 Signal extraction The mass distributions for the dilepton candidates that passed the selection described in the previous section are shown in Fig. 1. In all cases a clear J/ψ signal is observed over a small background. The data can be satisfactorily described in all cases by a combination of a Crystal-Ball function (CB) [33] and an exponential distribution, which represent the signal and the background from continuum dilepton production, respectively. The tail parameters of the CB distribution have been ﬁxed to the values obtained by ﬁtting simulated events. 3.4 Monte Carlo samples The dependence of exclusive photoproduction on the momentum transferred in the target vertex is modelled in STARLIGHT using an exponential distribution F(Q2) = e−bQ2, with the slope b taking the default value of 4 GeV−2 [32]. HERA measurements have shown that the value of this parameter depends on Wγ p [10]. This effect is particularly important for the mid-rapidity and the semi-backward sam- ples, where Wγ p is larger. To take this into account additional samples of exclusive J/ψ were produced using STARLIGHT with a value of b given by the H1 parameterisation from [10]. The number of candidates obtained from the ﬁtted tem- plate for exclusive J/ψ production has been corrected for the feed-down from exclusive production of ψ(2S) decay- ing into J/ψ + X [35]. This correction amounts to about 2% (4%) for the semi-backward and semi-forward (mid-rapidity) analyses. The correction factor to take into account the accep- tance and efﬁciency (ε × A) of the detector was computed using simulated events, as described in Sect. 3.4. This factor depends on the rapidity of the J/ψ and varies from around 3% to almost 9%. For the semi-backward sample the ε × A includes the efﬁciency of the V0C (around 60%) to detect the passing muon (because it is included in the trigger). The val- ues for the number of exclusive J/ψ extracted in the different data samples, as well as the corresponding ε × A factors and available luminosity, are summarised in Table 1. Note that, in order to make best use of the available statistics, both the central and the semi-forward (but not the semi-backward) samples have been sub-divided into two rapidity bins. 4.2 Estimation of systematic uncertainties The solid blue line is a ﬁt to a Crystal-Ball function plus an exponential distribution, where this last contribution is shown by a dotted red line ) 2 c (GeV/ -e + e m 2 2.5 3 3.5 4 4.5 ) 2 c Dielectron counts / (50 MeV/ 0 20 40 60 80 100 120 ALICE p-Pb = 5.02 TeV NN s -e + e → ψ J/ < 0.8 y -0.8 < ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (30 MeV/ 0 50 100 150 200 250 ALICE p-Pb = 5.02 TeV NN s - μ + μ → ψ J/ < 0.8 y -0.8 < ) 2 c Dimuon counts / (50 MeV/ ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (50 MeV/ 0 20 40 60 80 100 120 140 ALICE p-Pb = 5.02 TeV NN s - μ + μ → ψ J/ < 2.7 y 1.2 < ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (50 MeV/ 0 5 10 15 20 25 30 35 40 ALICE Pb-p = 5.02 TeV NN s - μ + μ → ψ J/ < -1.2 y -2.5 < ) 2 c (GeV/ - μ + μ m Fig. 1 Mass distributions of selected dileptons for the dielectron (upper left) and dimuon (upper right) samples for the central analy- sis and dimuon samples for the semi-forward (lower left) and semi- backward (lower right) analyses. In all cases the data are represented by points with error bars. The solid blue line is a ﬁt to a Crystal-Ball function plus an exponential distribution, where this last contribution is shown by a dotted red line the efﬁciency in simulations, which was then used to esti- mate a systematic uncertainty of 1%. There is also a small discrepancy between the efﬁciency in data and in simula- tions around the trigger threshold. This gives a contribution of 1.7% (1.3%) for the p–Pb (Pb–p) period. The addition in quadrature of these two effects yields the uncertainty on muon triggering. of measurements performed on simulations with those from real data [35] and amounts to 2% (3%) for the p–Pb (Pb–p) period. 4.2 Estimation of systematic uncertainties Several sources of systematic uncertainty have been studied. They are discussed below, while their contribution to the uncertainty on the measured cross sections are summarised in Table 2. Figure 2 shows the distribution of transverse momen- tum for dilepton pairs having mass (ml+l−) in the range 2.9 < ml+l−< 3.2 GeV/c2 for all the samples, except for the dimuon case in the mid-rapidity analysis where the lower boundwasraisedto3.0GeV/c2 tomakeuseofthebettermass resolutionofthischannel.Inallcasesthetransversemomenta populate the region below about 1 GeV/c, as expected from exclusive photoproduction off protons. These distributions are used to extract the number of exclusive J/ψ, within the stipulated mass range, in each of the measured rapidity inter- vals. An extended binned likelihood method is used in ﬁts of data to a sum of templates for signal and background obtained from data (see Sect. 3.3) and from Monte Carlo (see Sect. 3.4) samples. These templates are also shown in the ﬁgure. The uncertainty on the tracking efﬁciency in the TPC was estimated repeating the analyses using different track selections. Four different values for the minimal number of points and three values for the minimum number of TPC pads crossed by the track were used. The systematic uncer- tainty related to the selection of tracks at mid-rapidity was estimated from the spread of the variations with respect to the standard selection. It varies from 0.8 to 5.7%. The uncertainty related to the identiﬁcation of electrons and muons using their energy deposition in TPC has been obtained using an alternative selection based directly on the energy loss [36]. The difference between the two methods was used as an estimate of the systematic uncertainty, and was at most 1.3%. Cross contamination from muon pairs in the electron sample and vice versa was found to be negligible. The number of candidates from the continuum dilepton template, which is a free parameter in the ﬁt, was found always to be less than what is obtained, in the given mass range, from the exponential part of the ﬁt to the mass distri- bution. This exponential distribution includes both the exclu- The uncertainty on the single muon tracking efﬁciency in themuonspectrometerwasobtainedbycomparingtheresults 12 3 Eur. Phys. J. 4.2 Estimation of systematic uncertainties C (2019) 79 :402 402 Page 6 of 18 402 Page 6 of 18 ) 2 c (GeV/ -e + e m 2 2.5 3 3.5 4 4.5 ) 2 c Dielectron counts / (50 MeV/ 0 20 40 60 80 100 120 ALICE p-Pb = 5.02 TeV NN s -e + e → ψ J/ < 0.8 y -0.8 < ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (30 MeV/ 0 50 100 150 200 250 ALICE p-Pb = 5.02 TeV NN s - μ + μ → ψ J/ < 0.8 y -0.8 < ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (50 MeV/ 0 20 40 60 80 100 120 140 ALICE p-Pb = 5.02 TeV NN s - μ + μ → ψ J/ < 2.7 y 1.2 < ) 2 c (GeV/ - μ + μ m 2 2.5 3 3.5 4 4.5 ) 2 c Dimuon counts / (50 MeV/ 0 5 10 15 20 25 30 35 40 ALICE Pb-p = 5.02 TeV NN s - μ + μ → ψ J/ < -1.2 y -2.5 < Fig. 1 Mass distributions of selected dileptons for the dielectron (upper left) and dimuon (upper right) samples for the central analy- sis and dimuon samples for the semi-forward (lower left) and semi- backward (lower right) analyses. In all cases the data are represented by points with error bars. 4.2 Estimation of systematic uncertainties 2 Transverse momentum distributions of dileptons around the J/ψ mass for the dielectron (upper left) and dimuon (upper right) sam- ples for the central analysis and dimuon samples for the semi-forward (lower left) and semi-backward (lower right) analyses. In all cases the data are represented by points with error bars. The blue, magenta (dash) and green (dash-dot-dot) lines correspond to Monte Carlo templates for J/ψ coming from exclusive photoproduction off protons or off lead and continuous dilepton production respectively. The red (dash-dot) line is a template for dissociative and hadronic background obtained from data. The solid black line is the sum of all contributions the back-to-back topology condition in the SPD and TOF The efﬁciency of the V0C to detect one muon from the ) c (GeV/ T p Dielectron 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 ) c Dielectron counts / (100 MeV/ 0 10 20 30 40 50 60 = 5.02 TeV NN s ALICE p-Pb -e + e → ψ J/ < 0.8 y -0.8 < 2 c < 3.2 GeV/ -e + e m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. -e + e → γ γ +Pb γ ) c (GeV/ T p Dimuon 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 ) c Dimuon counts / (80 MeV/ 0 20 40 60 80 100 = 5.02 TeV NN s ALICE p-Pb - μ + μ → ψ J/ < 0.8 y -0.8 < 2 c < 3.2 GeV/ - μ + μ m 3.0 < Sum ψ Exclusive J/ Non-exclusive bkg. -μ + μ → γ γ +Pb γ ) c (GeV/ T p Dielectron 1.2 1.4 1.6 1.8 2 ) c (GeV/ T p Dielectron ) c (GeV/ T p Dimuon ) c Dimuon counts / (200 MeV/ ) c (GeV/ T p Dimuon 0 0.5 1 1.5 2 2.5 ) c Dimuon counts / (150 MeV/ 0 10 20 30 40 50 60 70 80 = 5.02 TeV NN s ALICE p-Pb - μ + μ → ψ J/ < 2.7 y 1.2 < 2 c < 3.2 GeV/ - μ + μ m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. 4.2 Estimation of systematic uncertainties There is also a 0.5% contribution from variations on the conditions required to match the trigger and the tracking information of a given muon. The uncertainties related to triggering in the muon spec- trometer have been evaluated as in [36]. The efﬁciency maps of the trigger chambers have been obtained using data. The statistical uncertainty on this procedure has been used to vary The two main contributions to the uncertainty on the trigger efﬁciency for the mid-rapidity analysis come from 3 3 Eur. Phys. J. C (2019) 79 :402 Page 7 of 18 402 ) c (GeV/ T p Dielectron 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 ) c Dielectron counts / (100 MeV/ 0 10 20 30 40 50 60 = 5.02 TeV NN s ALICE p-Pb -e + e → ψ J/ < 0.8 y -0.8 < 2 c < 3.2 GeV/ -e + e m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. -e + e → γ γ +Pb γ ) c (GeV/ T p Dimuon 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 ) c Dimuon counts / (80 MeV/ 0 20 40 60 80 100 = 5.02 TeV NN s ALICE p-Pb - μ + μ → ψ J/ < 0.8 y -0.8 < 2 c < 3.2 GeV/ - μ + μ m 3.0 < Sum ψ Exclusive J/ Non-exclusive bkg. -μ + μ → γ γ +Pb γ ) c (GeV/ T p Dimuon 0 0.5 1 1.5 2 2.5 ) c Dimuon counts / (150 MeV/ 0 10 20 30 40 50 60 70 80 = 5.02 TeV NN s ALICE p-Pb - μ + μ → ψ J/ < 2.7 y 1.2 < 2 c < 3.2 GeV/ - μ + μ m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. -μ + μ → γ γ ) c (GeV/ T p Dimuon 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 ) c Dimuon counts / (200 MeV/ 0 5 10 15 20 25 30 35 40 = 5.02 TeV NN s ALICE Pb-p - μ + μ → ψ J/ < -1.2 y -2.5 < 2 c < 3.2 GeV/ - μ + μ m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. -μ + μ → γ γ Fig. 4.2 Estimation of systematic uncertainties The uncertainty varies from 1.9 to 3.6% (see “signal extraction” in Table 2). Table 1 Number of measured exclusive J/ψ, value of the efﬁciency and acceptance correction factor, as well as integrated luminosity for each rapidity interval. These values are used in Eq. (1) to compute the measured cross sections Table 1 Number of measured exclusive J/ψ, value of the efﬁciency and acceptance correction factor, as well as integrated luminosity for each rapidity interval. These values are used in Eq. (1) to compute the measured cross sections Rapidity NJ/ψ ε × A Lint (nb−1) (1.9, 2.7) 73 ± 15 0.071 3.147 (1.2, 1.9) 98 ± 17 0.086 3.147 (0.0, 0.8) Dielectron 106 ± 16 0.029 6.915 Dimuon 196 ± 17 0.057 6.915 (−0.8, 0.0) Dielectron 117 ± 13 0.033 6.915 Dimuon 199 ± 20 0.063 6.915 (−2.5, −1.2) 76 ± 11 0.037 3.743 The polarization of the J/ψ coming from ψ(2S) feed- down is not known. The uncertainty on the amount of feed- down has been estimated by assuming that the J/ψ was either not polarised or that it was fully transversely or fully longitu- dinally polarised. This uncertainty is asymmetric and varies from + 1.0 to −1.4% (see “feed-down” in Table 2). The uncertainty on the measurement of the luminosity has a contribution of 1.6%, which is correlated between the p–Pb and Pb–p data-taking periods and, in addition, an uncorre- lated part of 3.3% (3.0%) in the p–Pb (Pb–p) conﬁguration [37]. For the mid-rapidity analysis, which has data from both p–Pb and Pb–p, the uncorrelated part of the uncertainty on the luminosity amounts to 2.3%. the mass dependence (2.0%) yields an uncertainty of 3.4%. The veto efﬁciency of the V0 detector can also be estimated ofﬂine using a more complex algorithm than that used in the online trigger. To estimate the uncertainty on the use of V0 to veto extra activity, the analyses were compared using the online V0 decision only and requiring (standard selection) in addition the ofﬂine decision, with the difference giving the systematic uncertainty. The uncertainty varies from 1.2% to 3.5%. Furthermore, the uncertainties on the TPC selection, parti- cleidentiﬁcation,ofﬂineV0vetoefﬁciencyandsignalextrac- tion are uncorrelated across rapidity. The uncertainty on the ψ(2S) feed-down is correlated for all rapidities. The uncer- tainties on muon tracking, matching and trigger efﬁciencies are mostly uncorrelated across rapidity, and for the purposes of this analysis we treat the uncertainties as fully uncorre- lated. 4.2 Estimation of systematic uncertainties -μ + μ → γ γ ) c (GeV/ T p Dimuon 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 ) c Dimuon counts / (200 MeV/ 0 5 10 15 20 25 30 35 40 = 5.02 TeV NN s ALICE Pb-p - μ + μ → ψ J/ < -1.2 y -2.5 < 2 c < 3.2 GeV/ - μ + μ m 2.9 < Sum ψ Exclusive J/ Non-exclusive bkg. -μ + μ → γ γ (Ge T p Dimuon Fig. 2 Transverse momentum distributions of dileptons around the J/ψ mass for the dielectron (upper left) and dimuon (upper right) sam- ples for the central analysis and dimuon samples for the semi-forward (lower left) and semi-backward (lower right) analyses. In all cases the data are represented by points with error bars. The blue, magenta (dash) and green (dash-dot-dot) lines correspond to Monte Carlo templates for J/ψ coming from exclusive photoproduction off protons or off lead and continuous dilepton production respectively. The red (dash-dot) line is a template for dissociative and hadronic background obtained from data. The solid black line is the sum of all contributions The efﬁciency of the V0C to detect one muon from the J/ψ decay, which is needed in the semi-backward analy- sis, was estimated using events from the p–Pb period, whose trigger did not include this V0C requirement. The procedure was cross checked using the forward dimuon sample used for the analysis described in [19]. The efﬁciency depends slightly on the mass range used to compute it. The addi- tion in quadrature of the statistical uncertainty (2.7%) and the back-to-back topology condition in the SPD and TOF detectors. The ﬁrst one was estimated using a data sample obtained using a zero-bias trigger (all bunch crossings taken). It amounts to 4.5%. The second one was taken from the anal- ysis of [35]. It amounts to (−9%, + 3.8)%, using a zero bias trigger to compute the efﬁciency and comparing the result with the efﬁciency from simulated events. 12 3 3 Eur. Phys. J. C (2019) 79 :402 402 Page 8 of 18 402 Page 8 of 18 algorithms used to determine the non-exclusive template (see Sect. 3.3). Furthermore, the value of the b parameter used in the production of the exclusive J/ψ template was varied, taking into account the uncertainties reported by H1 [10]. 5 Results 5.1 Cross sections for exclusive production of J/ψ in p–Pb collisions 5.1 Cross sections for exclusive production of J/ψ in p–Pb collisions 5.1 Cross sections for exclusive production of J/ψ in p–Pb collisions The measured cross section for the exclusive production of J/ψ in collisions of protons with lead nuclei is computed according to The measured cross sections are given in Table 3, and also include the value of the centre-of mass energy of the photon- proton system, ⟨Wγ p⟩, computed as the average of Wγ p over the rapidity interval weighted by the photoproduction cross section predicted by STARLIGHT. dσ dy = NJ/ψ ε × A · BR(J/ψ →l+l−) · Lint · y (1) (1) 4.2 Estimation of systematic uncertainties The ﬁrst uncertainty in the cross sections is statistical, the second is sys- tematic and the third comes from the uncertainty in the photon ﬂux Table 3 Measured differential cross sections dσ/dy for exclusive J/ψ photoproduction off protons in ultra-peripheral p–Pb (positive rapidity values) and Pb–p (negative rapidity values) collisions at √sNN = 5.02 TeV in the different rapidity intervals. Values of the photon ﬂux kdn/dk, the interval in Wγ p corresponding to the rapidity, the average Wγ p Rapidity dσ dy (μb) k dn dk Wγ p (GeV) ⟨Wγ p⟩(GeV) σ(γ + p →J/ψ + p)(nb) (1.9, 2.7) 6.9 ± 1.4 ± 0.6 163.6 ± 1.5 (40.8, 60.9) 50.4 42 ± 9 ± 4 ± 1 (1.2, 1.9) 8.7 ± 1.5 ± 0.6 140.2 ± 1.5 (60.9, 86.4) 73.1 62 ± 11 ± 4 ± 1 (0.0, 0.8) 10.6 ± 0.8 ± 1.2 104.3 ± 1.5 (105.5, 157.4) 129.9 101 ± 8 ± 11 ± 1 (−0.8, 0.0) 10.0 ± 0.8 ± 1.1 79.4 ± 1.5 (157.4, 234.9) 193.3 126 ± 10 ± 14 ± 2 (−2.5, −1.2) 7.1 ± 1.0 ± 0.5 36.5 ± 1.4 (286.9, 549.5) 391.2 194 ± 27 ± 15 ± 7 5.2 Cross sections for exclusive photoproduction of J/ψ off protons within the statistical uncertainties. As a cross-check, the pos- sible contribution from incoherent production of J/ψ off the Pb nucleus was investigated, and found to be negligible. In addition, the effect on the ε × A correction of varying the slope parameter b within the HERA limits was also found to be negligible. The relation between dσ/dy and the cross section for the photoproduction of J/ψ off protons, σ(γ + p →J/ψ + p), is given by Systematic effects related to noise or pileup events in the ZDC are estimated using randomly triggered events and are also found to be negligible. dσ dy = k dn dk σ(γ + p →J/ψ + p) (2) (2) where k = 0.5×MJ/ψ exp (−y) is the photon energy in the laboratory frame and kdn/dk the ﬂux of photons with energy k emitted by the lead nucleus. Using STARLIGHT, the ﬂux has been computed in impact parameter space and convo- luted with the probability of no hadronic interaction. 4.2 Estimation of systematic uncertainties The uncertainty in the ﬂux is obtained varying the radius of the lead nucleus, used in the nuclear form factor, by ± 0.5 fm, which corresponds to the nuclear skin thickness. 5 Results 4.2 Estimation of systematic uncertainties (This includes also the measurements from [19].) The trigger efﬁciency at mid-rapidity is correlated between the (0.0,0.8) and (−0.8,0.0) rapidity intervals. The trigger conditions associated with the upper limits in the activity in TOF, SPD and V0C have a negligible effect on the systematic uncertainty, because the limits are set well above the levels of activity produced by the signal in our sample. The mid-rapidity analysis offers two other possibilities to cross check the consistency of the results. One can compare the results of the cross section in the p–Pb and Pb–p peri- ods, and one can compare the results of the electron and the muon decay channels. The cross sections agreed in all cases The systematic uncertainty on the yield was obtained by varying the range of ﬁt to the transverse momentum template, the width of the binning and the selections and smoothing Table 2 Summary of the contributions to the systematic uncertainty, in percent, for the J/ψ cross section in the different rapidity intervals (1.9, 2.7) (1.2, 1.9) (0.0, 0.8) (−0.8, 0.0) (−2.5, −1.2) TPC track selection 5.7 1.2 0.9 0.8 3.3 Particle identiﬁcation – – 1.3 0.6 – Muon tracking efﬁciency 2.0 2.0 – – 3.0 Muon matching efﬁciency 0.5 0.5 – – 0.5 Trigger efﬁciency 2.0 2.0 +5.9 −10.1 +5.9 −10.1 1.6 V0C Trigger efﬁciency – – – – 3.4 Ofﬂine V0 veto efﬁciency 2.7 3.5 2.1 2.1 1.2 Signal extraction 3.6 2.2 2.1 1.9 3.0 Feed-down +0.0 −1.3 1.0 +0.6 −1.0 +1.0 −0.6 +0.0 −1.4 Luminosity uncorrelated 3.3 3.3 2.3 2.3 3.0 Luminosity correlated 1.6 1.6 1.6 1.6 1.6 Branching ratio [38] 0.6 0.6 0.4 0.4 0.6 Total 8.7 6.4 11.1 11.0 7.7 ntributions to the systematic uncertainty, in percent, for the J/ψ cross section in the different rapidity intervals (1 9 2 7) (1 2 1 9) (0 0 0 8) ( 0 8 0 0) ( 2 5 12 Eur. Phys. J. C (2019) 79 :402 Page 9 of 18 age 9 of 18 402 402 (⟨Wγ p⟩) and the extracted cross section σ(γ + p →J/ψ + p). 5.3 Energy dependence of the exclusive photoproduction of J/ψ off protons The Bjorken x value corresponding to Wγ p is also displayed on the top of the ﬁgure, see text for details 2 10 3 10 +p) (nb) ψ J/ → +p γ( σ 5 − 10 4 − 10 3 − 10 2 − 10 x ALICE ALICE (PRL113 (2014) 232504) Power-law fit to ALICE data H1 ZEUS LHCb pp (W+ solutions) LHCb pp (W- solutions) CCT JMRT NLO STARLIGHT param. NLO BFKL CGC (IP-Sat, b-CGC) 20 30 40 50 60 2 10 2 10 × 2 3 10 (GeV) p γ W 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Models / fit to data Fig. 3 (Upper panel) ALICE data (red symbols) on exclusive photoproduction of J/ψ off protons as a function of the centre-of-mass energy of the photon–proton system Wγ p, obtained in collisions of protons and lead nuclei at √sNN = 5.02 TeV, including results from [19], compared to a power-law ﬁt, to data from HERA [9,11], to the solutions from LHCb [39] and to theoretical models (see text). The uncertainties are the quadratic sum of the statistical and systematic uncertainties. (Lower panel) Ratio of the models shown in the upper panel to the power law ﬁt through the ALICE data points. The Bjorken x value corresponding to Wγ p is also displayed on the top of the ﬁgure, see text for details 2 10 3 10 +p) (nb) ψ J/ → +p γ( σ 5 − 10 4 − 10 3 − 10 2 − 10 x ALICE ALICE (PRL113 (2014) 232504) Power-law fit to ALICE data H1 ZEUS LHCb pp (W+ solutions) LHCb pp (W- solutions) CCT JMRT NLO STARLIGHT param. NLO BFKL CGC (IP-Sat, b-CGC) 20 30 40 50 60 2 10 2 10 × 2 3 10 (GeV) p γ W 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 Models / fit to data x (GeV) p γ W δ = 0.70 ± 0.05 with a correlation of −0.06 between both parameters. The quality of the ﬁt is χ2 = 1.21 for 7 degrees of freedom. The value of the exponent is compatible to that found using previous ALICE data [19], as well as with that found by HERA experiments [9,11]. duction scale M2 = 2.39 GeV2 [44] (NLO(BFKL)). These are shown as bands in the ﬁgure. 5.3 Energy dependence of the exclusive photoproduction of J/ψ off protons A third model, based on the colour dipole approach, and incorporating the energy depen- dence of geometrical ﬂuctuations of the proton structure in the impact parameter plane [45] is also shown (CCT). The models are in reasonable agreement with our data. Finally the STARLIGHT parameterisation relies on a power-law ﬁt to ﬁxed-target and HERA data. This model also agrees with our measurement. The comparison of ALICE measurements to data from other experiments as well as to the results from different models is also shown in Fig. 3. HERA [9,11] and ALICE data are compatible within uncertainties. LHCb measured the exclusive production of J/ψ in pp collisions, where the photon source can not be identiﬁed. Thus the extraction of the photoproduction cross section is not possible without fur- ther assumptions. For each measurement they reported two solutions [14] which also agree with ALICE measurements. ALICE measurements are also compared to theory in Fig. 3. The JMRT group [42] has two computations, one is based on the leading-order (LO) result from [8] with the addition of some corrections to the cross section, while the second includes also the main contributions expected from a next-to-leading order (NLO) result. The parameters of both models have been obtained by a ﬁt to the same data and their energy dependence is rather similar, so only the NLO version is shown. Recently, three new studies have appeared, describ- ing the W(γ p) dependence of the exclusive J/ψ cross section in terms of a colour dipole model [43] (CGC) or of the BFKL evolution of HERA values (HERA Fit 2) with a photopro- The comparison of ALICE measurements to data from other experiments as well as to the results from different models is also shown in Fig. 3. HERA [9,11] and ALICE data are compatible within uncertainties. LHCb measured the exclusive production of J/ψ in pp collisions, where the photon source can not be identiﬁed. Thus the extraction of the photoproduction cross section is not possible without fur- ther assumptions. For each measurement they reported two solutions [14] which also agree with ALICE measurements. 5.3 Energy dependence of the exclusive photoproduction of J/ψ off protons where y represents the width of the rapidity region where the measurement is performed and the branching ratios are BR(J/ψ →e+e−) = (5.97 ± 0.03)% and BR(J/ψ → μ+μ−) = (5.96 ± 0.03)% according to [38]. The values that go into this equation are listed in Table 1, while the measured cross sections are given in Table 3. Figure 3 shows all ALICE measurements, including those from [19], for the exclusive photoproduction of J/ψ off pro- tons as a function of Wγ p. The data cover the Wγ p range from 24 to 706 GeV, which corresponds roughly to three orders of magnitude in x, from ∼2 × 10−2 to ∼2 × 10−5. The error bars are the quadratic sum of the statistical and the total systematic uncertainties. For the mid-rapidity analysis the measurements in the muon and electron channels are computed independently. The cross sections in the rapidity range −0.8 < y < 0 (0 < y < 0.8) are 10.7 ± 1.3 (11.1 ± 1.8) μb for the electron channel and 9.6 ± 1.0 (10.4 ± 1.0) μb for the muon channel, where the errors represent the statistical uncertainty. These measurements have been averaged, weighting each cross sec- tion with its statistical uncertainty. The same ﬁgure shows the result of a χ2-ﬁt of a power law N(Wγ p/W0)δ to the full set of ALICE data, with W0 = 90.0 GeV as was done before in HERA analyses [40]. The ﬁt takes into account the statistical and systematic uncertain- ties, according to the technique used by H1 in [41]. The parameters found by the ﬁt are N = 71.8 ± 4.1 nb and 123 12 402 Page 10 of 18 Eur. Phys. J. C (2019) 79 :402 402 Page 10 of 18 Fig. 3 (Upper panel) ALICE data (red symbols) on exclusive photoproduction of J/ψ off protons as a function of the centre-of-mass energy of the photon–proton system Wγ p, obtained in collisions of protons and lead nuclei at √sNN = 5.02 TeV, including results from [19], compared to a power-law ﬁt, to data from HERA [9,11], to the solutions from LHCb [39] and to theoretical models (see text). The uncertainties are the quadratic sum of the statistical and systematic uncertainties. (Lower panel) Ratio of the models shown in the upper panel to the power law ﬁt through the ALICE data points. 6 Summary of Korea (NRF), Republic of Korea; Ministry of Education and Sci- entiﬁc Research, Institute of Atomic Physics and Romanian National Agency for Science, Technology and Innovation, Romania; Joint Insti- tute for Nuclear Research (JINR), Ministry of Education and Science of the Russian Federation and National Research Centre Kurchatov Insti- tute, Russia; Ministry of Education, Science, Research and Sport of the Slovak Republic, Slovakia; National Research Foundation of South Africa, South Africa; Swedish Research Council (VR) and Knut & Alice Wallenberg Foundation (KAW), Sweden; European Organization for Nuclear Research, Switzerland; National Science and Technology Development Agency (NSDTA), Suranaree University of Technology (SUT) and Ofﬁce of the Higher Education Commission under NRU project of Thailand, Thailand; Turkish Atomic Energy Agency (TAEK), Turkey; National Academy of Sciences of Ukraine, Ukraine; Science and Technology Facilities Council (STFC), United Kingdom; National Science Foundation of the United States of America (NSF) and United States Department of Energy, Ofﬁce of Nuclear Physics (DOE NP), United States of America. Acknowledgements The ALICE Collaboration would like to thank all its engineers and technicians for their invaluable contributions to the construction of the experiment and the CERN accelerator teams for the outstanding performance of the LHC complex. The ALICE Collaboration gratefully acknowledges the resources and support pro- vided by all Grid centres and the Worldwide LHC Computing Grid (WLCG) collaboration. The ALICE Collaboration acknowledges the following funding agencies for their support in building and running the ALICE detector: A. I. 6 Summary The ALICE Collaboration has measured the photoproduction of J/ψ mesons off protons in p–Pb interactions. New mea- surements, summarised in Table 3, at central, semi-backward and semi-forward rapidities are added to those previously given at forward and backward rapidities. Each rapidity inter- val corresponds to a given energy for exclusive photopro- duction in photon–proton interactions. The data agree with the previous ALICE measurements at forward and backward rapidities, with the LHCb results in pp interactions and with previous HERA measurements over a smaller energy range. The ALICE measurements are consistent with a power law dependence σ(γ p →J/ψp) ∼W δ γ p, with δ = 0.70 ± 0.05. Several models, based on different physics assumptions, [ ] g ALICE measurements are also compared to theory in Fig. 3. The JMRT group [42] has two computations, one is based on the leading-order (LO) result from [8] with the addition of some corrections to the cross section, while the second includes also the main contributions expected from a next-to-leading order (NLO) result. The parameters of both models have been obtained by a ﬁt to the same data and their energy dependence is rather similar, so only the NLO version is shown. Recently, three new studies have appeared, describ- ing the W(γ p) dependence of the exclusive J/ψ cross section in terms of a colour dipole model [43] (CGC) or of the BFKL evolution of HERA values (HERA Fit 2) with a photopro- 12 123 123 Eur. Phys. J. C (2019) 79 :402 Page 11 of 18 402 402 reproduced this behaviour within the current experimental uncertainties. The model predictions around and above 1 TeV are not very precise although all tend to go below the extrap- olation of the ﬁt, as shown in the lower panel of Fig. 3. This energy range will be reachable with the new LHC data from Run 2 and the data to be collected in Run 3 and Run 4. The data presented here, augmented with the results from future measurements, will be a powerful tool to better understand the role of saturation at the highest energies. 6 Summary Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation (ANSL), State Committee of Science and World Federation of Scientists (WFS), Armenia; Austrian Academy of Sciences and Nationalstiftung für Forschung, Technolo- gie und Entwicklung, Austria; Ministry of Communications and High Technologies, National Nuclear Research Center, Azerbaijan; Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), Uni- versidade Federal do Rio Grande do Sul (UFRGS), Financiadora de Estudos e Projetos (Finep) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil; Ministry of Science & Technol- ogy of China (MSTC), National Natural Science Foundation of China (NSFC) and Ministry of Education of China (MOEC), China; Ministry of Science and Education, Croatia; Centro de Aplicaciones Tecnológ- icas y Desarrollo Nuclear (CEADEN), Cubaenergía, Cuba; Ministry of Education, Youth and Sports of the Czech Republic, Czech Repub- lic; The Danish Council for Independent Research \| Natural Sciences, the Carlsberg Foundation and Danish National Research Foundation (DNRF), Denmark; Helsinki Institute of Physics (HIP), Finland; Com- missariat à l’Energie Atomique (CEA) and Institut National de Physique Nucléaire et de Physique des Particules (IN2P3) and Centre National de la Recherche Scientiﬁque (CNRS), France; Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) and GSI Helmholtzzentrum für Schwerionenforschung GmbH, Germany; Gen- eral Secretariat for Research and Technology, Ministry of Education, Research and Religions, Greece; National Research, Development and Innovation Ofﬁce, Hungary; Department of Atomic Energy Govern- ment of India (DAE), Department of Science and Technology, Govern- ment of India (DST), University Grants Commission, Government of India (UGC) and Council of Scientiﬁc and Industrial Research (CSIR), India; Indonesian Institute of Science, Indonesia; Centro Fermi - Museo Storico della Fisica e Centro Studi e Ricerche Enrico Fermi and Isti- tuto Nazionale di Fisica Nucleare (INFN), Italy; Institute for Innovative Science and Technology, Nagasaki Institute of Applied Science (IIST), Japan Society for the Promotion of Science (JSPS) KAKENHI and Japanese Ministry of Education, Culture, Sports, Science and Technol- ogy (MEXT), Japan; Consejo Nacional de Ciencia (CONACYT) y Tec- nología, through Fondo de Cooperación Internacional en Ciencia y Tec- nología (FONCICYT) and Dirección General de Asuntos del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie voor Weten- schappelijk Onderzoek (NWO), Netherlands; The Research Council of Norway, Norway; Commission on Science and Technology for Sus- tainable Development in the South (COMSATS), Pakistan; Pontiﬁcia Universidad Católica del Perú, Peru; Ministry of Science and Higher Education and National Science Centre, Poland; Korea Institute of Sci- ence and Technology Information and National Research Foundation Data Availability Statement This manuscript has associated data in a data repository. 6 Summary [Authors’ comment: The numerical values of the data points will be uploaded to HEPData.] Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecomm ons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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C (2019) 79 :402 Page 15 of 18 402 Page 15 of 18 402 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 1 A.I. ALICE Collaboration Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 Bogolyubov Institute for Theoretical Physics National Academy of Sciences of Ukraine Kiev Ukraine 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), Kolkata, India 4 dk i f l h i ibi k i 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), Kolkata, India 4 Budker Institute for Nuclear Physics, Novosibirsk, Russia 3 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (C 4 4 Budker Institute for Nuclear Physics, Novosibirsk, Russia 4 Budker Institute for Nuclear Physics, Novosibirsk, Russia 5 California Polytechnic State University, San Luis Obispo, CA, USA 5 California Polytechnic State University, San Luis Obispo, CA, USA California Polytechnic State University, San Luis 6 Central China Normal University, Wuhan, China 6 Central China Normal University, Wuhan, China 7 Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 7 Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 8 Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Havana, Cuba 9 Centro de Investigación y de Estudios Avanzados (CINVESTAV), Mexico City and Mérida, M 10 Centro Fermi-Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi’, Rome, Italy 11 11 Chicago State University, Chicago, IL, USA 11 Chicago State University, Chicago, IL, USA 12 China Institute of Atomic Energy, Beijing, China 12 China Institute of Atomic Energy, Beijing, China 13 Chonbuk National University, Jeonju, Republic of Korea 13 Chonbuk National University, Jeonju, Republic of Korea 14 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovakia 15 COMSATS I i f I f i T h l (CIIT) I l b d P ki 14 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatic 15 COMSATS Institute of Information Technology (CIIT), Islamabad, Pakistan 16 Creighton University, Omaha, NE, USA 16 Creighton University, Omaha, NE, USA 16 Creighton University, Omaha, NE, USA g y 17 Department of Physics, Aligarh Muslim University, Aligarh, India 17 Department of Physics, Aligarh Muslim University, Aligarh, India Physics, Aligarh Muslim University, Aligarh, Indi 18 Department of Physics, Pusan National University, Pusan, Republic of Kor 19 Department of Physics, Sejong University, Seoul, Republic of Korea 20 Department of Physics, University of California, Berkeley, CA, U 20 Department of Physics, University of California, Berkeley, CA, USA 21 Department of Physics, University of Oslo, Oslo, Norway 21 Department of Physics, University of Oslo, Oslo, Norway 123 12 123 402 Page 16 of 18 Eur. Phys. J. C (2019) 79 :402 402 Page 16 of 18 22 Department of Physics and Technology, University of Bergen, Bergen, Norway 23 Dipartimento di Fisica dell’Università ’La Sapienza’ and Sezione INFN, Rome, Italy 24 Dipartimento di Fisica dell’Università and Sezione INFN, Cagliari, Italy 25 Dipartimento di Fisica dell’Università and Sezione INFN, Trieste, Italy 26 Dipartimento di Fisica dell’Università and Sezione INFN, Turin, Italy 27 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Bologna, Italy 28 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Catania, Italy 29 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Padova, Italy 30 Dipartimento di Fisica ‘E.R. Caianiello’ dell’Università and Gruppo Collegato INFN, Saler 31 Dipartimento DISAT del Politecnico and Sezione INFN, Turin, Italy 32 Dipartimento di Scienze e Innovazione Tecnologica dell’Università del Piemonte Orientale and INFN Sezione di Torino, Alessandria, Italy 32 Dipartimento di Scienze e Innovazione Tecnologica dell’Università del Piemonte Orientale and INFN Sezione di Torino, Alessandria, Italy 33 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy 34 European Organization for Nuclear Research (CERN), Geneva, Switzerland 34 European Organization for Nuclear Research (CERN), Geneva, Switzerland y g g g g y p p 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 36 Faculty of Engineering and Science, Western Norway University of Applied Sciences, Bergen, Norway 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in P 37 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic 38 Faculty of Science, P.J. Šafárik University, Košice, Slovakia 38 Faculty of Science, P.J. 16 Creighton University, Omaha, NE, USA Šafárik University, Košice, Slovakia 39 Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universität Frankfurt, Frankfu e for Advanced Studies, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 39 Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universität Frankfurt, 39 Frankfurt Institute for Advanced Studies, Johann Wolfgang G 40 Gangneung-Wonju National University, Gangneung, Republic of Korea 40 Gangneung-Wonju National University, Gangneung, Republic of Korea 41 Department of Physics, Gauhati University, Guwahati, India 41 Department of Physics, Gauhati University, Guwahati, India p y y 42 Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany 43 42 Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany 43 mholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bon 42 Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische 43 Helsinki Institute of Physics (HIP), Helsinki, Finland 43 Helsinki Institute of Physics (HIP), Helsinki, Finland 44 High Energy Physics Group, Universidad Autónoma de Puebla, Puebla, Mexico 45 44 High Energy Physics Group, Universidad Autónoma de Puebla, Puebla, Mex 44 High Energy Physics Group, Universidad A 45 Hiroshima University, Hiroshima, Japan 46 Hochschule Worms, Zentrum für Technologietransfer und Telekommunikation (ZTT), Worms 47 , g ( ), , G y 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 47 Horia Hulubei National Institute of Physics and Nuclear Engineer 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 48 Indian Institute of Technology Bombay (IIT), Mumbai, India 48 Indian Institute of Technology Bombay (IIT), Mumbai, India 49 Indian Institute of Technology Indore, Indore, India 49 Indian Institute of Technology Indore, Indore, India 50 Indonesian Institute of Sciences, Jakarta, Indonesia 50 Indonesian Institute of Sciences, Jakarta, Indonesia 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 52 INFN, Sezione di Bari, Bari, Italy 53 INFN, Sezione di Bologna, Bologna, Ital 53 INFN, Sezione di Bologna, Bologna, Italy 54 INFN, Sezione di Cagliari, Cagliari, Italy 55 INFN, Sezione di Catania, Catania, Italy 56 INFN, Sezione di Padova, Padova, Italy 57 INFN, Sezione di Roma, Rome, Italy 5 58 INFN, Sezione di Torino, Turin, Italy 59 INFN, Sezione di Trieste, Trieste, Italy 60 Inha University, Incheon, Republic of Korea y p 61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des Particules p cléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des Particules 61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Phy 61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire 61 Institut de Physique Nucléaire d’Orsay (IPNO (IN2P3/CNRS), Université Paris-Sud, Université Paris-Saclay, Orsay, France (IN2P3/CNRS), Université Paris-Sud, Université Paris-Saclay, Orsay, France 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, Netherlands 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, Ne 64 Institute for Theoretical and Experimental Physics, Moscow, Russia 64 Institute for Theoretical and Experimental Physics, Moscow, Russia 65 Institute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovakia 65 Institute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovakia 66 Institute of Physics, Homi Bhabha National Institute, Bhubaneswar, India 66 Institute of Physics, Homi Bhabha National Institute, Bhubaneswar, India 67 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 67 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 68 Institute of Space Science (ISS), Bucharest, Romania 69 Institut für Kernphysik, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 69 Institut für Kernphysik, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 71 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 71 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 72 Instituto de Física, Universidad Nacional Autónoma de México, Mexico City, Mexico 72 Instituto de Física, Universidad Nacional Autónoma de México, Mexico City, Mexico 12 3 Eur. 16 Creighton University, Omaha, NE, USA Phys. J. 16 Creighton University, Omaha, NE, USA C (2019) 79 :402 Page 17 of 18 402 Page 17 of 18 402 402 73 iThemba LABS, National Research Foundation, Somerset West, South Africa 73 iThemba LABS, National Research Foundation, Somerset West, South Africa 74 Johann-Wolfgang-Goethe Universität Frankfurt Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany 74 Johann-Wolfgang-Goethe Universität Frankfurt Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany 75 Joint Institute for Nuclear Research (JINR), Dubna, Russia 75 Joint Institute for Nuclear Research (JINR), Dubna, Russia 76 Korea Institute of Science and Technology Information, Daejeon, Republic of Korea 76 Korea Institute of Science and Technology Information, Daejeon, Republic of Korea 76 Korea Institute of Science and Technology 77 KTO Karatay University, Konya, Turkey 78 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS-I 78 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes 78 Laboratoire de Physique Subatomique et 78 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS-IN2P3, Grenoble, France 79 Lawrence Berkeley National Laboratory Berkeley CA USA 79 Lawrence Berkeley National Laboratory, Berkeley, CA, USA 79 Lawrence Berkeley National Laboratory, Berkeley, CA, USA 80 Lund University Department of Physics, Division of Particle Physics, Lund, Sweden 80 Lund University Department of Physics, Division of Particle Physics, Lund, Sweden 81 Nagasaki Institute of Applied Science, Nagasaki, Japan 81 Nagasaki Institute of Applied Science, Nagasaki, Japan 82 Nara Women’s University (NWU), Nara, Japan 83 Department of Physics, National and Kapodistrian Univer 83 Department of Physics, National and Kapodistrian University of Athens, School of S 83 Department of Physics, National and Kapodistrian University of Athens, School of Science, Athens, Greece 84 National Centre for Nuclear Research, Warsaw, Poland 84 National Centre for Nuclear Research, Warsaw, Poland 85 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India National Institute of Science Education and Resea 85 National Institute of Science Education and Research, Homi Bhabha National Institute, Jatni, India ational Nuclear Research Center, Baku, Azerbaija 87 National Research Centre Kurchatov Institute, Moscow 87 National Research Centre Kurchatov Institute, Moscow, Russia 88 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 88 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89 89 Nikhef, National Institute for Subatomic Physics, Amsterdam, The Netherlands 89 Nikhef, National Institute for Subatomic Physics, Amsterdam, The Nether 90 NRC Kurchatov Institute IHEP, Protvino, Russia 90 NRC Kurchatov Institute IHEP, Protvino, Russia 91 NRNU Moscow Engineering Physics Institute, Moscow, Russia 91 NRNU Moscow Engineering Physics Institute, Moscow, Russia 92 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 92 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, 93 Nuclear Physics Institute of the Czech Academy of Sciences, ˇRežu Prahy, Czech Republic 93 Nuclear Physics Institute of the Czech Academy of Sciences, ˇRežu Prahy, Czech Re 94 Oak Ridge National Laboratory, Oak Ridge, TN, USA 94 Oak Ridge National Laboratory, Oak Ridge, TN, USA 95 Ohio State University, Columbus, OH, USA 96 Petersburg Nuclear Physics Institute, Gatchina, Russia 96 Petersburg Nuclear Physics Institute, Gatchina, Russia 97 Physics Department, Faculty of science, University of Zagreb, Zagreb, Croatia 98 97 Physics Department, Faculty of science, University of Zagreb, Zagreb, Croatia 98 98 Physics Department, Panjab University, Chandigarh, India 98 Physics Department, Panjab University, Chandigarh, India 99 Physics Department, University of Jammu, Jammu, India 99 Physics Department, University of Jammu, Jammu, India 100 Physics Department, University of Rajasthan, Jaipur, India 01 Physikalisches Institut, Eberhard Karls Universität Tübingen, Tübingen, Germany 101 Physikalisches Institut, Eberhard Karls Universität Tübingen, Tübingen, Germany 102 Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 102 Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 103 Physik Department, Technische Universität München, Munich, Germany 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany 105 Rudjer Boškovi´c Institute, Zagreb, Croatia 106 106 Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 106 Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 107 Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, Indi 07 Saha Institute of Nuclear Physics, Homi Bhabha National Institute, Kolkata, India 08 08 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 108 School of Physics and Astronomy, University of Birmingham, Birmingham, Uni 09 Sección Física, Departamento de Ciencias, Pontiﬁcia Universidad Católica del Perú, Lima, Peru 109 Sección Física, Departamento de Ciencias, Pontiﬁcia Universidad Católica del Pe 110 Shanghai Institute of Applied Physics, Shanghai, China 110 Shanghai Institute of Applied Physics, Shanghai, China 111 St. 145 Yonsei University, Seoul, Republic of Korea 145 Yonsei University, Seoul, Republic of Korea 145 Yonsei University, Seoul, Republic of Kore 144 Yale University, New Haven, CT, USA 144 Yale University, New Haven, CT, USA 144 Yale University, New Haven, CT, USA 16 Creighton University, Omaha, NE, USA C (2019) 79 :402 402 Page 18 of 18 402 Page 18 of 18 124 University of Cape Town, Cape Town, South Africa 124 University of Cape Town, Cape Town, South Africa 125 University of Houston, Houston, TX, USA 126 University of Jyväskylä, Jyväskylä, Finland 127 University of Liverpool, Liverpool, United Kingdom 127 University of Liverpool, Liverpool, United Kingdom 128 University of Tennessee, Knoxville, TN, USA 129 University of the Witwatersrand, Johannesburg, South Africa 129 University of the Witwatersrand, Johannesburg, South Africa 130 University of Tokyo, Tokyo, Japan 131 University of Tsukuba, Tsukuba, Japan 32 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 132 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 133 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France 133 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, Lyon, France 133 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurbanne, L y y y 34 Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France 134 Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France 135 Department de Physique Nucléaire (DPhN), Université Paris-Saclay Centre dÉtudes de Saclay (CEA), France 135 Department de Physique Nucléaire (DPhN), Université Paris-Saclay Centre dÉtudes de Saclay (CEA), IRFU, Saclay, France 136 Università degli Studi di Foggia, Foggia, Italy 137 136 Università degli Studi di Foggia, Foggia, Italy 137 Università degli Studi di Pavia and Sezione INFN, Pavia, Italy 137 Università degli Studi di Pavia and Sezione INFN, Pavia, Italy 138 Università di Brescia and Sezione INFN, Brescia, Italy 138 Università di Brescia and Sezione INFN, Brescia, Italy 139 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, India 139 Variable Energy Cyclotron Centre, Homi Bhabha National Institute, Kolkata, In 140 Warsaw University of Technology, Warsaw, Poland 140 Warsaw University of Technology, Warsaw, Poland 141 Wayne State University, Detroit, MI, USA 142 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https://openalex.org/W4309011020	https://www.hal.inserm.fr/inserm-03920076/file/fpubh-10-978627.pdf	English	null	Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic fibrosis population in France during 2020 (MUCONFIN)	Frontiers in public health	2,022	cc-by	13,759	To cite this version: Nadia Oubaya, Thibaud Pombet, Celine Delestrain, Natascha Remus, Benoit Douvry, et al.. Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic fibrosis population in France during 2020 (MUCONFIN). Frontiers in Public Health, 2022, 10, pp.978627. 10.3389/fpubh.2022.978627. inserm-03920076 Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic fibrosis population in France during 2020 (MUCONFIN) Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic fibrosis population in France during 2020 (MUCONFIN) Nadia Oubaya, Thibaud Pombet, Celine Delestrain, Natascha Remus, Benoit Douvry, Dominique Grenet, Harriet Corvol, Guillaume Thouvenin, Virginie Prulière-Escabasse, Hakima Mounir, et al. Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN) Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN) OPEN ACCESS EDITED BY Rona Macniven, University of New South Wales, Australia REVIEWED BY Sarvodaya Tripathy, Independent Researcher, Lucknow, India Estelle Michinov, University of Rennes 2–Upper Brittany, France CORRESPONDENCE Ralph Epaud ralph.epaud@chicreteil.fr †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Life-Course Epidemiology and Social Inequalities in Health, a section of the journal Frontiers in Public Health RECEIVED 26 June 2022 ACCEPTED 19 October 2022 PUBLISHED 14 November 2022 CITATION Oubaya N, Pombet T, Delestrain C, Remus N, Douvry B, Grenet D, Corvol H, Thouvenin G, Prulière-Escabasse V, Mounir H, Argoud D, Fretigne C, Costes L, Mackiewicz M-P, Jung C, Ahamada L, Lanone S, Maitre B, Bégot A-C and Epaud R (2022) Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN). Front. Public Health 10:978627. doi: 10.3389/fpubh.2022.978627 OPEN ACCESS EDITED BY Rona Macniven, University of New South Wales, Australia REVIEWED BY Sarvodaya Tripathy, Independent Researcher, Lucknow, India Estelle Michinov, University of Rennes 2–Upper Brittany, France CORRESPONDENCE Ralph Epaud ralph.epaud@chicreteil.fr †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Life-Course Epidemiology and Social Inequalities in Health, a section of the journal Frontiers in Public Health RECEIVED 26 June 2022 ACCEPTED 19 October 2022 PUBLISHED 14 November 2022 CITATION Oubaya N, Pombet T, Delestrain C, Remus N, Douvry B, Grenet D, Corvol H, Thouvenin G, Prulière-Escabasse V, Mounir H, Argoud D, Fretigne C, Costes L, Mackiewicz M-P, Jung C, Ahamada L, Lanone S, Maitre B, Bégot A-C and Epaud R (2022) Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN). Front. Public Health 10:978627. Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN) doi: 10.3389/fpubh.2022.978627 COPYRIGHT © 2022 O b P b t D l t i Nadia Oubaya1,2†, Thibaud Pombet3,4†, Celine Delestrain2,5,6,7†, Natascha Remus5,6, Benoit Douvry6,8, Dominique Grenet9, Harriet Corvol10,11, Guillaume Thouvenin10, Virginie Prulière-Escabasse2,6,12, Hakima Mounir3, Dominique Argoud3, Cédric Fretigne3, Laurence Costes3, Marie-Pierre Mackiewicz3, Camille Jung13, Laitissia Ahamada13, Sophie Lanone2,6,7, Bernard Maitre2,6,7,8, Anne-Cécile Bégot3 and Ralph Epaud2,5,6,7* 1Department of Public Health, AP-HP, Hôpitaux Henri-Mondor, Créteil, France, 2University Paris Est Créteil, INSERM, IMRB, Créteil, France, 3Laboratoire Interdisciplinaire de Recherche sur les Transformations des Pratiques Éducatives et des Pratiques Sociales (LIRTES)-EA7313, Université Paris-Est Créteil Val-de-Marne, Créteil, France, 4Faculté d’Éducation et de Formation, Institut Catholique de Paris (ICP), Paris, France, 5Centre Hospitalier Intercommunal de Créteil, Service de Pédiatrie Générale, Créteil, France, 6Centre des Maladies Respiratoires Rares (RESPIRARE®), CRCM, Créteil, France, 7Fédérations Hospitalo-Universitaires (FHU) Role of SENEscence in Chronic Diseases (SENEC), Créteil, France, 8Centre Hospitalier Intercommunal de Créteil, Service de Pneumologie, Créteil, France, 9Service de Pneumologie, CRCM-Centre de Transplantation Pulmonaire, Hôpital Foch, Suresnes, France, 10Assistance Publique-Hôpitaux de Paris, Hôpital Trousseau, Pediatric Pulmonary Department, Paris, France, 11Sorbonne Université, Institut National de la Santé et de la Recherche Médicale, Centre de Recherche Saint Antoine, Paris, France, 12Centre Hospitalier Intercommunal de Créteil, Service d’ORL, Créteil, France, 13Clinical Research Centre, Intercommunal Hospital of Créteil, Créteil, France Oubaya N, Pombet T, Delestrain C, Remus N, Douvry B, Grenet D, Corvol H, Thouvenin G, Prulière-Escabasse V, Mounir H, Argoud D, Fretigne C, Costes L, Mackiewicz M-P, Jung C, Ahamada L, Lanone S, Maitre B, Bégot A-C and Epaud R (2022) Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN). Front. Public Health 10:978627. doi: 10.3389/fpubh.2022.978627 COPYRIGHT © 2022 Oubaya, Pombet, Delestrain, Remus, Douvry, Grenet, Corvol, Thouvenin, Prulière-Escabasse, Mounir, Argoud, Fretigne, Costes, Mackiewicz, Jung, Ahamada, Lanone, Maitre, Bégot and Epaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Background: Most of the studies on cystic ﬁbrosis (CF) focused on SARS-CoV-2 prevalence and suggested a low incidence of infection in this population. 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TYPE Original Research PUBLISHED 14 November 2022 DOI 10.3389/fpubh.2022.978627 TYPE Original Research PUBLISHED 14 November 2022 DOI 10.3389/fpubh.2022.978627 Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN) We aimed to assess the impact of the pandemic and related lockdown measures implemented in May 2020 in response to the ﬁrst wave of SARS-CoV-2 infection on healthcare access, health, and behavior in CF patients. Methods: A national questionnaire opened online from May 15th, 2020 to June 11th, 2020 was completed by 751 CF-patients, aged 14 years and over. It comprised questions about access to healthcare, anxiety and depression, smoking, alcohol, drug and psychotropic drug consumption, adherence to CF treatment, and constraints. A semi-structured comprehensive interview was performed no later than 1 month after the end of the lockdown in 16 CF-patients. 01 frontiersin.org Frontiers in Public Health Oubaya et al. 10.3389/fpubh.2022.978627 Results: The mean age of the population was 28.0 [interquartile range (IQR) 20.0–37.0] years old. More than 75% of in-person consultations scheduled during the lockdown were canceled. Alternatively, 27% were postponed, and telehealth consultations were proposed and accepted in almost 40% of cases. More than 75% of the scheduled physiotherapy sessions were canceled and replaced mainly by self-drainage. Annual follow-up clinic visits were consistently postponed whereas required hospitalizations at CF centers for exacerbation were maintained in most cases. While 43.2% CF-patients had signs of anxiety, 51.0% presented symptoms of depression, both associated with increased use of psychotic medications and inversely correlated to COVID-19 prevalence. Among the lower and lower middle classes, very little medical information was obtained or requested by the patient, participation to sports or other activities was low, while excessive home conﬁnement and isolation were more frequent. In contrast, in the upper middle and upper classes, individuals solicitated help to their CF centre, had more physical activities, and maintained contact with friends or families. Conclusion: The ﬁrst lockdown in France had only minimal impact on the management care of CF-patients but was associated with increased symptoms of anxiety and depression, together with behavioral changes that varied with social class. Trial registration: NCT04463628. KEYWORDS cystic ﬁbrosis, healthcare, COVID-19, lockdown, anxiety Results: The mean age of the population was 28.0 [interquartile range (IQR) 20.0–37.0] years old. More than 75% of in-person consultations scheduled during the lockdown were canceled. Alternatively, 27% were postponed, and telehealth consultations were proposed and accepted in almost 40% of cases. More than 75% of the scheduled physiotherapy sessions were canceled and replaced mainly by self-drainage. Introduction of standard protocols and the centralization of services delivered by highly trained, multidisciplinary teams have contributed to prevention of the progressive deterioration of respiratory function described in the older CF literature. Despite apparent similarities in access to guideline-based care at an accredited CF Centre, evidence is conﬂicting regarding the potential role socioeconomic status, level of education but also racial and ethnic disparities as shown from a study in a CF centre in New York City (USA) (5, 6). The discovery of a new form of pneumonia in early December 2019 in Wuhan, Hubei Province, followed by the rapid spread of the virus in China and across all continents, has drastically changed the face of healthcare throughout the world (1). By March 2020, France had the second highest number of SARS-CoV-2 infections and the greatest number of deaths in Europe, which led the French authorities to initiate a strict lockdown from March 17th to May 10th, 2020. Pandemic viral illnesses are challenging for patients with pre-existing lung disease. Although it has been shown that virus pandemic such as H1N1 may cause signiﬁcant morbidity in patient with CF (7, 8), the impact of SARS-CoV-2 infection on these patients was uncertain at the time of the ﬁrst wave. Nevertheless, co-morbidities such as underlying respiratory problems were identiﬁed as risk factors for severe COVID- 19 disease, and given the serious respiratory complications caused by viral infections (7, 9), CF-patients were expected to be at higher risk of severity. Surprisingly, the ﬁrst reports in Europe suggested a lower impact overall than initially feared for these patients (6, 10). Since then, several publications have shown, probably due to a higher degree of contagiousness of the virus, that SARS-CoV-2 is not a benign disease for Cystic ﬁbrosis (CF) is the most common autosomal recessive disease that leads to early mortality in Caucasians, and aﬀects around 7,500 patients in France (2). Lung disease remains the major cause of morbidity and mortality in CF, with a progressive decline of lung function due to a vicious cycle of airway infections and inﬂammation (3). In France, the national newborn screening program, established in 2002, has been associated with the accreditation of specialized CF care centres (CRCM: Centre de Ressources et de Compétences de la Mucoviscidose). Impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behavior of the cystic ﬁbrosis population in France during 2020 (MUCONFIN) Annual follow-up clinic visits were consistently postponed whereas required hospitalizations at CF centers for exacerbation were maintained in most cases. While 43.2% CF-patients had signs of anxiety, 51.0% presented symptoms of depression, both associated with increased use of psychotic medications and inversely correlated to COVID-19 prevalence. Among the lower and lower middle classes, very little medical information was obtained or requested by the patient, participation to sports or other activities was low, while excessive home conﬁnement and isolation were more frequent. In contrast, in the upper middle and upper classes, individuals solicitated help to their CF centre, had more physical activities, and maintained contact with friends or families. Conclusion: The ﬁrst lockdown in France had only minimal impact on the management care of CF-patients but was associated with increased symptoms of anxiety and depression, together with behavioral changes that varied with social class. Trial registration: NCT04463628. Trial registration: NCT04463628. KEYWORDS KEYWORDS cystic ﬁbrosis, healthcare, COVID-19, lockdown, anxiety Introduction These centers are covering the entire population of patients in France based on their geographic location and are dedicated to the close follow-up of CF- patients from diagnosis to adulthood (4). The implementation Frontiers in Public Health frontiersin.org 02 10.3389/fpubh.2022.978627 Oubaya et al. characteristics (gender, age, education, employment, marital status), (2) access to healthcare during lockdown deﬁned as follows: cancellation or rescheduling of consultations (with the doctor or the physiotherapist) by the healthcare professional or by the patient, cancellation by the patient of telehealth, cancellation or rescheduling by the hospital or by the patient of hospitalisations (planned or not) and change in the route of administration of antibiotics (oral administration instead of intravenous), (3) Compliance to medical treatment and airway clearance as assessed by the adherence score (12). The results were categorized as good compliance if the number of “yes” = 0, minor non-compliance for 1 or 2 “yes” and non-compliance for 2 or 3 “yes”; (4) Assessment of anxiety and depression: participants were asked to complete the 7-Item Generalized Anxiety Disorder Scale (GAD-7) (13) and the validated French version of 9-item Patient Health Questionnaire Depression Scale (PHQ-9) (14, 15). The total GAD-7 score ranges from 0 to 21, with a cut oﬀof 10 indicating the presence of signiﬁcant anxiety symptoms. GAD-7 scores of 5, 10, and 15 represent cut- oﬀpoints for mild, moderate, and severe anxiety, respectively. The PHQ-9 score is composed of nine depressive symptom items listed in the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSMIV) for depression and ranges from 0 to 27 (14). PHQ-9 scores of 5, 10, 15, and 20 represent cut-oﬀpoints for mild, moderate, moderately severe, and severe depression, respectively (14); (4) Assessment of quality of life using the speciﬁc CF CFQ 14 questionnaire that was adapted to the context of lockdown; (5) Knowledge and concern about COVID-19 and the declared prevalence of COVID-19 infections suspected or conﬁrmed. “COVID-19 dangerous for me,” “COVID-19 dangerous for relatives,” constraints of barrier measures, constraints of lockdown, were assessed using a scale from 1 to 10, with 1 being “very low constraining” and 10 being “very constraining.” all people in this patient group (11). Design and population This is a French national multicentre cross-sectional study including both quantitative and qualitative analysis. The inclusion criteria were as follows: CF-patients with a chloride sweat test >60 mEq/L and/or 2 CFTR gene variants, 14 years- old and over, followed at one of the French CF reference centres (CRCM), covered by the national health insurance system, and willing to participate to the study (non-opposition from the patient if aged >18 years and non-opposition from the parents if the patient is <18 years old). Qualitative study The questionnaire was complemented by a semi-structured comprehensive interview (16, 17) performed by Visio conference between the end of the lockdown and up to 1 month later. A sample of 16 CF-patients representative of the French population (with regard to age, sex and occupation) according to the data produced by the French Cystic Fibrosis Registry was selected from three centers: mixed (Créteil), pediatric (Trousseau, Paris) and adult (Foch, Suresnes). The diﬀerent topics discussed during the interview focused on the ﬁrst lockdown period and included the following: experience with the disease, treatment, care, accessibility to medical facilities, modalities and organization of work or schooling, lockdown conditions (with family or friends), organization of daily life, sports, and social networks. The interview ended with the collection of socio-demographic data. Introduction Nonetheless, it rapidly became evident that the impact of COVID-19 on CF-patients was not limited to the consequences of the infection but also resulted from the necessary measures taken to limit the spread of the disease such as isolation, quarantine, social distancing and community containment. These measures, particularly the national lockdown, aﬀected daily life due to the closure of school, shops and others, but also aﬀected the hospitals, which had to profoundly change their procedures and almost entirely focus on the care of COVID-19 patients. This forced transformation was at the expense of other critical functions, including the management of chronic diseases such as CF. Thus, the eﬀects of the pandemic were far reaching and may have aﬀected daily life, work performance, access to care, and mental ﬁtness. In this context, the present study more speciﬁcally addresses the impact of the COVID-19 pandemic and associated lockdown measures on the management, health, and behaviors of CF- patients during the ﬁrst wave of COVID-19 in France. Our results are based on a national survey from patients over 14 years old using web questionnaires. This quantitative study was completed by a qualitative study including representative CF-patients who were interviewed by social science and humanities researchers. Secondary outcomes The secondary outcomes included: (1) Compliance to treatment plans and airway clearance, (2) Assessment of anxiety and depression, (3) Assessment of quality of life, (4) Knowledge and concern about COVID-19 and the declared prevalence of COVID-19 infections suspected or conﬁrmed, (5) The experience and social representations of the lockdown in CF- patients as assessed by qualitative qualitative methodology), (6) The impact of the lockdown on social inequality (assessed by qualitative methodology). Primary outcome Primary outcome The primary outcome was the reduction to healthcare access. The primary outcome was the reduction to healthcare access. Data analysis working hypothesis and the conditions for carrying out the interview. Each interview was fully transcribed, and the analysis of the interviews was led by two members of the team. Interviews were subjected to content analysis, in particular to a thematic analysis 22. Characteristics, such as the occupation, were crossed with the last diploma to inform socially diﬀerentiated practices. Trends were identiﬁed according to three social classes: working or lower class (workers, employees), middle class (intermediate professions) and upper class (executives and higher intellectual professions, company directors). Students were deﬁned by their parents’ class. Individuals belonging to the middle class were diﬀerentiated based on their diploma. Thus, we created the lower-middle class (2 years post-graduation) and the upper-middle class (3 and 4 years post-graduation). Quantitative analysis The results were reported according to the STROBE guidelines for observational studies. Sociodemographic characteristics of the population were described using numbers (percentages) for categorical variables and mean ± standard deviation (SD) or median [interquartile range (IQR)], as appropriate, for quantitative variables. For the primary outcome, the proportion of patients with a reduction in healthcare access during lockdown was described by percentage and 95% conﬁdence interval. The other outcomes were described similarly than sociodemographic characteristics (2, 18). Associations between the primary outcome and, respectively, age, sex, occupation, and geographic area (Grand Est region vs. rest of the territory) and between age and, respectively, anxiety/depression, treatment, alcohol and smoking behaviors were assessed using Chi2 tests or Fisher’s exact tests. Maps were built to represent anxiety, depression scores and concern and constraints about COVID-19; median scores were used in each geographic area. A map of the prevalence of COVID-19 in the general population in France at time of the questionnaire survey was built as a reference to visually compare scores between geographic areas, taking into account the prevalence of COVID-19 in these areas. All signiﬁcance tests were two-tailed, and the threshold for statistical signiﬁcance level was set to 5%. All analyses were performed with Stata software (v16.0 StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC.) and R software (R Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2020). Quantitative study CF-patients had to ﬁll out an online questionnaire. The study was advertised with the support of all the CRCM and the patient association “Vaincre la Mucoviscidose” via websites and social networks such as Facebook and Twitter, as well as via emails sent by the CRCM. Patients could access the questionnaire using a generic link available in the announcement of the study. The questionnaire was open from May 15th, 2020 to June 11th, 2020 and comprised the following parts: (1) Sociodemographic Frontiers in Public Health frontiersin.org 03 Oubaya et al. 10.3389/fpubh.2022.978627 TABLE 1 Baseline characteristics of the study population. Sample size Assuming 80% patients would have reduced health access, i.e., 20% with health access, 1,200 patients were needed to estimate this primary outcome with a precision of ±2%, with a two-tailed alpha risk of 5%. The inclusions were stopped after a month even though the sample size was not reached as the responses became scarce and the time period became far from the end of lockdown. Outcomes No. (%) Total (N = 725) Age, med [IQR] 28.0 [20.0; 37.0] Female 453 (62.5) Occupation Private sector 156 (21.7%) National or public company 126 (17.5%) Self-employed 26 (3.6%) Seeking a ﬁrst job 17 (2.4%) Seeking a job (have already work) 46 (6.4%) Retired 12 (1.7%) Homemaker 60 (8.3%) Student (including high school) 203 (28.2%) Other 74 (10.3%) Frontiers in Public Health Consequences of the COVID-19 pandemic and associated lockdown measures on healthcare organization TABLE 2 Use of care by cystic ﬁbrosis (CF) patients during the ﬁrst lockdown period. lockdown period. No. (%) Scheduled consultation in CF centre during lockdown period 414 (57.7) Canceled by patient 20 (4.8) Canceled by the hospital (no rescheduling or telehealth oﬀered) 40 (9.7) Telehealth oﬀered by the hospital but refused by patient 4 (1.0) Telehealth oﬀered by the hospital and accepted by patient 165 (39.9) Postponed with new appointment at the hospital 111 (26.8) Maintained in-person at the hospital 102 (24.6) Scheduled physiotherapy sessions 333 (46.5) Canceled by patient 95 (28.5) Canceled by the physiotherapist-no telehealth/online program oﬀered 90 (27.0) Online program oﬀered by the physiotherapist and accepted by patient 7 (2.1) Telehealth oﬀered by the physiotherapist and accepted by patient 11 (3.3) Telehealth or program oﬀered by the physiotherapist and refused by patient 4 (1.20) Postponed with new appointment at the hospital 34 (10.2) Maintained at home or physiotherapist oﬃce 115 (34.5) Scheduled annual review in CF centre during lockdown period 105 (14.7) Canceled by patient 5 (4.8) Canceled by the hospital (no rescheduling or telehealth oﬀered) 21 (20.0) Telehealth oﬀered by the hospital but refused by patient 0 (0.0) Telehealth oﬀered by the hospital and accepted by patient 15 (14.3) Postponed with new appointment in-person at the hospital 56 (53.3) Maintained at the hospital 13 (12.4) Hospitalization required during the lockdown 48 (6.7) Replaced by day hospital 4 (8.3) Performed in another unit (not CF centre) 7 (14.6) Postponed 6 (12.5) Maintained 33 (68.8) Intravenous antibiotic required during the lockdown 66 (9.2) Performed at home 51 (77.3) Replaced by oral antibiotic treatment 2 (3.0) Postponed 0 (0.00) Performed at hospital 19 (28.8) Hospitalization required during the lockdown 48 (6.7) Replaced by day hospital 4 (8.3) More than 75% of consultations scheduled during the lockdown were canceled (Tables 2, 3-interview comments verbatim A5, A9). However, telehealth was alternatively proposed and accepted in almost 40% of cases, whereas 27% of scheduled consultations were postponed. Overall, 87.7% CF-patients [CI 95%: 84.1; 90.5] were oﬀered access to consultations. The trend varied depending on the geographic location (Supplementary Table 1) [Grand Est region (64.0%) vs. rest of the territory (89.2%); p < 0.001] or the age of patients [under 18 (95.2%) vs. adults (86.3%); p = 0.05], and it did not vary depending on sex [male (90.2%) vs. female (85.7%); p = 0.20] or occupation [working (87.8%) vs. Consequences of the COVID-19 pandemic and associated lockdown measures on healthcare organization seeking for a job/retired (87.3%) vs. student (88.8) vs. others (84.6); p = 0.92]. More than 75% of the scheduled physiotherapy sessions were canceled (mostly by the physiotherapist), and only few telehealth or online programs were proposed (Table 3, A1), resulting in the use of self-drainage in a signiﬁcant number of cases (Table 3, B1). While some patients indicated that they found it diﬃcult to be away from their physiotherapists during the conﬁnement (Table 3, B7), especially when they needed reassurance from these professionals (Table 3, B1), others indicated that they beneﬁted from the advice given by their center physiotherapist or that they initiated new activities such as yoga or home exercise to stay active (Table 3, B2–6). As predicted, the routine annual follow-up visits were consistently canceled or postponed in most cases. In contrast, hospitalizations required for symptom exacerbation were maintained in most of the cases in the CF centres. The required intravenous administration of antibiotics was performed at home in more than 75% of patients. Although the qualitative questionnaire did not speciﬁcally include any questions regarding this speciﬁc topic, CF-patients who underwent qualitative interviews had no diﬃculty getting the needed prescription drugs during the lockdown period (Table 3, C6). Qualitative analysis Baseline characteristics and geographic location of the study population are summarized in Table 1. Within a month, we collected 751 completed questionnaires, with 725 being Each interview was summarized by the interviewer. It allowed a pre-analysis of the interview with regard to Frontiers in Public Health 04 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 Theme A: In relation to the medical institution There was nothing done by remote conference, and so uh she tells me uh now we cancel here, you seem stable uh in relation to the information I was giving her...” (I6) A12: “Exactly, um, in no case, um I don’t know if the other patients of Foch um [her center], formulate it as I do, in no case do we feel abandoned by the medical staﬀ, never, and that’s important.” (I13) A13: “It’s true that I was very anxious because of my profession... how was it going to go, how was I going to manage... I had called the CRCM several times... to ask them, already before 16 March, if I should stop [to work]” (I8) A14: “It’s true that my doctors... told me that there had been a few cases of CF who had had COVID-19, that it hadn’t been as severe as that... that they hadn’t developed serious forms, so it’s true that that reassured me” (I8). A15: “Well, as time goes by, this question is always present because, um, knowing whether we will develop more severe symptoms quickly. It’s true that it’s frightening, it accentuates the anxiety” (I8) A16: “a WhatsApp group where the doctor sent messages basically to parents and patients... with the latest information. It’s always been quite reassuring, the most important feedback I got was that there were a hundred (patients) CF who had... been infected and about ten who were in, there were no deaths, as far as I know, and a dozen or so ended up in intensive care... but all the intensive care where the people for whom the coronavirus was a problem were patients who had had transplants... so we weren’t told about the coronavirus as a threat speciﬁcally to cystic ﬁbrosis” (I5). A17: “my doctor used to tell all his patients ‘seeing the state of information on this pandemic, here’s what to do, here’s what not to do, and if you have a concern, here’s the person to contact”’ (I2). A18: “she distributed information... it’s mostly by e-mail that I receive information... how to deal vis-a-vis your work, under what conditions you yourself could stop, if in terms of work, working from home was not possible... some information on hygiene measures, masks, how you could get them” (I2). Concerns about COVID-19 exploitable (Supplementary Figure 1). The mean age of the studied population was 28.0 years-old [IQR 20.0–37.0], with a predominance of women (62.5%). Students were highly represented (28%), and among patients of working age, the private sector was the most represented (22%). Homemakers represented 8% of the CF-patients. For qualitative analysis, 16 CF-patients were solicited, and 15 were interviewed. Numerous CF-patients reported that their center initially provided insuﬃcient information about speciﬁc risk of COVID- 19 for CF-patients (Table 3, A3, A4, C9). Nevertheless, this improved overtime through interactions with physicians and coordinating nurses or through direct information from medical institutions, such as via WhatsApp groups or email diﬀusion (Table 3, A5–6, A8–12, A16, A18). As a result, answers to Frontiers in Public Health 05 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 TABLE 3 Verbatim from 9 representative patient’s interviews. Theme A: In relation to the medical institution A7: “I went to a lot of diﬀerent hospitals because I rebelled a little bit, I didn’t agree at all with the care, it didn’t suit me” (I6). A8: “I decided after 2 weeks... I said stop the TV because you’re going to go crazy... one never learns things from the news... I told my pulmonologist, ‘Listen to me, I’m referring to you, so if there are things you think I need to know, you have to tell me”’ (I6) A8: “I decided after 2 weeks... I said stop the TV because you’re going to go crazy... one never learns things from the news... I told my pulmonologist, ‘Listen to me, I’m referring to you, so if there are things you think I need to know, you have to tell me”’ (I6) A9: “And so it reassured her that I knew how to take care of myself, she reassured me about my own competence by telling me that I shouldn’t worry because I was managing my own care by myself etc.” (I6) A10: “And then, I contacted my CRCM by email saying, well, I don’t understand, we haven’t heard any news, so I was a bit panicked in fact, saying, ‘what’s going on and what in fact are we doing?”. And so then I had my [coordinating] nurse who got in touch with me and she told me that they had decided to respond to all requests but to answer only if we had questions and not to communicate with us if in fact we didn’t have any.” (I6) A11: “here, they told me that it’s here that you cancel all medical appointments and uh I didn’t uh really understand because in fact, I told him, but uh you cancel all appointments, but then how were we going to do it really and at the time in fact one was not doing remote conferencing really, it was not even possible in the medical ﬁeld, in fact it was always necessary to move and me, I am already nearby now and I always had to go to Foch etc. Theme A: In relation to the medical institution A1: “I had called the physio’s oﬃce and it actually went to voicemail and I listened to the voicemail, they said the oﬃce was going to be closed [during Lockdown]” (I7). A2: “I did some research [on the impact of COVID-19 on cystic ﬁbrosis], but in fact didn’t ﬁnd any... I went on the Internet...but I couldn’t ﬁnd any information, so I was disappointed” (I7). A3: “At the beginning, I must admit that I felt a bit lost, in the sense that our doctors did not call us to tell us what we had to do, how we were going to protect ourselves, plus at the beginning, we did not have a mask... I’m married, so, my husband, what can he do, can he go out shopping or not, can he maybe be near others or not, knowing that if he catches the virus and brings it home and I get it...how does it work ﬁnally, me, well, I was a little bit confused at ﬁrst” (I9). A4: “He told me that it was really serious, that I should be careful, that I shouldn’t go out, that it was necessary for someone to go shopping for me, but afterwards, well, we didn’t go into as much detail on the subject because they were really busy during that time, which I can understand, so they don’t have a lot of time to devote to us, we can’t stay on the phone for an hour, to get many explanations, so, well, that was okay, at ﬁrst” (I9). A5: “There were planned [consultations] during the conﬁnement, but in the end everything was canceled, on the one hand, on my side. I didn’t feel like coming to the hospital, I was afraid. The anxiety was terrible. Afterwards, I was oﬀered telephone calls, so it was more convenient for me and for them too. It allows you to have follow-ups so it also reassured me in that sense. And now [after deconﬁnement], I have face-to-face appointments again.” (I15) A6: “I had questions, I asked them... he answered on a medical level, he was really available, any time, me, sometimes I sent emails in the middle of the night, yes because I had questions” (I6). A7: “I went to a lot of diﬀerent hospitals because I rebelled a little bit, I didn’t agree at all with the care, it didn’t suit me” (I6). Theme A: In relation to the medical institution B1: “I’m still very independent in this respect, so I know how to do my physiotherapy, I know how to take care of myself, I took care of myself for 2 months without any problem, but I must admit that at the end of 2 months, I still needed to see my physiotherapist again, if only for the purpose ﬁnally to reassure myself, the fact that he [her physiotherapist] comes and tells me, ‘It’s OK, you’ve done your physiotherapy well’, it’s more for me, to reassure me” (I9). (Continued) 06 Frontiers in Public Health frontiersin.org 10.3389/fpubh.2022.978627 Oubaya et al. TABLE 3 (Continued) B2: “What I told myself is that yoga is pretty good, it’s an activity that’s not hyper physical and at the same time it’s based on breathing, so I said to myself that maybe it could do me some good and um then the fact of breathing well could also take away, if you have anxieties, some anxieties, ﬁnally, it can in fact calm the body and the mind. So I started from that principle, saying to myself that in any case it could be good for me.”(19) B2: “What I told myself is that yoga is pretty good, it’s an activity that’s not hyper physical and at the same time it’s based on breathing, so I said to myself that maybe it could do me some good and um then the fact of breathing well could also take away, if you have anxieties, some anxieties, ﬁnally, it can in fact calm the body and the mind. So I started from that principle, saying to myself that in any case it could be good for me.”(19) B3: “It’s all going to be ﬁne now [interview conducted after deconﬁnement] with these physiotherapy sessions. But eﬀectively, not having a physiotherapist, not being able to do the exercise rehabilitation sessions with the equipment like at the physiotherapist’s, and then to have gained weight at the same time, it was a bit diﬃcult” (I13). B4: “Sports is physical activity, it’s also a moment I spend with other people. So then [during the conﬁnement], I wasn’t moving, and I was all alone” (I1). B5: “I contacted my physiotherapist, she gave me some tips on how to do sports in the garden, a bit anywhere. I followed her advice, it’s fun at ﬁrst, but you quickly let the rhythm go” (I15). Theme A: In relation to the medical institution Theme C: psychosocial adjustment to COVID-19 pandemic and associated lockdown measures Theme C: psychosocial adjustment to COVID-19 pandemic and associated lockdown measures wee: Yes, we talk to them often, we even created a WhatsApp group so that we can keep in touch and everything, but with the conﬁnement, as there are e pathologies, we can’t all see each other in fact.” (I7) C2: “So I said to myself, frankly, what I should do is not go out because I don’t really have any information, especially as they said that those who... those who have a lung disease are already at risk of it being complicated for them with the coronavirus. (I7) C3: “I wash my hands quite a lot but [with COVID-19], I washed my hands all the time, all the time, as soon as I touched something that came from outside, like the mail, a parcel, in fact I spent my life washing my hands” (I9) C4: “I kept telling my husband, when he went out, ‘you put your mask on, you took your mask, you took your alcohol gel’... I kept telling him, ‘don’t forget to wash your hands as soon as you touch a door handle, there you go, then you use your alcohol gel.” (I9). C5: “I say to myself, they don’t understand the message, so there comes a time when you can’t be in conﬂict all the time, you can’t try to change people’s lives, so I just let it go, the problem is that I think it isolates me” (I9) C6: “I didn’t want to go to the pharmacy because I said to myself, maybe there are sick people coming to get their medicine... I didn’t want my husband to go either because it was tricky, so the pharmacist was very nice and very accommodating... I sent her my prescription by email and then she came to deliver my medicine to my house” (I9) C7: “The problem is that sometimes what I reproach people for is that they manage to make me feel guilty, in fact, um, I know that my father-in-law wanted to come to the house even during the lockdown. He wanted to come to the house for a drink etc., so I told him no and I could see that he wasn’t happy and that... Theme A: In relation to the medical institution B6: “So in fact I started to do quite a lot of sports and above all to surpass myself in fact... so I did quite a lot of sports... I followed quite a few live streams in fact, from people who did, who before, did things for a fee, for example, yoga and things like that, and me, I took advantage of this, in fact, to enrich myself with a lot of things that I would have loved to do but that in fact usually either you have to travel for or pay a certain amount of money for etc... and so I decided to eat up as many things as possible that I wanted... it was brilliant” (I6). B7: “So at the beginning, um, there we were, we went on a bit of a tour, um, all together, nibbling in front of the TV, um, there we were. So um it’s true that at the beginning we um it was a bit of a mess at home. I don’t go to a gym because, um, I’d have to be followed by a coach etc. And then, well, it’s not cheap either, so um, so no gym, um, I walk. And um during the conﬁnement, zero sports to be honest, zero sports and um and then um not long ago I started cycling a bit again, the stationary bike...”. (I13) B8: “So yes, in my spare time, before the conﬁnement, I used to do contemporary dance. Otherwise, I’ve always been a bit sporty, so I like running, sports, I don’t stop doing sports because I love it.” (I15) B9: “Yes, so we used to do sports um at home, whether it was with my mother or just me on my own, um and plus as the weather was good and we had a garden, it’s true that it was nice so we could be outside. So yes, I...I continued at home, um and it’s true that we also used small equipment such as elastics or weights and all that. Theme A: In relation to the medical institution So it’s true that it allowed us to keep uh to keep up an activity in fact, to have classes anyway so uh no on that, it was also in the end uh ﬁnding a rhythm that helped.” (I8) B9: “Yes, so we used to do sports um at home, whether it was with my mother or just me on my own, um and plus as the weather was good and we had a garden, it’s true that it was nice so we could be outside. So yes, I...I continued at home, um and it’s true that we also used small equipment such as elastics or weights and all that. So it’s true that it allowed us to keep uh to keep up an activity in fact, to have classes anyway so uh no on that, it was also in the end uh ﬁnding a rhythm that helped.” (I8) B10: “I started (sports) again with a friend who wanted to get back into it and that’s it, we’d meet every other day at 4pm, we’d do sports for an hour, an hour and a half... on Skype... it wasn’t anything that required going outside, we were in our respective rooms’ (I5). it was nice so we could be outside. So yes, I...I continued at home, um and it’s true that we also used small equipment such as elastics or weights and all that. So it’s true that it allowed us to keep uh to keep up an activity in fact, to have classes anyway so uh no on that, it was also in the end uh ﬁnding a rhythm that helped.” (I8) B10: “I started (sports) again with a friend who wanted to get back into it and that’s it, we’d meet every other day at 4pm, we’d do sports for an hour, an hour and a half... on Skype... it wasn’t anything that required going outside, we were in our respective rooms’ (I5). B10: “I started (sports) again with a friend who wanted to get back into it and that’s it, we’d meet every other day at 4pm, we’d do sports for an hour, an hour and a half... on Skype... it wasn’t anything that required going outside, we were in our respective rooms’ (I5). Theme A: In relation to the medical institution so the problem is that it makes me feel guilty because I say to myself, um well I’m, I’m in... I’m the bad guy. I’m the bad guy, I tell him not to come, so because of me he doesn’t see his son, um, so in a way it’s not nice, well... that’s how I experienced it anyway.” (I9). C8: “my mother took it upon herself to be exposed to the virus (notably because she was in charge of doing the shopping for her, her daughter and her grandson), in fact, so when she returned home, potentially, we had to wait a fortnight to be sure... well, that’s what my pulmonologist used to tell me, to make sure that no one got it, so in fact, each time it necessitated pushing back on when I could see my son, so it lasted a very long time, and then he (the pulmonologist) started talking to me about the fact that in fact there was no end date for knowing when I would see my son again (...) as my pulmonologist used to say, it was that you had to wait until you had the tests” to see your son again (I6) C9: “basically, I had to do everything I had forbade myself to do during, for my part, over two and a half months” (I6). C10: “at the pharmacy, we are in contact with a lot of sick people, so I’m going to wait for the [contamination]rate to decrease a little”(I1). C11: “I already know that I am very vulnerable, very fragile, so I did not go out as soon as the lockdown was announced.” (I15) C12: “No, I don’t go out in public places. Ok so, I can walk my dog but I don’t go out to places where I can meet people, it still scares me in any case.” C12: “No, I don’t go out in public places. Ok so, I can walk my dog but I don’t go out to places where I can meet people, it still scares me in any case.”(I15) (Continued) (Continued) Frontiers in Public Health 07 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 TABLE 3 (Continued) C13: “we clean more, we clean between each patient, the equipment, also each time, I change my gown” (I8). C13: “we clean more, we clean between each patient, the equipment, also each time, I change my gown” (I8). Theme A: In relation to the medical institution Individuals with CF were less worried about the risks from their treatment, as only 9.5 and 14% considered that CF medications were putting them at greater risk of COVID-19 or COVID-19 complications, respectively (Supplementary Table 2). Unexpectedly, CF-patients living in the Southwest of France (where low case numbers were reported at the time of the ﬁrst wave) were signiﬁcantly more likely to be very concerned about becoming ill with SARS- CoV-2 infection compared to those living in the Northeast of France (with high prevalence of SARS-CoV-2 cases at the time of the ﬁrst lockdown) (Figure 1). On a constraint scale from 1 to 10, with 1 being “very low” and 10 being “very high,” the constraint of barrier measures was 2 [0–5 IQR] while that from the lockdown was 5 [2–8 IQR]. Results are shown for each of the French regions in Figure 1. Figure 1. Theme A: In relation to the medical institution Color code: dark gray, PC; light gray, LMC; very light gray, UMC; and white, UC. (Supplementary Table 2). Analysis of the 725 validated questionnaires (Table 4) showed that 43.2% of the CF-patients had signs of anxiety based on the GAD-7 questionnaire, with 25.9, 11.3, and 6.0% having mild, moderate or severe anxiety, respectively, whereas 51.0% presented signs of depression based on the PHQ-9 questionnaire, with 29.8, 14.6, 4.4, and 2.2% having mild, moderate, moderately severe and severe depression, respectively. These anxiety symptoms were also reported in interviews and were mostly related to the perceived higher risk of COVID-19 for CF-patients (Table 3, A5, A13, A15, B2). Anxiety and depression prevalence (mild, moderate or severe) was lower in the 14–18 years-old group compared to older CF-patients [anxiety: 32.5%, (14–18 years) vs. 43.3% (18–25 years), 48.4%, (25–35 years) and 43.0% ≥35 years; p = 0.049; depression: 44.7% (14–18 years) vs. 55.6% (18–25 years), 56.1% (25–35 years) and 46.6% ≥35 years; p = 0.066]. Furthermore, the prevalence and severity of anxiety and depression were inversely correlated with the prevalence of COVID-19 cases during the ﬁrst lockdown (Figure 1). In line with the increase in anxiety and depression with age, a signiﬁcant increase in smoking, consumption of alcohol and psychotic medications, and sleep disorders was found in older patients (Table 4). Assessing treatment adherence using a score of compliance/6 indicated a median [IQR] score of 1.0 (1.0–3.0) with 21% of the CF-patients having good compliance, 53% minor non-compliance and 25% non-compliance (Table 5). Interestingly, a signiﬁcant diﬀerence was observed with age, with a median score of compliance/6 of 1.0 [0.0–2.0] in older patients (>35 years old) as compared with that in younger patients [1.0 (1.0–3.0) for <18, 2.0 (1.0–2.5) for 18–25 and 2.0 (1.0–3.0) for 25–35 years old; p = 0.0004], contrasting with the higher percentage of “good compliance” in the older group (>35 years old) the questionnaires showed that CF-patients had signiﬁcant knowledge about the SARS-CoV-2 infection. Nonetheless, 65% of CF-patients believed that they were at higher risks of COVID-19, and 85% thought that they were more likely to have COVID-19 complications (Supplementary Table 2; Table 3, A2, A14, C4, C16). This contrasted with the small number of CF-patients who were infected, as only 74 among those responding to the questionnaire had symptoms suggestive of COVID-19 (10%), and among the 20 who were tested, only one was positive. Theme A: In relation to the medical institution C14: “Um, afterwards it’s true that my doctors in [her center] told me that uh there had been a few cases of CF who had uh COVID-19 that it hadn’t been as severe as it had been, that it was rather maintained, that it had not developed serious forms. So it’s true that that reassured me, um, to be able to allow myself, um, it’s true that so me, I live with my parents...” (I8) 5: “we didn’t put any more precautions than that or extreme precautions inside the house, it’s just maybe that we had less contact but that’s all” (I8). C16: “I said, ‘well, it’s not complicated, we all respect the lockdown... what we’re going to do is have face-to-face aperitifs’. We’re two houses side by side with a hedge that crosses between, so we made a plan of who’s going into the courtyard of which house, and we each arrive with our table, our chairs, our bottle and our glass, and we don’t share anything at all, and we limit ourselves to half an hour. So it was me who set this up, it lasted half an hour, we were very happy, it was Saturday evenings, the following Saturday, it lasted an hour, then it went on longer... and the seventy year old neighbor was very happy” (I2). C17: “Nobody comes into my home [Laughs]... but otherwise, we go for a walk, so I go with my mask and everything, I don’t touch anything, in the street I don’t sit anywhere. When someone who doesn’t have a mask passes by, I try to avoid them without appearing too hysterical either, but I’m really careful that... that when they talk to me, they have a mask, they have... in any case the people I agree to see are people who have good hygiene rules, who have known me since I was a child and who know what to do, wash their hands, put on a mask and everything.” (I4) I1: LMC, F 26 yo; I2: UC, M, 52 yo; I3: LMC, 59 yo; I4: UC, F,16 yo; I5: UC, M,18 yo; I6: LMC, F, 31 yo; I7: PC, F,17 yo; I8: UMC, F, 27 yo; I9: LMC, F, 38 yo; I13: UC, F, 50 yo; I, Interview; F, female; M, male; yo, years old; PC, Popular category; LMC, Lower middle category; UMC, Upper middle category; UC, Upper category. Frontiers in Public Health I1: LMC, F 26 yo; I2: UC, M, 52 yo; I3: LMC, 59 yo; I4: UC, F,16 yo; I5: UC, M,18 yo; I6: LMC, F, 31 yo; I7: PC, F,17 yo; I8: UMC, F, 27 yo; I9: LMC, F, 38 yo; I13: UC, F, 50 yo; I, Interview; F, female; M, male; yo, years old; PC, Popular category; LMC, Lower middle category; UMC, Upper middle category; UC, Upper category. Color code: dark gray, PC; light gray, LMC; very light gray, UMC; and white, UC. Psychological consequences of the COVID-19 pandemic and associated lockdown measures “COVID-19 dangerous for me,” “COVID-19 dangerous for relatives,” constraints from barrier measures, and constraints from lockdown were assessed using a scale of 1 to 10, with 1 being “very low” and 10 being “very high.” Results are represented for each of the French geographic areas, with colors that correspond to the values in an intuitive manner (higher values are darker blue, while lower values are lighter blue). Psychological consequences of the COVID-19 pandemic and associated lockdown measures The majority of patients (418/703; 59.5%) did not feel diﬀerently about their health status compared to 3 months earlier, whereas 161 (22.9%) felt that their health improved and 124 (17.6%) reported health degradation Frontiers in Public Health 08 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 FIGURE 1 Health impact of the COVID-19 epidemic and associated lockdown measures on cystic ﬁbrosis patients during the ﬁrst wave of COVID-19 according to diferent geographic regions of France. Assessment of anxiety and depression: participants were asked to complete the 7-Item Generalized Anxiety Disorder Scale (GAD-7) (13) and the validated French version of the 9-item Patient Health Questionnaire Depression Scale (PHQ-9) (14, 15). The total GAD-7 score ranges from 0 to 21, with a cut of of 10 indicating the presence of signiﬁcant anxiety symptoms. The PHQ-9 score is composed of nine depression symptom items listed in the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSMIV) for depression and ranges from 0 to 27 (14). The median score is indicated for each metropolitan French geographic area. “COVID-19 dangerous for me,” “COVID-19 dangerous for relatives,” constraints from barrier measures, and constraints from lockdown were assessed using a scale of 1 to 10, with 1 being “very low” and 10 being “very high.” Results are represented for each of the French geographic areas, with colors that correspond to the values in an intuitive manner (higher values are darker blue, while lower values are lighter blue). FIGURE 1 Health impact of the COVID-19 epidemic and associated lockdown measures on cystic ﬁbrosis patients during the ﬁrst wave of COVID-19 according to diferent geographic regions of France. Assessment of anxiety and depression: participants were asked to complete the 7-Item Generalized Anxiety Disorder Scale (GAD-7) (13) and the validated French version of the 9-item Patient Health Questionnaire Depression Scale (PHQ-9) (14, 15). The total GAD-7 score ranges from 0 to 21, with a cut of of 10 indicating the presence of signiﬁcant anxiety symptoms. The PHQ-9 score is composed of nine depression symptom items listed in the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSMIV) for depression and ranges from 0 to 27 (14). The median score is indicated for each metropolitan French geographic area. Social inequalities during COVID-19 pandemic and associated lockdown with 33.5%, more than double that observed in younger patients (15.6 for <18, 15.3 for 18–25, 15.3 for 25–35 years old, p < 0.001) (Table 5). Moreover, non-compliance was associated with the presence of anxiety (32.8% in patients with anxiety vs. 20.1% in the non-anxious group; p < 0.0001) or depression symptoms (32.3% in patients with depression symptoms vs. 18.4% in the non-depressive group; p < 0.0001). Among the working population, 131 (44.9%) stopped working, 91 (31.2%) reduced their hours of work, and 70 (24.0%) worked more than before lockdown. Regarding the place of work, 176 (81.5%) worked from home, 29 (13.4%) were already home-based prior to the lockdown and 11 (5.1%) were still going to their place of work. Among the students, 192 (96.0%) Frontiers in Public Health 09 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 TABLE 4 Anxiety and depression during lockdown period. Age (years) [14–18] [18–25] [25–35] ≥35 p-value N = 725 N = 117 N = 153 N = 223 N = 232 Anxiety (GAD-7) N = 717 N = 114 N = 150 N = 223 N = 230 0.09 No (0–4) 408 (56.8) 77 (67.5) 85 (56.7) 115 (51.6) 131 (57.0) Mild (5–9) 186 (25.9) 29 (25.4) 37 (24.7) 62 (27.8) 57 (24.8) Moderate (10–14) 81 (11.3) 5 (4.4) 18 (12.0) 27 (12.1) 31 (13.5) Severe (>15) 43 (6.0) 3 (2.6) 10 (6.7) 19 (8.5) 11 (4.8) Depression (PHQ-9) N = 720 N = 114 N = 151 N = 223 N = 232 <0.001* No (0–4) 352 (48.9) 63 (55.3) 67 (44.4) 98 (44.0) 124 (53.5) Mild (5–9) 215 (29.8) 30 (26.3) 33 (21.9) 75 (33.6) 77 (33.2) Moderate (10–14) 105 (14.6) 18 (15.8) 33 (21.9) 35 (15.7) 19 (8.2) Moderately severe (15–19) 32 (4.4) 2 (1.8) 13 (8.6) 8 (3.6) 9 (3.9) Severe (>20) 16 (2.2) 1 (0.9) 5 (3.3) 7 (3.1) 3 (1.3) Participants were asked to complete the 7-Item Generalized Anxiety Disorder Scale (GAD-7) (13) and the validated French version of 9-item Patient Health Questionnaire Depression Scale (PHQ-9) (14, 15). The total GAD-7 score ranges from 0 to 21, with a cut oﬀof 10 indicating the presence of signiﬁcant anxiety symptoms. GAD-7 scores of 5, 10, and 15 represent cut-oﬀpoints for mild, moderate, and severe anxiety, respectively. Social inequalities during COVID-19 pandemic and associated lockdown The PHQ-9 score is composed of nine depressive symptom items listed in the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSMIV) for depression and ranges from 0 to 27 (14). PHQ-9 scores of 5, 10, 15, and 20 represent cut-oﬀpoints for mild, moderate, moderately severe, and severe depression, respectively (14). Numbers in brackets refer to percentage. Chi square test with Moderately severe and Severe categories combined due to small numbers. Participants were asked to complete the 7-Item Generalized Anxiety Disorder Scale (GAD-7) (13) and the validated French version of 9-item Patient Health Questionnaire Depression Scale (PHQ-9) (14, 15). The total GAD-7 score ranges from 0 to 21, with a cut oﬀof 10 indicating the presence of signiﬁcant anxiety symptoms. GAD-7 scores of 5, 10, and 15 represent cut-oﬀpoints for mild, moderate, and severe anxiety, respectively. The PHQ-9 score is composed of nine depressive symptom items listed in the Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSMIV) for depression and ranges from 0 to 27 (14). PHQ-9 scores of 5, 10, 15, and 20 represent cut-oﬀpoints for mild, moderate, moderately severe, and severe depression, respectively (14). Numbers in brackets refer to percentage. Chi square test with Moderately severe and Severe categories combined due to small numbers. BLE 5 Impact of COVID-19 pandemic and associated lockdown measures on treatment, alcohol, and smoking behaviors. Frontiers in Public Health Discussion In this qualitative and quantitative study, we show that the ﬁrst lockdown did not signiﬁcantly impact care as provided by CF centres. However, despite the low number of CF- patients diagnosed with COVID-19, they developed signiﬁcant worsening symptoms of anxiety and depression during the ﬁrst lockdown. One common observation in patients with chronic disease was an increase in anxiety and depression symptoms. In the general population, results from surveys conducted early in the COVID-19 pandemic (March–June 2020) have shown increased prevalence of mental health symptoms, especially among young adults (25–29). People with CF are two to three times more likely to experience depression, anxiety, or both, compared to people in the general population (18, 30, 31). In France, data from the CF registry identiﬁed about 8% of patients with depression symptoms (diagnosed or followed), increasing with age, from 4% in adolescents to 16% in subjects older than 35 years old (2). The results of our study suggest that anxiety and depression symptoms were signiﬁcantly increased in CF-patients during the ﬁrst lockdown in France, and an increase with age was also observed. This resulted in an increase in sleep disorders, together with increased alcohol and psychotropic medication use. Surprisingly, anxiety and depression symptoms were more frequent in geographic areas where COVID-19 prevalence was low, while they were less important in areas with higher prevalence, suggesting that these symptoms do not correlate with the real risk of disease in this population. In France, as in other developed countries, healthcare systems faced two major issues. The ﬁrst was the saturation of hospital infrastructures that have been largely oriented toward outpatient and day hospital settings over the last 10 years, and the physical and mental exhaustion of the healthcare workforce. The second was the reorganization of all the structures, procedures and workforce of the hospitals toward conﬁgurations almost entirely focused on caring for COVID- 19 patients. In France, consultations fell by 40% among general practitioners and by 50% among specialists since the beginning of the pandemic, even when accounting for the surge of tele- consultations (19, 20). Concurrently, hospitals were asked to postpone consultations and surgeries considered to be non- urgent. In this context, one could have feared a disruption of the management of chronic diseases, such as CF, due to the delaying of care procedures and clinic visits, increasing the risk for morbidity and mortality. Social inequalities during COVID-19 pandemic and associated lockdown Age (years) Total [14–18] [18–25] [25–35] ≥35 p-value N = 725 N = 117 N = 153 N = 223 N = 232 N (%) N (%) N (%) N (%) N (%) Smoking behaviors Initiate/Increase 10 (28.6) 0 (0.0) 2 (22.2) 6 (40.0) 2 (22.2) 0.94 Stable 12 (34.3) 1 (50.0) 3 (33.3) 4 (26.7) 4 (44.4) Decrease/Stop 13 (37.1) 1 (50.0) 4 (44.4) 5 (33.3) 3 (33.3) Alcohol behaviors Initiate/Increase 64 (16.4) 0 (0.0) 10 (9.4) 20 (15.8) 34 (23.5) 0.04 Stable 150 (38.4) 5 (38.5) 41 (38.7) 49 (38.6) 55 (37.9) Decrease/Stop 177 (45.3) 8 (61.5) 55 (51.9) 58 (45.7) 56 (38.6) Psychotropic Medications Initiate/Increase 26 (34.7) 0 (0.0) 1 (20.0) 9 (42.9) 16 (37.2) 0.02 Stable 41 (54.7) 4 (66.7) 3 (60.0) 8 (38.1) 26 (60.5) Decrease/Stop 8 (10.7) 2 (33.3) 1 (20.0) 4 (19.1) 1 (2.3) Sleep disorders Initiate/Increase 254 (59.5) 27 (57.5) 61 (66.3) 81 (56.3) 85 (59.0) 0.034 Stable 146 (34.2) 17 (36.2) 24 (26.1) 51 (35.4) 54 (37.5) Decrease/Stop 27 (6.3) 3 (6.38) 7 (7.6) 12 (8.3) 5 (3.5) Compliance Good compliance 147 (21.1) 17 (15.6) 23 (15.3) 33 (15.3 74 (33.5) <0.0001 Minor non-compliance 372 (53.5) 59 (54.1) 89 (59.3) 121 (56.0) 103 (46.6) Non-compliance 177 (25.4) 33 (30.3) 38 (25.3) 62 (28.7) 44 (19.9) remained schooled (online classes as all schools were closed in France at that moment). remained schooled (online classes as all schools were closed in France at that moment). or requested by the patients (Table 3, A1). These patients felt “lost” (Table 3, A2-3), forced to manage their care on their own, and plagued by feelings of anxiety (Table 3, A5-7). In the upper-middle and upper categories, some CF-patients did not hesitate to call their CF centre several times for information, providing reassurance (Table 3, A13). Second, regarding the relationship to sport and physical therapy (Table 3, B), CF- patients from the lower-middle category reported doing little or Based on content analysis, diﬀerences by social categories among the 3 major themes of concern emerged from the interviews (Table 3, A–C). First, regarding the relationship to medical institutions, we observed that among the working and lower-middle social categories, very little information and guidance in relation to the COVID-19 pandemic was obtained Frontiers in Public Health 10 frontiersin.org 10.3389/fpubh.2022.978627 Oubaya et al. no sport, either before conﬁnement or after (Table 3, B7). Social inequalities during COVID-19 pandemic and associated lockdown Finally, intravenous antibiotic treatments were mostly performed at home without delay, which is also a common practice in CF-patients. This low impact of the ﬁrst wave of the pandemic on CF care in France, but also in other countries with similar organization, is likely due to the availability of CF Centres that were able to adjust and initiate rapid changes in care delivery (4, 22). This may have facilitated the preservation of usual care for most CF-patients, even though adult pulmonary specialists were not readily available due to their taking care of COVID-19 patients. In the absence of available vaccines, social distancing is one of the main tools for preventing the transmission of SARS-CoV- 2. As CF increases the risk of viral and bacterial respiratory infections, CF-patients are acutely aware of the beneﬁts of practicing social distancing (23, 24). This can explain that constraint from this barrier measure was reported to be low in our study. In contrast, more patients were aﬀected by the restrictions from the lockdown. Social inequalities during COVID-19 pandemic and associated lockdown Not moving was perceived in this group as reinforcing the feeling of being alone and isolated (Table 3, B4). These CF-patients attested to their diﬃculty being away from their physiotherapists during lockdown (Table 3, B1–7). In CF-patients from more privileged categories status, the conﬁnement was an opportunity to ﬁnd new activities to practice physical exercise (Table 3, B9, B10). The third theme that emerged from the interviews related to psychosocial adjustment to COVID-19 pandemic and associated lockdown measures. CF-patients from the lowest social categories mostly over-conﬁned themselves, with some not getting out at all during the lockdown (Table 3, C1–2, C6, C10–12). In contrast, some CF-patients belonging to the upper- middle and upper categories declared that they did not take additional precautions during the conﬁnement (Table 3, C14, 15) but tried to meet outside and for some of them, even had get-together between neighbors (Table 3, C16). fewer hospital admissions, the proportion of non-elective and intensive care unit admissions increased, as did the 30-day liver-related mortality. In the present study, almost 2/3 of the consultations were maintained or replaced by telehealth and almost all necessary hospitalizations occurred (mostly in their aﬃliated centers). Consultations for physiotherapy were more aﬀected as they were canceled by physiotherapist or CF- patients. However, self-drainage was used as a substitute as it is a common practice in CF. Finally, intravenous antibiotic treatments were mostly performed at home without delay, which is also a common practice in CF-patients. This low impact of the ﬁrst wave of the pandemic on CF care in France, but also in other countries with similar organization, is likely due to the availability of CF Centres that were able to adjust and initiate rapid changes in care delivery (4, 22). This may have facilitated the preservation of usual care for most CF-patients, even though adult pulmonary specialists were not readily available due to their taking care of COVID-19 patients. fewer hospital admissions, the proportion of non-elective and intensive care unit admissions increased, as did the 30-day liver-related mortality. In the present study, almost 2/3 of the consultations were maintained or replaced by telehealth and almost all necessary hospitalizations occurred (mostly in their aﬃliated centers). Consultations for physiotherapy were more aﬀected as they were canceled by physiotherapist or CF- patients. However, self-drainage was used as a substitute as it is a common practice in CF. Frontiers in Public Health Discussion Poor treatment adherence is common in CF (35, 36) and is likely to be worsened by decrease access to healthcare caregivers/structures, increased fears of SARS- CoV-2 contamination when having to get medications in pharmacies or increased anxiety/depression symptoms. Smith et al. (31) reported that depression symptoms in children were signiﬁcantly associated with lower rates of adherence to airway clearance, whereas there was no signiﬁcant association between patient’s adherence to medical treatment/airway clearance and the PHQ-9 or GAD-7 scores of patients in the Turkish study. In our study, the presence of anxiety/depression symptoms during lockdown was associated with poor treatment adherence. A trend similar to that of COPD (disease with higher median age) was observed with only 20% non-compliance; however, this did not seem to be due to increased anxiety or depressive symptoms. Our study has also some strengths. This was a national multicentre study with participating patients from all CF centres in France (only Corse region was underrepresented because of the absence of CF centre). The short duration of the recruitment period avoided bias in the context of a very rapidly evolving pandemic situation. Furthermore, the study used both quantitative and qualitative methodologies, which helped understanding all aspects of the COVID-19 pandemic and the resulting lockdown. In conclusion, COVID-19 pandemic and associated lockdown measures in France had only minimal impact on the care management of CF-patients. The adaptability of well-structured CF centres in establishing short-term measures during the pandemic may be a model for the development of similar organization for other chronic lung diseases. However, the higher risk for anxiety and depression in CF-patients reported here needs to be considered when developing new models for care during future sanitary crisis. Several social science studies have shown that the health crisis caused by the COVID-19 pandemic is compounded by a major social crisis, having accentuated the inequalities between all social category, gender and generation (37–39). Explanations are multifactorial, including high frequency of chronic diseases (comorbidity being critical in case of COVID-19), type of employment (service jobs, precarious jobs), impossibility of telecommuting or crowded housing (40). During the SARS-CoV-2 pandemic, it has been emphasized that the most precarious populations were more aﬀected by COVID-19, notably because they accumulate several factors that cause the spread of the virus (41). Discussion A study compared 2 cohorts of subjects in the hospital for management of liver disease before (December 2019 to February 2020) and during (March to May 2020) the COVID-19 pandemic in Austria (21). The authors assessed patients’ perceptions on quality of care by telesurvey (cohort 1) and written questionnaire (cohort 2), but also by trends in elective and non-elective admissions. During this time, more than 90% had contact, either by telehealth or in-person visits. However, 57% reported that contacting their physician during the pandemic was diﬃcult to impossible, and despite This discrepancy may also be explained by concern about the impact of COVID-19 on the healthcare system. Blendon et al. (32) undertook a survey of residents from Hong Kong, Taiwan, Singapore, and the United States to understand the public reaction to the use of widespread quarantine. In this survey, 44 and 69% reported that they were very worried about Frontiers in Public Health 11 frontiersin.org Oubaya et al. 10.3389/fpubh.2022.978627 10.3389/fpubh.2022.978627 not being able to get the healthcare or prescription drugs they needed during the quarantine period, respectively. The present study has several limitations. Subjects responding on a voluntary basis could limit the external validity, there were only self-reported measures with no objective measures of disease management. The questionnaires were available online with a generic link, and we could not verify that all responders were CF-patients > 14 years old. However, an information letter was provided with the questionnaire, explaining the selection criteria, and requiring for the responders to enter their age on the questionnaire, which prevented those under 14 to ﬁll out the remaining questionnaire. Even though the number of patients included was below expectations, as the observed health access was better than expected, the precision of the estimation was good and the study powered enough to assess other outcomes and evaluate associations. The impact of COVID-19 and associated measures on mental disorders during the lockdown is not limited to CF-patients. Portuguese patients with rheumatoid arthritis experienced signiﬁcant worsening of their symptoms, along with anxiety and depression during the ﬁrst conﬁnement (21). Similar trends were reported in patients with COPD during lockdown with signiﬁcantly intensiﬁed symptoms and disorders of mental illness such as fear, anxiety and depression (33). Interestingly, this was associated with increased adherence to using their preventive inhalers in more than one fourth of the subjects (34). Data availability statement The original contributions presented in the study are included in the article/Supplementary materials, further inquiries can be directed to the corresponding author/s. Discussion Among them, three factors, often cumulative, reinforce social inequalities in healthcare: diﬀerences in access to care, inequalities in exposure risk and diﬀerences in vulnerability to disease (42). Interestingly, these factors were of moderate impact in our study across social categories. The very tight relationship between CF-patients and their CF healthcare team at the center may have helped maintain good level of care independently of social disparities, as there are no charges for the patient, even during the health crisis. In terms of access to care, according to the French Health Insurance registry, practice visits declined by 40% and specialist visits by 50% in the general population, while a signiﬁcant decrease in access to care for CF-patients was not observed, even in the context of precarity. Funding This study was performed under the frame of the REMEDIA project funded by the European Union’s Horizon 2020 Research and Innovation Program under grant no: 874753. Ethics statement This study was carried out in accordance with the Declaration of Helsinki, good clinical practice, and French legislation on clinical research. The study was approved by the Ethics Committee [Comité de Protection des Personnes (CPP) Est, 15/05/2020] (CPP N◦20.05.11). Informed consent was obtained from all patients > 18 years old and from parents for patients <18 prior to inclusion. ClinicalTrials.gov Identiﬁer: NCT04463628. Frontiers in Public Health frontiersin.org frontiersin.org 12 Oubaya et al. 10.3389/fpubh.2022.978627 Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh. 2022.978627/full#supplementary-material Author contributions questionnaires. We are grateful to Cécile Hoﬀart for clinical research coordination assistance, Martine Torres (manuscript) and Sequoya Frey (verbatims) for English editing assistance. NO performed the statistical analysis, wrote the original draft of the manuscript, and has veriﬁed all data. TP wrote and reviewed the manuscript and participated to the quantitative interviews and analysis. CD designed the study, wrote and reviewed the manuscript, and has veriﬁed all data. NR, BD, DG, HC, GT, and VP-E designed the questionnaire and reviewed the manuscript. HM, DA, CF, LC, and M-PM performed and analyzed the quantitative interviews. CJ and LA designed the statistical analysis strategy and scripts for the statistical analyses. BM, SL, A-CB, and RE conceived and designed the study, reviewed the manuscript, and veriﬁed all data. RE is the principal investigator of the MUCONFIN study, study guarantor and attests that all listed authors meet authorship criteria, and that no others meeting the criteria have been omitted. All authors contributed to the article and approved the submitted version. Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. SUPPLEMENTARY TABLE 2 OVID-19 disease and beliefs about the risk of COVID-19 infection. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. References 1. Zhu H, Wei L, Niu P. The novel coronavirus outbreak in Wuhan, China. Glob Health Res Policy. (2020) 5:6. doi: 10.1186/s41256-020-0 0135-6 ﬁbrosis: an international observational study. J Cyst Fibros. (2021) 20:25–30. doi: 10.1016/j.jcf.2020.11.021 7. Viviani L, Assael BM, Kerem E, group EHNs. Impact of the A (H1N1) pandemic inﬂuenza (season 2009-2010) on patients with cystic ﬁbrosis. J Cyst Fibros. (2011) 10:370–6. doi: 10.1016/j.jcf.2011.06.004 2. Registre français de la mucoviscidose –Bilan des données 2019 (Vaincre la Mucoviscidose, 2021). 3. Elborn JS. Cystic ﬁbrosis. Lancet. 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SUPPLEMENTARY TABLE 1 Repartition of CF patients among the diferent geographic metropolitan regions of France. Acknowledgments SUPPLEMENTARY FIGURE 1 Flow chart of the study. We are very grateful to the patients who took time to ﬁll in questionnaires or undergo interviews. We are grateful to National Clinical Research Programme (PNRC) and the Clinical Research Centre (CRC) of the CHIC for their valuable help in the elaboration of the questionnaires. We thank Vaincre La Mucoviscidose, the Filière MUCOVICIDOSE-CFTR, the CHIC communication department and all the CRCMs in France for their support and their assistance in the distribution of the SUPPLEMENTARY FIGURE 2 Number of patients hospitalized for COVID-19 on May 24th, 2020 in the diferent geographic regions of France. Number of patients hospitalized for COVID-19 on May 24th, 2020 in the diferent geographic regions of France. 10. Corvol H, de Miranda S, Lemonnier L, Kemgang A, Gaubert MR, Chiron R, et al. 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https://openalex.org/W3209540485	https://europepmc.org/articles/pmc8556965?pdf=render	English	null	Comparison of mini-open repair system and percutaneous repair for acute Achilles tendon rupture	BMC musculoskeletal disorders	2,021	cc-by	7,812	Abstract Background: To reduce incision complications, minimally invasive operative approaches for treatment with acute Achilles tendon rupture have been developed, such as Mini-open repair and percutaneous repair. Which technique is the better surgical option? In the present study, we compared the two surgical procedures— modified Mini-open repair versus percutaneous repair—in the treatment of acute Achilles tendon rupture. Methods: From January 2016 to November 2018, 68 matched patients with acute Achilles tendon rupture were divided into treatment group (Mini-open with modified Ma-Griffith technique) and control group (the Ma–Griffith technique). The patients were then treated with different surgical techniques and followed up for no less than 24 months, and the functional outcome scores and complications were retrospectively evaluated. Results: The mean follow-up time in Mini-open repair group was 29.0±2.9 months, and that in control group was 27.9±2.9 months (P=0.147). The Mini-open repair group showed reliably higher American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Score and Achilles tendon Total Rupture Score (ATRS) than the control group in functional assessment (95.0±3.8 vs. 92.3±5.3, P=0.000; 93.8±3.8 vs. 90.9±4.5,P=0.000). There was no cases of sural nerve injury in Mini-open repair group, whereas the percutaneous repair group had 5 cases of the same (P=0.027). No significant differences were found in the calf circumference (32.3±3.9 vs. 31.8±3.6) (P=0.564), range of motion of the ankle (51.3±4.8 vs. 50.5±4.2, P=0.362), or wound complications (34/0 vs. 34/0) (P=1.000) between the two groups at the end of the follow-up time. However, the percutaneous repair group had a shorter average operating time (23.1±5.2 min) than that of the Mini-open repair group (27.7±4.3 min) (P=0.000). Conclusions: Acute Achilles tendon ruptures may be treated successfully with a new Mini-open repair system or percutaneous repair technique. However, the Mini-open repair system may represent a superior surgical option, since it offers advantages in terms of direct visual control of the repair, AOFAS Ankle-Hindfoot Score, Achilles tendon Total Rupture Score and risk of sural nerve palsy. Study design: Case-control studies, Level of evidence, 3. Study design: Case-control studies, Level of evidence, 3. Keywords: Acute Achilles tendon rupture, Minimally invasive, Percutaneous, Mini-open Comparison of mini-open repair system and percutaneous repair for acute Achilles tendon rupture Yong Li1, Qiang Jiang1, Hua Chen2, Hongkui Xin1, Qing He1 and Dike Ruan1 Yong Li1, Qiang Jiang1, Hua Chen2, Hongkui Xin1, Qing He1 and Dike Ruan1 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Acute rupture of the Achilles tendon is one of the most common types of tendon ruptures in the human body [1]. This type of rupture commonly occurs at the location of the tendon with poor blood supply—that is, 2 cm to 6 cm above the insertion site. Because of the imperfections of Correspondence: liyong807@163.com 1 Department of Orthopedics Surgery, The 6th Medical Center of Chinese PLA General Hospital, No. 6, Fucheng Road, Haidian District, Beijing 100048, People’s Republic of China Full list of author information is available at the end of the article Correspondence: liyong807@163.com 1 Department of Orthopedics Surgery, The 6th Medical Center of Chinese PLA General Hospital, No. 6, Fucheng Road, Haidian District, Beijing 100048, People’s Republic of China Full list of author information is available at the end of the article Li et al. BMC Musculoskelet Disord (2021) 22:914 https://doi.org/10.1186/s12891-021-04802-8 Li et al. BMC Musculoskelet Disord (2021) 22:914 https://doi.org/10.1186/s12891-021-04802-8 © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 2 of 9 the Achillon device and no differences in rerupture rate, sural nerve injury, return to sports, or American Orthopaedic Foot and Ankle Society (AOFAS) score compared with open repair. Although the design of the Achillon device is ingenious, one of the disadvantages of Achillon device is that suture crossing is cumber- some, and the crossing sutures may cut through the Achilles tendon [21]. It affects the tensile strength of the Achilles tendon after repair. This device requires at least 6 sutures, and there should be at least 6 knots at the broken end. As a result, it would increase suture reactivity, which can affect postoperative recovery and Achilles tendon function. In 2010, the another Mini- open Repair System (PARS, Arthrex, Inc, Naples, FL) has been available. This device is similar to the Achil- lon device, but includes nonlocking and locking sutures to better grasp the tendon ends and potentially improve the strength of the repair. Although the PARS reduces the complications related to wounds and sural nerve entrapment, it is still relatively complex in the proce- dure, and also requires longer operating time. surgical techniques [2, 3] and close relationship with the paratenon and plantar fascia [4], the optimal treatment of acute Achilles tendon ruptures is still under debate [5]. Patients and methodsh This control-matched study was conducted at the Navy General Hospital of PLA. Using the database and the medical records (between January 2016 and Novem- ber 2018), 34 patients with acute Achilles tendon rup- ture treated with Mini-open repair were matched to 34 patients treated with percutaneous minimally invasive anastomosis. The age(±5), sex, and BMI(±5) were similar and well-matched in both groups. This study approved by the ethical committee of the Navy General Hospital of PLA in 2016. All experiments were performed in accord- ance with relevant guidelines and regulations. Both the surgical interventions described in study were imple- mented as standard-of-care at hospital. The patients can withdraw from the study at any time without discrimina- tion or retaliation, and the medical treatment and rights and interests will not be affected. Regardless of any patient who refuses to participate in the study, he/she will still receive appropriate surgical treatment. Sample-size estimation was based on what was needed to detect the difference in complications in the groups. We estimated that more than 30 patients in each group were enough to detect a 20% difference in AOFAS score or ATRS between groups, with the alpha set at 0.05 and beta at 0.1. An additional 10 % of total participants was planned for each group to make up for possible loss. All patients read the detailed information sheet and signed a written con- sent form.h The criteria for inclusion in this study were as follows: (1) Patients with acute, closed Achilles tendon rupture; (2) A positive Thompson test; (3) Presence of pitting as assessed by observation and palpation between the two broken ends of the Achilles tendon; and (4) Complete rupture of the Achilles tendon as observed by ultrasonic examination. By contrast, patients with incomplete rup- ture of the Achilles tendon or open injury, patients with a repair time of more than 2 weeks, and patients with incomplete clinical data were excluded. Mini‑open repair grouph surgical procedures—Mini-open repair versus percuta- neous repair—in the treatment of acute Achilles tendon rupture. The process was as follows. (1) Establishment of the sur- gical incision. The patient was placed in the prone posi- tion, and epidural anesthesia was applied. The end of the Achilles tendon was subsequently exposed by making an approximately 2–3 cm transverse incision at the level of tendon rupture (Fig. 2a). The proximal end of the Achil- les tendon was also clamped and pulled out with hemo- static forceps. The channel instrument was inserted into the epitenon of the Achilles tendon along the fibers of the Achilles tendon. The instrument was then repeat- edly pushed and pulled to achieve blunt separation of the proximal Achilles tendon and fascia (Fig. 2b). A longitu- dinal skin incision measuring approximately 5 mm was made along the guide holes on both sides of the proxi- mal end of the tendon (Fig. 2c). The subcutaneous tissue was bluntly separated with hemostatic forceps (to pro- tect the sural nerve from damage). (2) Establishment of the proximal suture channel. Two-sided tapered sleeves and center guides were placed along the proximal guide hole, and the suture channel was established (Fig.2d). (3) Suturing of the ruptured Achilles tendon proximally and distally. The physician threaded the needle once along the center guide on both sides while pulling the hemo- static forceps distally. The physician then adjusted the orientation of the guide needle and threaded the needle again without pulling the hemostatic forceps distally. The channel instrument was subsequently withdrawn, and the proximal suture was pulled out (Fig. 2e). Suturing of the ruptured Achilles tendon was completed proximally (Fig. 2f). Moreover, suturing of the ruptured Achilles ten- don was completed distally by using the same method (Fig. 2g) as that for proximal suturing. (4) Anastomosis of the ruptured Achilles tendon distally and proximally. Tension was placed on the two ends of the suture, which were knotted for fixation (Fig. 2h). The broken ends were sutured with absorbable Vicryl Suture 3-0 to strengthen the anastomosis of the broken ends. The incision was sutured successively, and the long leg was fixed in plaster. The operation was completed. Postoperative care and rehabilitation (1) Non–weight-bearing equinus cast in place (0–2 weeks after surgery). A cast in a 20° to 25 ° “equi- nus” position was applied after both procedures and a below the knee gravity equinus cast was applied for approximately 2 weeks. (1) Non–weight-bearing equinus cast in place (0–2 weeks after surgery). A cast in a 20° to 25 ° “equi- nus” position was applied after both procedures and a below the knee gravity equinus cast was applied for approximately 2 weeks. To reduce incision complications, minimally invasive operative approaches have been developed, such as per- cutaneous repair and Mini-open repair. Percutaneous suture technique is widely used by many surgeons in Achilles tendon repair, but sural nerve injury remains a problem. Sural nerve entrapment is one of the most common complications after percutaneous surgery [6–9]. The careful placement of stab incisions to expose the nerve so as to avoid it has been advocated. In addi- tion, in order to reduce the risk of sural nerve injury, some surgeons use curved ring forceps [10, 11] or shap- ing Kirschner wires [12] for assistance, but it remains a challenge to prevent the sural nerve from being punc- tured or entrapment. In 2019, Carmont and Maffulli reported the results about percutaneous Bunnel type repairs for the treatment of acute Achilles tendon rup- tures [13]. The rate of sural nerve damage remains as high as 6.8%. To reduce incision complications and nerve damage, various limited-open repair techniques have been developed recently [14–18]. According to the Kakiuchi’s suture method [12], Assal et al developed a device, later known as the Achillon® System™, and they published a prospective review of 87 patients treated for acute Achilles tendon rupture using this device in 2002 [19]. The invention of Achillon is a step forward in Mini-open treatment of the Achilles tendon. A meta- analysis [20] reported fewer wound complications with Which technique is the better surgical option for treatment with acute Achilles tendon rupture? In the present study, we used a new Mini-Open Achilles ten- don repair system with modified Ma-Griffith tech- nique (Fig. 1) [22, 23]. This suture system was based on the Bunnell suture method, which was different from the Achillon system. This new device requires at least 2 sutures and 2 knots at the broken end. In this retro- spective control-matched study, we compared the two Fig. 1 The principle of two kinds of minimally invasive anastomosis of Achilles tendon. A Mini-open repair system. B Percutaneous minimally invasive anastomosis (Ma and Griffith’s technique) Fig. 1 The principle of two kinds of minimally invasive anastomosis of Achilles tendon. A Mini-open repair system. B Percutaneous minimally invasive anastomosis (Ma and Griffith’s technique) Li et al. BMC Musculoskelet Disord (2021) 22:914 Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 3 of 9 Percutaneous repair (the Ma–Griffith technique) Percutaneous Achilles tendon repair was performed in this study in accordance with the Ma–Griffith technique (Fig. 1). The patient was placed in a prone position, and a tourniquet was applied. The specific Ma–Griffith tech- nique used in this study has been referred to in previous studies [24–26]. We incorporate the benefits of some new percutaneous repair approaches to minimize sural nerve damage. (2) “Walker boot” period/muscle strength recovery period (3–10 weeks after surgery). After 2 weeks, the below the knee cast was removed and the patient began to ambulate in a “walker boot”, range- Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 4 of 9 Fig. 2 Operative approaches with Mini-open repair system. a The position relationship between sural nerve and Achilles tendon. B Establishment of the proximal surgical channel. c The epitenon is cut and proximal pilot hole is formed. d: Suturing of the ruptured Achilles tendon (AT) proximally. e The proximal suture was pulled out. f The proximal Achilles tendon was sutured and sural nerve was avoided successfully. g Suturing of the ruptured Achilles tendon distally. h Anastomosis of the ruptured Achilles tendon (AT) distally and proximally Fig. 2 Operative approaches with Mini-open repair system. a The position relationship between sural nerve and Achilles tendon. B Establishment of the proximal surgical channel. c The epitenon is cut and proximal pilot hole is formed. d: Suturing of the ruptured Achilles tendon (AT) proximally. e The proximal suture was pulled out. f The proximal Achilles tendon was sutured and sural nerve was avoided successfully. g Suturing of the ruptured Achilles tendon distally. h Anastomosis of the ruptured Achilles tendon (AT) distally and proximally of-motion movements for the ankle were practiced, the leg muscles were strengthened, and normal gait was gradually restored. During the next 3-4 weeks the angle of the “walker boot” was gradually changed to a neutral position. The “walker boot” period was maintained for at least 8 weeks. ually restore motor function. Attempts were made to achieve full movement at the ankle. The ATRS [27, 28] and AOFAS [29, 30] ankle–hindfoot scale score were used to evaluate the clinical outcome at the last follow-up. (4) The patients were followed up for no less than 24 months, and the functional outcome scores and complications were retrospectively evaluated. (3) Muscle strengthening period (10 weeks after sur- gery and beyond). Ten weeks after surgery, ankle flexibility and leg muscle strength were improved to increase the stability of the lower limbs and to grad- (4) The patients were followed up for no less than 24 months, and the functional outcome scores and complications were retrospectively evaluated. Functional evaluation was based on the clinical AOFAS score and ATRS along with other find- Resultsh Ankle-Hindfoot Score and ATRS than the control group in functional assessment (95.0±3.8 vs. 92.3±5.3, P=0.000; 93.8±3.8 vs. 90.9±4.5, P=0.000). There was no cases of sural nerve injury in Mini-open repair group, whereas the percutaneous repair group had 5 cases of the same (P=0.027). No significant differences were found in the calf circumference (32.3±3.9 vs. 31.8±3.6) (P=0.564), range of motion of the ankle (51.3±4.8 vs. 50.5±4.2, P=0.362), or the number of wound necrosis or infec- tion (34/0 vs. 34/0) (P=1.000) between the two groups at the end of the follow-up time. However, the percuta- neous repair group had a shorter average operating time (23.1±5.2 min), compared with the Mini-open repair group (27.7±4.3 min) (P=0.000). No cases of sural nerve injury in the Mini-open repair group were reported, but five such cases were found in the percutaneous repair group (P=0.027). Ankle-Hindfoot Score and ATRS than the control group in functional assessment (95.0±3.8 vs. 92.3±5.3, P=0.000; 93.8±3.8 vs. 90.9±4.5, P=0.000). There was no cases of sural nerve injury in Mini-open repair group, whereas the percutaneous repair group had 5 cases of the same (P=0.027). No significant differences were found in the calf circumference (32.3±3.9 vs. 31.8±3.6) (P=0.564), range of motion of the ankle (51.3±4.8 vs. 50.5±4.2, P=0.362), or the number of wound necrosis or infec- tion (34/0 vs. 34/0) (P=1.000) between the two groups at the end of the follow-up time. However, the percuta- neous repair group had a shorter average operating time (23.1±5.2 min), compared with the Mini-open repair group (27.7±4.3 min) (P=0.000). No cases of sural nerve injury in the Mini-open repair group were reported, but five such cases were found in the percutaneous repair group (P=0.027). The baseline information and demographics of both groups are listed in Table 1. A total of 68 patients were enrolled in the Mini-open repair group with an average age of 32.3±6.9 y (range, 21–42 y) and the percutaneous repair group with an average age of 30.5±7.1 y (range, 18–40 y).h The follow-up data were summarized, and func- tional results were evaluated in both groups (Table 2). All patients in both groups were available for follow- up, with a mean follow-up time of 29.0±2.9 months in group A and 27.9±2.9 months in group B (P=0.147). Resultsh The Mini-open repair group showed reliably higher AOFAS Table 1 Baseline characteristics of both groups a P-value as determined by the Paired Samples t-Test; b The Chi-square test was used for the comparison of rates; C Fisher’s exact test was used when one or more expected values are less than 5 Variable Mini-open repair group Percutaneous repair group P value Age (years) 32.3±6.9 30.5±7.1 0.253a Gender(M/F) 31/3 31/3 1.000b Side(L/R) BMI (kg/m2) Diabetes mellitus (%) Smoking (%) Alcohol use (%) Corticosteroids (%) Peripheral vascular disease (%) 19/15 24.3±2.7 5.9 32.4 55.9 5.9 8.8 18/16 23.5±3.4 11.8 29.4 50.0 8.8 11.8 1.000b 0.302a 0.673c 0.793b 0.627b 1.000c 1.000c Table 1 Baseline characteristics of both groups Multivariate analysis was performed to analyze the relationship. The age, BMI, Operating time, hospital stay, ATRS and ROM of ankle joint were taken as independent variables, while AOFAS was taken as dependent variables for linear regression analysis. The results were shown in the Supplement Tables 1 and 2. Statistical analysis SPSS 23 0 y SPSS 23.0 was used for statistical analysis. The two groups were compared with respect to sex, age, follow- up time, operating time, hospital stay, calf circumference, AOFAS score, ATRS score, number of wound compli- cations, sural nerve injury, and ankle ROM. Statistical analysis was conducted by an independent statistician not directly involved in the study. The Paired Samples t-Test, the results of which were expressed as the mean and standard deviation (SD), was used for the quantita- tive data analysis with equal variance assumed between the two groups. The Chi-square test was used to assess the qualitative data between the two groups. A P value of less than 0.05 was considered significant. a P-value as determined by the Paired Samples t-Test b The Fisher two-sided exact test was used for the comparison of rates c The Fisher one-sided exact test was used for the comparison of rates Percutaneous repair (the Ma–Griffith technique) Functional evaluation was based on the clinical AOFAS score and ATRS along with other find- (3) Muscle strengthening period (10 weeks after sur- gery and beyond). Ten weeks after surgery, ankle flexibility and leg muscle strength were improved to increase the stability of the lower limbs and to grad- Li et al. BMC Musculoskelet Disord (2021) 22:914 Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 5 of 9 Table 2 Comparison of the main follow-up data for both groups of patients a P-value as determined by the Paired Samples t-Test b The Fisher two-sided exact test was used for the comparison of rates c The Fisher one-sided exact test was used for the comparison of rates Variable Mini-open repair group percutaneous repair group P value Average operating time(min) 27.7±4.3 23.1±5.2 0.000a Follow-up time(months) 29.0±2.9 27.9±2.9 0.147a Calf circumference 32.3±3.9 31.8±3.6 0.564a Re-rupture (n) 0 0 1.000b Palpable knot (n) 5 8 0.355b Scar tissue adhesions (n) 0 2 0.493b Wound necrosis (n) 0 0 1.000b Superficial infection (n) 0 0 1.000b Deep infection (n) 0 0 1.000b AOFAS score 95.0±3.8 92.3±5.3 0.000a ATRS score 93.8±3.8 90.9±4.5 0.000 a Sural nerve palsy (n) 0 5 0.027c Ankle ROM(°) 51.3±4.8 50.5±4.2 0.362a Table 2 Comparison of the main follow-up data for both groups of patients ings, such as the length of the scar, neurologic defi- cit, calf circumference, and range of motion of the ankle. Whether there was no deep vein thrombosis or sural nerve injury was based on color Doppler ultrasound and electromyography. Discussionh The choice of treatment for acute Achilles tendon rup- ture remains a challenge for surgeons. Despite hundreds of publications in the medical literature on the sub- ject of acute rupture of the Achilles tendon, its optimal treatment remains under debate. One study from the Li et al. BMC Musculoskelet Disord (2021) 22:914 Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 6 of 9 Netherlands has described that although open repair (65%) was the most common surgical technique and Bunnell sutures (55%) were mostly applied, trauma sur- geons and orthopaedic surgeons differed significantly on surgical technique (p= 0.001), suturing technique (p= 0.002) [31]. Surgical treatment can effectively reduce the rate of re-rupture and can lead to early functional recovery with exercise [32]. However, open surgery usu- ally requires a long operative incision (average length of approximately 10 cm) and requires too much shedding of the Achilles tendon tissue, which can affect postopera- tive recovery [33]. Minimally invasive repair for Achilles tendon rupture has become widely applied to avoid long surgical incision, soft tissue necrosis, infection, and other related complications [34, 35]. Ma et al. introduced the use of percutaneous minimally invasive suture repair for Achilles tendon rupture [36]. Mini-open repair includes medial and lateral percutaneous–minimally invasive incisions and suture of the ruptured proximal tendon with a modified Bunnell suture and a diatal box suture [37]. Khan, R. J. et al. [6] concluded that compared with open surgical techniques, percutaneous techniques led to reductions in re-ruptures and overall complication rate. To better reconstruct the continuity of the tendon ends and reduce the risk of complications, the proper- ties of the open and percutaneous techniques were com- bined [12]. An increasing number of orthopedic surgeons currently prefer to perform Mini-open procedures with surgical aid devices (Fig. 3) such as Tenolig [35, 38, 39], Achillon, PARS [34], or the Dresden instrument [19, 25, 40, 41] and at times integrate the method with ultra- sound-guided approaches. In the present study, we used a new minimally inva- sive and direct visual control of Achilles tendon suture system (Fig. 1). This Mini-open repair system was based on the Bunnell suture method, which was dif- ferent from the Achillon system (Fig. 2). The proximal end of Achilles tendon is sutured with three transverse crosses of sutures by using an eccentric sleeve. We sim- plified the complex steps of Achillon and PARS repair Fig. Discussionh This Mini-open repair sys- tem has reduced suture knots, lowering the foreign body sensation of the suture knot and keloid after recovery and consequently improving the function and appearance of hindfoot. Our results suggest that both Mini-open repair and percutaneous repair can achieve satisfactory functional outcomes in patients with Achilles tendon. However, the Mini-open repair group showed reliably higher AOFAS Ankle-Hindfoot Score and ATRS than the control group in Function assessment (95.0±3.8 vs. 92.3±5.3, P=0.000; 93.8±3.8 vs. 90.9±4.5,P=0.000). The functional results are comparable to the results in several other investigations using open and mini-open repair techniques [44–47]. Calder et al. treated 25 patients by using an Achillon Achilles tendon suture device, and the follow-up AOFAS score was as high as 98.4 points [48]. Chen et al. performed Mini-open repair repair in 41 patients [22], and 90.5 was the reported AOFAS score 12 months after surgery. Multivariate analysis was per- formed to analyze the relationship. The results in the supplement tables showed that in the Mini-open repair group, the R2 value of in the model is 0.699 and it meant that the age, BMI, Operating time, hospital stay, ATRS and ROM of ankle joint could explain 69.9 % variations in AOFAS. The model was tested by the F-test (F=11.967, P<0.05), indicating that at least one of age, BMI, operat- ing time, hospital stay, ankle range of motion and ATRS Sural nerve palsy is one of the most important compli- cations of minimally invasive repair of Achilles tendon rupture. A recent meta-analysis [49] suggests that sural nerve palsy is still a considerable complication of MIS. Initially, under the Ma–Griffith percutaneous technique, a sural nerve palsy rate reaching 60% has been reported. Haji et al. found that applying this technique [7] results in up to 10.5% transient sural nerve injury rate. Suther- land et al. treated 31 patients with this minimally invasive percutaneous suturing method, 5 of whom developed sural nerve injury [50]. In 2011, Taglialavoro et al. [51] reported that Tenolig group showed a lower risk of dam- age to the sural nerve compared to the Ma and Griffith technique(2/30 VS. 4/30). In the current study, the results of our classic percutaneous surgery were similar to those in other studies, with 5 cases of sural nerve injury. Discussionh treated 25 patients by using an Achillon Achilles tendon suture device, and the follow-up AOFAS score was as high as 98.4 points [48]. Chen et al. performed Mini-open repair repair in 41 patients [22], and 90.5 was the reported AOFAS score 12 months after surgery. Multivariate analysis was per- formed to analyze the relationship. The results in the supplement tables showed that in the Mini-open repair group, the R2 value of in the model is 0.699 and it meant that the age, BMI, Operating time, hospital stay, ATRS and ROM of ankle joint could explain 69.9 % variations in AOFAS. The model was tested by the F-test (F=11.967, P<0.05), indicating that at least one of age, BMI, operat- ing time, hospital stay, ankle range of motion and ATRS repair group, the R2 value of model was 0.619 and it also passed the F-test (F=7.649, P<0.05), indicating that the age, BMI, a model could explain 61.9% of the variation in AOFAS. and decreased the number of knots, facilitating the gliding of the Achilles tendon with the surrounding tis- sue after long-term repair and reducing the formation of keloids [42, 43]. So, this Mini-open repair system can reduce the risk of suture reactivity and make the bro- ken end smoother. The aforementioned methods are the important factors affecting long-term functional recov- ery after Achilles tendon repair. In vitro studies showed no significant reduction in suture strength, although the number of stitches was decreased. After the proximal and distal sutures were tied, the strength of repair suffi- ciently met the requirements of early functional exercise. A study [23] performed a biomechanical comparison of the Mini-open repair repair system and three common Achilles tendon restoration techniques (Achillon, PARS, Krackow) in an in vitro model via a progressive rehabili- tation program. Mini-open repair can achieve reliable suture strength with fewer stitches and knots, as strong as that of the open Krackow restoration, but weaker than those of the Achillon and PARS techniques. To a certain extent, the greater tensile strength of the suture used, the stronger the tensile strength of the Mini-open repair suture structure. Therefore, the repaired Achilles tendon exhibits high tensile strength, allows early functional exercise, and requires skills that can be easily mastered with a short learning curve. Discussionh The sural nerve injury caused by this method may be mainly attributed to the nerve not being fully exposed during the operation and the suture needle being placed blindly, hence the risk of a direct puncture injury to the nerve. An increased risk of direct sural nerve injury or indirect irritation by sutures exists particularly when needles are pierced laterally into the proximal portion of the Achil- les tendon. Simultaneously, it leads to tethering of the fascia cruris to the tendon. Therefore, minimally invasive Achilles tendon surgery should aim to avoid sural nerve injury. In this study, modified Mini-open repair was used to establish the suture channel so that the sural nerve was located outside the suture channel before sutur- ing (Fig. 2), and the injury was effectively avoided dur- ing suture threading. In this study, no sural nerve injury occurred in the Mini-open repair group. On the basis of the results, Mini-open repair as a surgical option may be preferable to percutaneous repair for the treatment of acute Achilles tendon rupture because the former avoids damage to the sural nerves. This study has certain limitations in control-matched designs. Selection bias was not avoided, considering that the surgical treatment to be performed was determined by the orthopedic surgeon. Although the two groups of patients were matched, only the sex, age and BMI of the patients were matched in the groups to maintain a suf- ficient number of patients. Discussionh 3 Photographs and simplified schematic diagrams illustrating the different repair constructs and suture configurations. a,a’ Achillon repair, b,b’ PARS repair, c,c’ Mini-open repair Fig. 3 Photographs and simplified schematic diagrams illustrating the different repair constructs and suture configurations. a,a’ Achillon repair, b,b’ PARS repair c c’ Mini open repair ig. 3 Photographs and simplified schematic diagrams illustrating the different repair constructs and suture configurations. a, ARS repair, c,c’ Mini-open repair Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 7 of 9 and decreased the number of knots, facilitating the gliding of the Achilles tendon with the surrounding tis- sue after long-term repair and reducing the formation of keloids [42, 43]. So, this Mini-open repair system can reduce the risk of suture reactivity and make the bro- ken end smoother. The aforementioned methods are the important factors affecting long-term functional recov- ery after Achilles tendon repair. In vitro studies showed no significant reduction in suture strength, although the number of stitches was decreased. After the proximal and distal sutures were tied, the strength of repair suffi- ciently met the requirements of early functional exercise. A study [23] performed a biomechanical comparison of the Mini-open repair repair system and three common Achilles tendon restoration techniques (Achillon, PARS, Krackow) in an in vitro model via a progressive rehabili- tation program. Mini-open repair can achieve reliable suture strength with fewer stitches and knots, as strong as that of the open Krackow restoration, but weaker than those of the Achillon and PARS techniques. To a certain extent, the greater tensile strength of the suture used, the stronger the tensile strength of the Mini-open repair suture structure. Therefore, the repaired Achilles tendon exhibits high tensile strength, allows early functional exercise, and requires skills that can be easily mastered with a short learning curve. This Mini-open repair sys- tem has reduced suture knots, lowering the foreign body sensation of the suture knot and keloid after recovery and consequently improving the function and appearance of hindfoot. Our results suggest that both Mini-open repair and percutaneous repair can achieve satisfactory functional outcomes in patients with Achilles tendon. However, the Mini-open repair group showed reliably higher AOFAS Ankle-Hindfoot Score and ATRS than the control group in Function assessment (95.0±3.8 vs. 92.3±5.3, P=0.000; 93.8±3.8 vs. 90.9±4.5,P=0.000). The functional results are comparable to the results in several other investigations using open and mini-open repair techniques [44–47]. Calder et al. Abbreviations Abbreviations AT: Achilles tendon; ATRS: Achilles tendon Total Rupture Score; AOFAS: Ameri- can Orthopedic Foot and Ankle Society; SD: standard deviation; BMI: body mass index. 4. Stecco C, Corradin M, Macchi V, Morra A, Porzionato A, Biz C, et al. Plantar fascia anatomy and its relationship with Achilles tendon and paratenon. J Anat. 2013;223(6):665–76. 4. Stecco C, Corradin M, Macchi V, Morra A, Porzionato A, Biz C, et al. Plantar fascia anatomy and its relationship with Achilles tendon and paratenon. J Anat. 2013;223(6):665–76. 5. Maffulli N, Peretti GM. Treatment decisions for acute Achilles tendon ruptures. Lancet. 2020;395(10222):397–8. 5. Maffulli N, Peretti GM. Treatment decisions for acute Achilles tendon ruptures. Lancet. 2020;395(10222):397–8. Availability of data and materials The datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request. 16. Keller A, Ortiz C, Wagner E, Wagner P, Mococain P. Mini-open tenorrhaphy of acute Achilles tendon ruptures: medium-term follow-up of 100 cases. Am J Sports Med. 2014;42(3):731–6. Funding This research was supported by the new technology & new business projects of Navy General Hospital of Chinese PLA. g 14. Sliwa M. Percutaneous and minimally invasive Achilles tendon repair - review of surgical techniques. Pol Orthop Traumatol. 2014;79:92–6. 15. Demetracopoulos CA, Gilbert SL, Young E, Baxter JR, Deland JT. Limited- open achilles tendon repair using locking sutures versus nonlocking sutures: an in vitro model. Foot Ankle Int. 2014;35(6):612–8. Acknowledgments We acknowledge Jia Liu in Chinese Center for Disease Control and Prevention for statistical assistance. 10. Park CH, Na HD, Chang MC. Clinical outcomes of minimally invasive repair using ring forceps for acute achilles tendon rupture. J Foot Ankle Surg. 2021;60(2):237–41. Declarations 17. Baltes TPA, Zwiers R, Wiegerinck JI, van Dijk CN. Surgical treatment for midportion Achilles tendinopathy: a systematic review. Knee Surg Sports Traumatol Arthrosc. 2017;25(6):1817–38. Competing interests The authors declare that they have no competing interests. 21. Ismail M, Karim A, Shulman R, Amis A, Calder J. The Achillon achilles tendon repair: is it strong enough? Foot Ankle Int. 2008;29(8):808–13. Consent for publication Not Applicable. 20. Alcelik I, Saeed ZM, Haughton BA, Shahid R, Alcelik JC, Brogden C, et al. Achillon versus open surgery in acute Achilles tendon repair. Foot Ankle Surg. 2018;24(5):427–34. Authors’ contributions Dr Yong Li had full access to all of the data in the study and takes respon- sibility for the integrity of the data and the accuracy of the data analysis. Study design: YL, QJ and DR. Acquisition of data: YL, QJ, HX, HC. Analysis and interpretation of the data: YL, HC, QH, DR. Writing: YL. Statistical analysis: YL, QH, DR. YL and QJ contributed equally to this study. 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The online version contains supplementary material available at https://doi. org/10.1186/s12891-021-04802-8. 6. Khan RJ, Fick D, Keogh A, Crawford J, Brammar T, Parker M. Treatment of acute achilles tendon ruptures. A meta-analysis of randomized, con- trolled trials. J Bone Joint Surg Am. 2005;87(10):2202–10. 7. Haji A, Sahai A, Symes A, Vyas JK. Percutaneous versus open tendo achillis repair. Foot Ankle Int. 2004;25(4):215–8. Additional file 1: Supplement Table 1. Multivariate analysis results in Mini-open repair group p ; ( ) 8. Rippstein PF, Jung M, Assal M. Surgical repair of acute Achilles tendon rupture using a “mini-open” technique. Foot Ankle Clin. 2002;7(3):611–9. 9. Majewski M, Rohrbach M, Czaja S, Ochsner P. Avoiding sural nerve injuries during percutaneous Achilles tendon repair. Am J Sports Med. 2006;34(5):793–8. Conclusions In conclusion, our results suggest that satisfactory func- tional outcomes can be obtained for both treatment meth- ods. This new Mini-open repair system is easy to operate and the guide instrument of the system is placed deep into Li et al. BMC Musculoskelet Disord (2021) 22:914 Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 8 of 9 the paratenon, preventing the risk of a subcutaneous nerve being trapped in the suture itself. Mini-open repair may be the superior surgical option, given its advantages in terms of direct visual control of the repair, AOFAS Ankle-Hind- foot Score, ATRS and risk of sural nerve palsy. the paratenon, preventing the risk of a subcutaneous nerve being trapped in the suture itself. Mini-open repair may be the superior surgical option, given its advantages in terms of direct visual control of the repair, AOFAS Ankle-Hind- foot Score, ATRS and risk of sural nerve palsy. Received: 2 July 2021 Accepted: 19 October 2021 Ethics approval and consent to participate All procedures performed in this study involving human participants were approved by the Ethical Committee of Navy General Hospital (6th Medical Center of PLA General Hospital), which followed the ethical standards of the institutional and national research committee and the 1964 Helsinki Declara- tion and its later amendments (IRB No.: ECNGH-2016040). 18. Ceccarelli F, Berti L, Giuriati L, Romagnoli M, Giannini S. Percutaneous and minimally invasive techniques of Achilles tendon repair. Clin Orthop Relat Res. 2007;458:188–93. 19. Assal M, Jung M, Stern R, Rippstein P, Delmi M, Hoffmeyer P. Limited open repair of Achilles tendon ruptures: a technique with a new instrument and findings of a prospective multicenter study. J Bone Joint Surg Am. 2002;84(2):161–70. Author details 1 Author details 1 Department of Orthopedics Surgery, The 6th Medical Center of Chinese PLA General Hospital, No. 6, Fucheng Road, Haidian District, Beijing 100048, People’s Republic of China. 2 Department of Orthopedics, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100048, People’s Republic of China. 22. Chen H, Ji X, Zhang Q, Liang X, Tang P. Channel-assisted minimally invasive repair of acute Achilles tendon rupture. J Orthop Surg Res. 2015;10:167. 23. Qi H, Ji X, Cui Y, Wang L, Chen H, Tang P. Comparison of channel-assisted minimally invasive repair and 3 common Achilles tendon restoration techniques. Exp Ther Med. 2019;17(2):1426–34. Received: 2 July 2021 Accepted: 19 October 2021 24. Karabinas PK, Benetos IS, Lampropoulou-Adamidou K, Romoudis P, Mav- rogenis AF, Vlamis J. Percutaneous versus open repair of acute Achilles tendon ruptures. Eur J Orthop Surg Traumatol. 2014;24(4):607–13. 25. Hsu AR, Jones CP, Cohen BE, Davis WH, Ellington JK, Anderson RB. Clinical outcomes and complications of percutaneous achilles repair system Li et al. BMC Musculoskelet Disord (2021) 22:914 Page 9 of 9 versus open technique for acute achilles tendon ruptures. Foot Ankle Int. 2015;36(11):1279–86. 40. Gigante A, Moschini A, Verdenelli A, Del Torto M, Ulisse S, de Palma L. Open versus percutaneous repair in the treatment of acute Achilles tendon rupture: a randomized prospective study. Knee Surg Sports Traumatol Arthrosc. 2008;16(2):204–9. 26. Tejwani NC, Lee J, Weatherall J, Sherman O. Acute achilles tendon ruptures: a comparison of minimally invasive and open approach repairs followed by early rehabilitation. Am J Orthop. 2014;43(10):E221–5. 41. Amlang MH, Christiani P, Heinz P, Zwipp H. The percutaneous suture of the Achilles tendon with the Dresden instrument. Oper Orthop Trauma- tol. 2006;18(4):287–99. 27. Nilsson-Helander K, Sward L, Silbernagel KG, Thomee R, Eriksson BI, Karlsson J. A new surgical method to treat chronic ruptures and reruptures of the Achilles tendon. Knee Surg Sports Traumatol Arthrosc. 2008;16(6):614–20. 42. Frosch S, Buchhorn G, Hawellek T, Walde TA, Lehmann W, Hubert J. Comparison of the double loop knot stitch and Kessler stitch for Achilles tendon repair: A biomechanical cadaver study. PLoS One. 2020;15(12):e0243306. 28. He SK, Liao JP, Huang FG. Higher rate of postoperative complications in delayed achilles tendon repair compared to early Achilles tendon repair: a meta-analysis. J Invest Surg. 2020:1–7. 43. Bekler HI, Beyzadeoglu T, Gokce A, Servet E. [Aseptic drainage associated with polyglactine sutures used for repair of Achilles tendon ruptures]. Acta Orthop Traumatol Turc 2008, 42(2):135-138. Author details 1 A modified technique of percutaneous repair of ruptured Achilles tendon. Orthop Traumatol. 1998;7(4):288–95. 51. Taglialavoro G, Biz C, Mastrangelo G, Aldegheri R. The repair of the Achil- les tendon rupture: comparison of two percutaneous techniques. Strateg Trauma Limb Reconstr. 2011;6(3):147–54. 51. Taglialavoro G, Biz C, Mastrangelo G, Aldegheri R. The repair of the Achil- les tendon rupture: comparison of two percutaneous techniques. Strateg Trauma Limb Reconstr. 2011;6(3):147–54. 37. Del Buono A, Volpin A, Maffulli N. Minimally invasive versus open surgery for acute Achilles tendon rupture: a systematic review. Br Med Bull. 2013;109(1):45–54. 38. Martinelli B. Percutaneous repair of the Achilles tendon in athletes. Bull Hosp Jt Dis. 2000;59(3):149–52. 38. Martinelli B. Percutaneous repair of the Achilles tendon in athletes. Bull Hosp Jt Dis. 2000;59(3):149–52. Author details 1 y 29. Jaakkola JI, Beskin JL, Griffith LH, Cernansky G. Early ankle motion after tri- ple bundle technique repair vs. casting for acute Achilles tendon rupture. Foot Ankle Int. 2001;22(12):979–84. 44. Mukundan C, El Husseiny M, Rayan F, Salim J, Budgen A. “Mini-open” repair of acute tendo Achilles ruptures--the solution? Foot Ankle Surg. 2010;16(3):122–5. 30. Meulenkamp B, Stacey D, Fergusson D, Hutton B, Mlis RS, Graham ID. Protocol for treatment of Achilles tendon ruptures; a systematic review with network meta-analysis. Syst Rev. 2018;7(1):247. 45. Vadala A, De Carli A, Vulpiani MC, Iorio R, Vetrano M, Scapellato S, et al. Clinical, functional and radiological results of Achilles tenorraphy surgically treated with mini-open technique. J Sports Med Phys Fitness. 2012;52(6):616–21. 31. Dams OC, van den Akker-Scheek I, Diercks RL, Wendt KW, Zwerver J, Reininga IHF. Surveying the management of Achilles tendon ruptures in the Netherlands: lack of consensus and need for treatment guidelines. Knee Surg Sports Traumatol Arthrosc. 2019;27(9):2754–64. 46. Vadala A, Lanzetti RM, Ciompi A, Rossi C, Lupariello D, Ferretti A. Functional evaluation of professional athletes treated with a mini-open technique for achilles tendon rupture. Muscles Ligaments Tendons J. 2014;4(2):177–81. 32. Jiang N, Wang B, Chen A, Dong F, Yu B. Operative versus nonoperative treatment for acute Achilles tendon rupture: a meta-analysis based on current evidence. Int Orthop. 2012;36(4):765–73. 33. Kadakia AR, Dekker RG, Ho BS. Acute achilles tendon ruptures. J Am Acad Orthop Surg. 2017;25(1):23–31. 47. Ling SKK, Slocum A, Lui TH. 5-year results of the 1.5cm incision Achilles tendon repair. Foot (Edinb). 2017;33:35–8. 34. Clanton T, Stake IK, Bartush K, Jamieson MD. Minimally invasive achilles repair techniques. Orthop Clin North Am. 2020;51(3):391–402. 48. Calder JDF. Early, active rehabilitation following mini-open repair of Achilles tendon rupture: a prospective study. Br J Sports Med. 2005;39(11):857–9. 35. Biz C, Cerchiaro M, Belluzzi E, Bragazzi NL, Guttry GD, Ruggieri P. Long term clinical–functional and ultrasound outcomes in recreational athletes after achilles tendon rupture :ma and griffith versus tenolig. Medicina. 2021;57(10):1073. 49. Gatz M, Driessen A, Eschweiler J, Tingart M, Migliorini F. Open versus minimally-invasive surgery for Achilles tendon rupture: a meta-analysis study. Arch Orthop Trauma Surg. 2020. 50. Sutherland A, Maffulli N. A modified technique of percutaneous repair of ruptured Achilles tendon. Orthop Traumatol. 1998;7(4):288–95. 36. Ma GW, Griffith TG. Percutaneous repair of acute closed ruptured achilles tendon: a new technique. Clin Orthop Relat Res. 1977;128:247–55. 50. Sutherland A, Maffulli N. Publisher’s Note 39. Lacoste S, Feron JM, Cherrier B. Percutaneous Tenolig((R)) repair under intra-operative ultrasonography guidance in acute Achilles tendon rupture. Orthop Traumatol Surg Res. 2014;100(8):925–30. 39. Lacoste S, Feron JM, Cherrier B. Percutaneous Tenolig((R)) repair under intra-operative ultrasonography guidance in acute Achilles tendon rupture. Orthop Traumatol Surg Res. 2014;100(8):925–30. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W2118832531	https://hal.science/hal-01438941/document	English	null	The UV-optical colour dependence of galaxy clustering in the local universe	Monthly Notices of the Royal Astronomical Society	2,010	cc-by	14,587	The UV-optical colour dependence of galaxy clustering in the local universe Yeong-Shang Loh, Robert Michael Rich, Sebastien Heinis, Ryan Scranton, Ryan P. Mallery, Samir Salim, D. Christopher Martin, Ted Wyder, Stephane Arnouts, Tom Barlow, et al. To cite this version: Yeong-Shang Loh, Robert Michael Rich, Sebastien Heinis, Ryan Scranton, Ryan P. Mallery, et al.. The UV-optical colour dependence of galaxy clustering in the local universe. Monthly Notices of the Royal Astronomical Society, 2010, 407 (1), pp.55–70. 10.1111/j.1365-2966.2010.16908.x. hal-01438941 To cite this version: Yeong-Shang Loh, Robert Michael Rich, Sebastien Heinis, Ryan Scranton, Ryan P. Mallery, et al.. The UV-optical colour dependence of galaxy clustering in the local universe. Monthly Notices of the Royal Astronomical Society, 2010, 407 (1), pp.55–70. 10.1111/j.1365-2966.2010.16908.x. hal-01438941 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01438941 https://hal.science/hal-01438941v1 Submitted on 19 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License doi:10.1111/j.1365-2966.2010.16908.x Mon. Not. R. Astron. Soc. 407, 55–70 (2010) ⋆E-mail: yeongloh@astro.ucla.edu The UV–optical colour dependence of galaxy clustering in the local universe Yeong-Shang Loh,1⋆R. Michael Rich,1 S´ebastien Heinis,2 Ryan Scranton,3 Ryan P. Mallery,1 Samir Salim,4 D. Christopher Martin,5 Ted Wyder,5 St´ephane Arnouts,2 Tom A. Barlow,5 Karl Forster,5 Peter G. Friedman,5 Patrick Morrissey,5 Susan G. Neff,6 David Schiminovich,7 Mark Seibert,5 Luciana Bianchi,8 Jose Donas,9 Timothy M. Heckman,2 Young-Wook Lee,10 Barry F. Madore,11,12 Bruno Milliard,9 Alex S. Szalay2 and Barry Y. Welsh13 1Department of Physics and Astronomy, University of California, Los Angeles, CA 90095-1562, USA 2Department of Physics and Astronomy, The Johns Hopkins University, Homewood Campus, Baltimore, MD 21218, USA 3Department of Physics and Astronomy, University of Pittsburgh, 3941 O’Hara St., Pittsburgh, PA 15260, USA 4National Optical Astronomy Observatory, 950 North Cherry Avenue, Tucson, AZ 85719, USA 5California Institute of Technology, MC 405-47, 1200 East California Boulevard, Pasadena, CA 91125, USA 6Laboratory for Astronomy and Solar Physics, NASA Goddard Space Flight Center, Greenbelt, MD 20771, USA 7Department of Astronomy, Columbia University, New York, NY 10027, USA 8Center for Astrophysical Sciences, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA 9Laboratoire d’Astrophysique de Marseille, BP 8, Traverse du Siphon, 13376 Marseille Cedex 12, France 10Center for Space Astrophysics, Yonsei University, Seoul 120-749, Korea 11Space Sciences Laboratory, University of California at Berkeley, 601 Campbell Hall, Berkeley, CA 94720, USA 12Observatories of the Carnegie Institution of Washington, 813 Santa Barbara St., Pasadena, CA 91101, USA 13Space Sciences Laboratory, University of California at Berkeley, 601 Campbell Hall, Berkeley, CA 94720, USA Accepted 2010 April 22. Received 2010 April 20; in original form 2009 April 8 Accepted 2010 April 22. Received 2010 April 20; in original form 2009 April 8 ⋆E-mail: yeongloh@astro.ucla.edu C⃝2010 The Authors. Journal compilation C⃝2010 RAS 1 INTRODUCTION With the advent of the Sloan Digital Sky Survey (SDSS; York et al. 2000) and its value-added galaxy catalogues, it has been possible to study the subject of galaxy bimodality and its relationship to fundamental properties, such as stellar mass and star formation history (e.g. Kauffmann et al. 2003; Schiminovich et al. 2007; Salim et al. 2007). The broad division of galaxies into star-forming discs and quiescent early-type galaxies is the fundamental principle of Hubble’s tuning fork system of classiﬁcation and is well established. In a plot of optical g −r colour versus Mr, red galaxies deﬁne a clear sequence, while the locus of blue galaxies is broadened into the so- called blue cloud. The red sequence has been shown to maintain its integrity with look-back time (Bower et al. 1992) and has grown in mass since redshift ∼1. Studies by Bell et al. (2004), Blanton (2006), Faber et al. (2007) and Brown et al. (2008) argue that the stellar mass contained within the red population has increased by roughly a factor of 2 in half the Hubble time. The current paradigm of structure formation assumes that galax- ies are assembled in dark matter haloes. The dependence of the clustering on galaxy properties may provide clues to the baryonic processes that are important to galaxy formation and evolution. The dependence of galaxy clustering on galaxy type has been known since the earliest studies of extragalactic astronomy (Hubble 1936; Zwicky et al. 1968). In the modern era of large-scale galaxy surveys, Davis & Geller (1976) showed that the angular auto-correlation of ellipticals has a steeper power-law slope than those of spirals. Re- cent redshift surveys using the Two-Degree Field (2dF) Galaxy Survey and SDSS conﬁrms these earlier results and the apparent bimodal nature of galaxy clustering (Madgwick et al. 2003; Bu- davari et al. 2003; Zehavi et al. 2005; Li et al. 2006a; Wang et al. 2007). Studies using SDSS have further revealed that galaxy colour is the property most predictive of local environment. Blanton et al. (2005b) found that at ﬁxed luminosity and colour, density does not correlate with surface brightness nor the Sersic index, and argue that morphological properties of galaxies are less closely related to environment than their star formation history, and are traced by broad-band optical colours. [See Park et al. (2007) for an alterna- tive analysis and point of view.] Li et al. 1 INTRODUCTION (2006a) found that the dependence of clustering on optical g −r colour and D4000 is much stronger than structural parameters like concentration and surface brightness, and extend to 5 h−1 Mpc, beyond what is expected from the localized halo paradigm of structure formation. They concluded that at ﬁxed stellar mass, the clustering properties of the surround- ing dark matter haloes are correlated with the colour of the selected galaxies. They further argued that different physical processes may be required to explain environmental trends in star formation, dis- tinct from those established by galaxy structure. A signiﬁcant breakthrough in expressing this blue/red dichotomy occurred when photometry from the Galaxy Evolution Explorer (GALEX), notably the near-ultraviolet (NUV) band, was matched with SDSS photometry (Martin et al. 2007; Wyder et al. 2007; Schiminovich et al. 2007; Salim et al. 2007). When the diagram is plotted using NUV−r as the colour, two clear sequences emerge: the familiar red sequence and a new blue sequence in place of the ’blue cloud’ of optical studies. Between the blue and red sequences there are galaxies present in a so-called green valley. Many of these are spectroscopically classiﬁed Type II active galactic nuclei (AGN) (Rich et al. 2005; Martin et al. 2007; Salim et al. 2007). Faber et al. (2007), Martin et al. (2007) and Schiminovich et al. (2007) propose several paths by which galaxies might transition from the blue to the red sequence. The presence of AGN in the green valley suggests that AGN activity is associated with a quenching of star formation (Hopkins et al. 2006, 2007; Silk & Rees 1998). Other paths from the blue to the red sequence might, hypothetically, in- volve gas-rich mergers of blue galaxies (Toomre & Toomre 1972), or virial shock heating of cold gas streams (Dekel & Birnboim 2006). The red sequence might consolidate in luminosity via dissi- pationless mergers of red galaxies, or low-luminosity blue galaxies might acquire bulges through mergers with starbursts, retaining sufﬁcient mass to land the evolved galaxy on the red sequence. However, the green valley might also be populated by casual visi- tors – red galaxies that acquire gas and form stars or feed a central AGN. Following this brief burst of star formation, these galax- ies might ultimately return to the red sequence from which they started. ABSTRACT We measure the UV-optical colour dependence of galaxy clustering in the local Universe. Using the clean separation of the red and blue sequences made possible by the NUV −r colour–magnitude diagram, we segregate the galaxies into red, blue and intermediate ‘green’ classes. We explore the clustering as a function of this segregation by removing the dependence on luminosity and by excluding edge-on galaxies as a means of a non-model dependent veto of highly extincted galaxies. We ﬁnd that ξ(rp, π) for both red and green galaxies shows strong redshift-space distortion on small scales – the ‘ﬁnger-of-God’ effect, with green galaxies having a lower amplitude than is seen for the red sequence, and the blue sequence showing almost no distortion. On large scales, ξ(rp, π) for all three samples show the effect of large-scale streaming from coherent infall. On scales of 1 h−1 Mpc < rp < 10 h−1 Mpc, the projected auto- correlation function wp(rp) for red and green galaxies ﬁts a power law with slope γ ∼1.93 and amplitude r0 ∼7.5 and 5.3, compared with γ ∼1.75 and r0 ∼3.9 h−1 Mpc for blue sequence galaxies. Compared to the clustering of a ﬁducial L∗galaxy, the red, green and blue have a relative bias of 1.5, 1.1 and 0.9, respectively. The wp(rp) for blue galaxies display an increase in convexity at ∼1 h−1 Mpc, with an excess of large-scale clustering. Our results suggest that the majority of blue galaxies are likely central galaxies in less massive haloes, while red and green galaxies have larger satellite fractions, and preferentially reside in virialized structures. If blue sequence galaxies migrate to the red sequence via processes like mergers or quenching Y.-S. Loh et al. 56 that take them through the green valley, such a transformation may be accompanied by a change in environment in addition to any change in luminosity and colour. Key words: methods: statistical – galaxies: clusters: general – galaxies: elliptical and lentic- ular – galaxies: evolution. some relationship to environment and to the dark matter haloes in which the galaxies reside. In this study, we investigate the cluster- ing environment of the blue and red sequences, and for the green valley. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 2.1 GALEX and SDSS data The ultraviolet imaging portion of the data set is from the GALEX that was launched in 2003 April (Martin et al. 2005; Morrissey et al. 2005, 2007). GALEX obtains wide ﬁeld imaging in both the far-UV (FUV; centred at 1540 Å) and the near-UV (NUV, 2300 Å) over a 1.◦2 diameter ﬁeld of view, with 5 arcsec images. Here, we use data from the Medium Imaging Survey (MIS); these images are ≈1500 s in duration reaching NUV ≈23 mag, covering an orbital shadow crossing. The MIS pointing that deﬁnes our sample targets the North Galactic Cap, which overlaps the SDSS spectroscopic footprint; this part of the program was designed from studies cross- matching SDSS and GALEX data. The data set used for our current analysis is from the Galaxy Release 3 (GR3) which is available from the Multi-mission Archive at STScI (MAST). The GALEX pipeline uses SEXTRATOR (Bertin & Arnouts 1996) to detect sources and measure ﬂuxes. We use the ‘MAG_AUTO’ output from SEXTRATOR as our default ﬂux measurement; it is essentially a Kron (1980) magnitude with an elliptical aperture. In order to measure the correlation function, a random sample needs to be constructed to normalize the galaxy pair counts. Angular sampling completeness as a function of position in the sky encoded in the footprint server is used to generate random samples of density roughly 50 times the galaxy density. We adopt the method proposed by Li et al. (2006a) where we assign each galaxy in our sample to a random position on the sky but keep all other attributes the same (e.g. redshift, colour, magnitude). This random sample by construc- tion has the same redshift distribution as the original sample, and thus does not smooth out the redshift structure like those gener- ated via the luminosity function. As noted in Li et al. (2006a), this approach works well in surveys with a wide-angular sky coverage (e.g. much larger than the typical LSS), and with small variation in survey depth. We note here that our random sample would inherit the redshift correlation function of the colour–magnitude distribution of the parent galaxy sample; only spatial distribution has been ran- domized, hence any excess in clustering must be due to positional differences. Because our analysis requires each galaxy to have a spectroscopic redshift, we start our cross-matching with a galaxy from the SDSS Main spectroscopic survey. 2.1 GALEX and SDSS data For each SDSS galaxy, we search for the closest GALEX detection within 4 arcsec radius from the location of the SDSS spectroscopic ﬁbre. Only GALEX sources within 0.◦55 of the tile centre ﬁeld-of-view (FOV) are retained, since astrometry degrades towards the periphery of the FOV (a problem which will be resolved in later releases) and the incidence of artefacts increases as well. After the matching of GALEX and SDSS sources, we further trim the sample to create a statistically complete data set following the procedure of Wyder et al. (2007). We will refer the reader to their table 1 for the full details. Here, we list a few essential parameters and the minor modiﬁcations we employed: 14.0 < r < 17.6, 0.03 < z < 0.25, σr < 0.2, zconf > 0.67, GALEX exposure time t > 750 s and 16.0 < NUV < 23.0.1 All magnitudes are AB magnitudes and corrected for Galactic foreground extinction. In the SDSS spectroscopic survey, no two galaxies with separa- tion θ less than 55 arcsec can both be assigned spectroscopic ﬁbres for observation on any given observing plate. Hence, a large fraction of galaxy pairs with θ < 55 arcsec are missing. We correct for this ‘ﬁbre-collision’ problem by using the observed angular correlation function, a method ﬁrst suggested by Li et al. (2006a), and described in detail in the companion paper by Heinis et al. (2009). In brief, the observed projected two-point angular correlation function for both the photometric sample wph(θ) and the spectroscopic sample wsp(θ) is measured and used to construct the pair weighting ratio: 2.3 GALEX–SDSS overlapping footprint In order to statistically deﬁne our combined GALEX–SDSS survey, we need to have the understanding of the angular sampling function of the two surveys, which varies across different regions of the sky. GALEX’s MIS survey consists of overlapping circular tiles (radius 0.◦55), while the SDSS spectroscopic survey is a combination of circular spectroscopic plates, but with ﬁbre placement based on a rectangular imaging survey that runs along great circles. To com- bine the footprints of both surveys, we use the GESTALT footprint server. GESTALT2 uses a hierarchical pixelization system, enabling one to encode observations of arbitrary geometry while tagging in- formation about completeness and the masking of artefacts. We ﬁrst encode the GALEX MIS survey using GESTALT. We then ob- tain the detailed observational footprint of the SDSS LSS sample from the NYU-VAGC web site. The SDSS footprint is expressed as a set of disjoint polygons using the software MANGLE (Hamilton & Tegmark 2002) which takes into account the complex angular mask and geometry of the SDSS survey. We convert these polygons into the pixelization scheme of GESTALT and consider the inter- section of the two surveys. There are approximately 490 deg2 in the GALEX–SDSS overlapping footprints after masking for holes, bright stars and satellite trails, and excluding defects. 1Our faint-end limit of 23.0 is about 0.5 mag fainter than those employed by Wyder et al. (2007). 1 INTRODUCTION In this paper, we will consider the colour dependence of the two-point physical correlation function of galaxies using samples constructed from GALEX, augmented with redshift and optical data from SDSS. In particular, we use the natural separation from the NUV −r colour to assign galaxies into three subsamples of red, green and blue galaxies. Our study complements recent work by Heinis et al. (2009) who investigate the physical clustering of galax- ies as a function of star formation history in the local universe, as well as earlier studies by Milliard et al. (2007), Heinis et al. (2007) and Basu-Zych et al. (2008) who investigate the angular-correlation function of rest-frame UV-selected galaxies and their evolution. We measure the auto-correlation function (ACF) of each of the differ- ent subsamples of galaxies, as well as the CCF (CCF) between the subsamples. In Section 2, we describe in detail the data used in this analysis. In Section 3 we describe the method for estimating cor- relation functions. We present our results in Section 4, and discuss their implications for the nature of green valley galaxies and the In Salim et al. (2007), a plot of mass against speciﬁc star forma- tion rate (SFR) reveals a clear division between lower mass, star- forming, blue sequence galaxies and more massive AGN, which are not detected in large numbers until stellar mass M ∼3 × 1010 M⊙. The process responsible for populating the green valley and for potentially contributing to evolution from blue to red must bear C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 57 UV–optical clustering 2.3 GALEX–SDSS overlapping footprint formation of red sequence galaxies. We summarize our ﬁndings in Section 6. formation of red sequence galaxies. We summarize our ﬁndings in Section 6. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 2http://nvogre.phyast.pitt.edu:8080/gestalt_tutorial/ 3.1.2 Cross-correlation Related to the ACF is the CCF between two classes of galaxies. The CCF ξ1,2(r), measures the clustering of one type of galaxy around another. ξ1,2(r) is essentially the probability of ﬁnding a galaxies of type 1 around a galaxy of type 2 as a function of separation r. For our analysis, we use the cross-correlation version of the classical Davis & Peebles (1983) estimator: For a galaxy survey with a well-deﬁned angular selection func- tion, ξ can be estimated using an optimal estimator like the Landy & Szalay (1993) estimator: ξLS = DD −2DR + RR RR (3) ξ1,2 = D1D2 D1R2 −1 (7) (7) (3) where D1D2 are the normalized counts of cross-pairs, while D1R2 are cross-pairs of type 1 with random galaxies having the same redshift distributions as galaxies of type 2. where DD, DR and RR are normalized counts of galaxy pairs in the data–data, data–random and random–random catalogues.4 The Landy & Szalay (1993) estimator is preferred because it is rela- tively insensitive to the size of the random catalogue and to edge corrections (Kerscher et al. 2000). Recently, the CCF has been used extensively in clustering stud- ies of galaxy properties (Zehavi et al. 2005; Wang et al. 2007; Coil et al. 2008; Chen 2009; Padmanabhan et al. 2009) since the ACF alone tells us little about how galaxies of various types relate to one another. Two populations of galaxies may both have compara- ble correlation strength, and yet be physically unrelated if they are spatially segregated. Speciﬁcally, the CCF may be used in conjunc- tion with the ACFs of the galaxies to glean information as to how they mix statistically. For example, if the two populations are well mixed, i.e. they are consistent with being drawn stochastically from their respective ACF equally, their projected CCF w1,2 p (rp) would follow that of the geometric mean of their ACFs Because we observe galaxies in redshift and projected space and not in physical space, in practice, the correlation function is ﬁrst measured on a two-dimensional grid of separations: π along the line of sight (redshift space) and rp for angular separation on the sky. In addition to providing information about the underlying mass distribution through the amplitude of the clustering signals, the two-dimensional correlation function ξ(π, rp) contains additional information about the dynamics of the galaxies (Peebles 1980). (2) (2) dP = n[1 + ξ(r)]dV , In practice, we integrate along the line-of-sight direction out to π = 30 h−1 Mpc. This is large enough to include almost all cor- related pairs but also stable enough to suppress noise from distant uncorrelated pairs. The projected correlation function can in turn be related to the real-space correlation function ξ(r), where n is the mean number density of galaxy sample. For the last 40 years, the correlation function has served as the primary method for cosmologists to quantify the clustering properties of galaxies from large-scale surveys (Totsuji & Kihara 1969; Peebles 1980). If the underlying density distribution is Gaussian, then the correlation function fully describes all statistical properties of a given distribu- tion. Since early cosmological models are often based on primor- dial Gaussian dark matter density ﬁelds, the use of the correlation function leads to a straightforward comparison between empirical studies on the statistical distribution of galaxies with such models. Recently, with the widespread adoption of a standard -dominated cosmology (Ostriker & Steinhardt 1995; Riess et al. 1998; Perlmutter et al. 1999; Spergel et al. 2007), the correlation function is used instead to probe the growth of structure in the universe and the range of formation scenarios for varying kinds of galaxies.3 The correlation length – the amplitude from a power-law correlation function – provides information on the mass of dark matter haloes in which the various galaxies reside, linking observation with theo- retical description of structure formation (Bower et al. 2006; Croton et al. 2006) wp(rp) = 2 ∞ 0 rdrξ(r)(r2 −rp)−1/2. (5) (5) If the real-space correlation function follows a power law ξ(r) = (r/r0)γ , we can infer its parameters: the correlation length r0 and the power-law slope γ from the best-ﬁtting power law to wp(rp) using the following deprojection: wp(rp) = rp rp r0 −γ 1 2 γ −1 2 γ 2 . (6) (6) 3 METHODOLOGY 3.1.1 Real-space correlation 3.1.1 Real-space correlation Because redshift-space distortion only affects the line-of-sight com- ponent of ξ(π, rp), we can recover the true space correlation function ξ(r) by following the standard procedure of computing the projected correlation function: 3.1.2 Cross-correlation At small projected separations rp random motions within virialized overdensity (e.g. clusters of galaxies) cause an elongation along the line-of-sight (π direction) known as the ‘ﬁnger-of-God’ effect. On large scales, coherent streaming of galaxies into potential wells causes an apparent compression of structure along the line-of-sight (Sargent & Turner 1977; Kaiser 1987; Hamilton 1992). Various studies have used ξ(π, rp) to extract cosmological and dynamical information (e.g. Tinker 2007). w1,2 p (rp) = w1,1 p (rp)w2,2 p (rp). (8) (8) On the other hand, if the populations were segregation between the populations and do not distribute evenly in all space, e.g. they sit on different haloes, the amplitude of w1,2 p (rp) would be lower than that of w1,2 p (rp) known in the literature as stochastic anti-bias (e.g. Blanton 2000; Swanson et al. 2008). 3The use of correlation function to probe the growth of structure predates the advent of cold dark matter, especially in the study of faint blue galaxies (e.g. Efstathiou et al. 1991). Y.-S. Loh et al. Y.-S. Loh et al. 58 3The use of correlation function to probe the growth of structure predates the advent of cold dark matter, especially in the study of faint blue galaxies (e.g. Efstathiou et al. 1991). 4Normalized counts of galaxy pairs are weighted by the selection function of the galaxies involved. 2.2 SDSS large-scale structure sample Considerable effort has been invested by the SDSS team to prepare the redshift data for large-scale structure (LSS) studies. This sec- ondary data set known as the New York University Value Added Catalogue (NYU-VAGC) is documented in Blanton et al. (2005a) and available from the NYU web site. Proprietary versions of this catalogue have been used by many groups within the SDSS col- laboration for various investigations of clustering and luminosity function of galaxies. We match the GALEX–SDSS catalogue con- structed above with the SDSS LSS DR5 sample. The version used for our analysis includes all of the detailed radial and angular se- lection functions for the various statistical subsamples used in the previous analyses (e.g. Zehavi et al. 2005). This sample is ideal because we can compare our result with that of Zehavi et al. (2005) which was based solely on selection from optical criteria. F(θ) = wph(θ) + 1 wsp(θ) + 1 F(θ) = wph(θ) + 1 wsp(θ) + 1 (1) (1) as a function of separation. Empirically, Heinis et al. (2009) ﬁnd F(θ) ∼3 for θ < 55 arcsec and zero otherwise for the all GALEX– SDSS cross-matched galaxies. We apply this correction to each of the red, green and blue subsamples equally. 2http://nvogre.phyast.pitt.edu:8080/gestalt_tutorial/ C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 3The use of correlation function to probe the growth of structure predates the advent of cold dark matter, especially in the study of faint blue galaxies (e.g. Efstathiou et al. 1991). 4Normalized counts of galaxy pairs are weighted by the selection function of the galaxies involved. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 3.1 The two-point correlation function In brief, the two-point ACF ξ(r) measures the excess probability of ﬁnding a galaxy pair with separation r from a random galaxy distribution, wp(rp) = 2 ∞ 0 dπξ(rp, π). (4) (4) dP = n[1 + ξ(r)]dV , (2) 3.1.3 Bootstrap errors We estimate the errors for the correlation measurements using a modiﬁed bootstrap (Efron 1981) method for spatial statistics known 4Normalized counts of galaxy pairs are weighted by the selection function of the galaxies involved. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 59 UV–optical clustering as marked-point bootstrap (Loh 2008). We ﬁrst estimate the contri- bution of each galaxy to the correlation function – the marks: in units with Hubble constant H0 = 100 h km s−1 Mpc−1, i.e. Mr ≡ Mr −5 log(h = 1). Compared with the optical CMD (e.g. g −r versus Mr), the NUV −r colour axis has a higher dispersive power. The scatter plot shows two clear sequences: red and blue, and an intermediate ‘valley’ of ‘green’ population. Conventional optical diagrams only display a single red sequence with an extended blue cloud. Hence, diagnostic diagrams like Fig. 1 separate galaxies into three natural groupings: (1) a red sequence of bulge-dominated galaxies with old stellar systems, (2) a blue sequence of star-forming systems consisting of mainly late-type galaxies and (3) galaxies in a ‘green valley’ that in principle might exhibit transitional properties (Martin et al. 2007; Schiminovich et al. 2007). Detailed luminosity functions and physical properties analysis of these galaxies derived from this UV–optical CMD has been reported elsewhere (Wyder et al. 2007; Martin et al. 2007; Salim et al. 2007; Schiminovich et al. 2007). Here, we investigate the clustering properties of these three subpopulations of galaxies, separated by the two tilted horizontal lines, using the two-point ACF, as well as their relation to each other using the CCF. Our results are not sensitive to 0.25 mag shift in the oblique equation used to classify the galaxies. ξi(r) = n j=1,j̸=i φ{\|xi −xj\| ∈(r −dr, r + dr)} (9) (9) such that such that ξ(r) = n i=1 ξi (10) ξ(r) = n i=1 ξi (10) where n is the number of galaxies and φ is the chosen correlation estimator (e.g. ξLS). Hence, the mark ξi associated with galaxy xi is the number of excess pairs at a distant r from xi. These ξi are (roughly) independent, and identically distributed, and can be used to replicate B bootstrap samples by the sampling with replacement. 4.1 Dust Dust content within each galaxy can modify their broad-band colours, hence the resultant CMD, as galaxies move from one group to another (Martin et al. 2007; Schiminovich et al. 2007). This is a concern, as it might affect our estimate of the correlation func- tion by either increasing the scatter between groups or biasing the result in a systematic but unknown manner. Indeed, a fraction of the green valley galaxies are dusty star-forming galaxies whose in- trinsic colour would have placed them on the blue sequence in the absence of dust (Wyder et al. 2007). However, the available proce- dures for dust correction are highly uncertain. They give inconsis- tent results depending on whether one uses a primarily photometric approach (Salim et al. 2007; Johnson et al. 2006) or one based on spectroscopic indices (Kauffmann et al. 2003). The former, for example, has the side effect of reducing the red sequence number counts substantially (Schiminovich et al. 2007; Heinis et al. 2009), while the latter approach requires spectroscopy of modest signal- to-noise ratio (S/N), which is not available for part of our sample. We chose this method over the conventional jackknife methods because of the fragmented nature of the GALEX–SDSS footprint. Our bootstrap errors are consistent with the jackknife errors esti- mated by excluding one GALEX tile at a time. We note here that jackknife errors are known to overestimate the variance on small scale, and often bias the overall correlation estimates (Norberg et al. 2009). Since bootstrap is a form of internally estimated errors, and internally estimated errors are known not to reproduce externally estimated errors accurately (Norberg et al. 2009), our clustering results should be treated with caution. 3.1.3 Bootstrap errors An estimate of the correlation function ξ ∗ j can be computed from each of the jth bootstrap samples. We use B = 999 bootstrap samples for our analysis. The distribution of ξ ∗(ri) can be use to estimate the error bars of the correlation function at each separation ri. For an equivalent of 1σ errors, we ranked ξ ∗(ri) and take the 159th and 840th for the lower and upper error bounds. Because the ξ errors for the ri are correlated, when we estimate parameters for the two- parameter power-law model (equation 6), we reﬁtted each bootstrap sample to obtain a two-dimensional distribution of the parameters. 4 DEPENDENCE OF CLUSTERING ON UV–OPTICAL COLOUR Fig. 1 shows the NUV −r colour–magnitude diagram (CMD) in the local universe. All absolute magnitudes are k-corrected using the K_CORRECT program by Blanton & Roweis (2007) and are quoted Figure 1. NUV −r versus Mr CMD of galaxies in the local Universe. The left-hand panel shows the full distribution. The middle panel shows the distribution drawn from face-on galaxies with b/a > 0.6, used to correct for the effect of dust on the CMD. The right-hand panel shows the distribution of edge-on/face-on ratio overlay on to the CMD contours. The green valley is dominated by the region of the CMD with high relative fraction of edge-on galaxies. By limiting ourselves to galaxies with Mr < −20.3 (dashed vertical lines) for the volume-limited sample, we also eliminate region of the CMD with the highest fraction of edge-on galaxies. Figure 1. NUV −r versus Mr CMD of galaxies in the local Universe. The left-hand panel shows the full distribution. The middle panel shows the distribution drawn from face-on galaxies with b/a > 0.6, used to correct for the effect of dust on the CMD. The right-hand panel shows the distribution of edge-on/face-on ratio overlay on to the CMD contours. The green valley is dominated by the region of the CMD with high relative fraction of edge-on galaxies. By limiting ourselves to galaxies with Mr < −20.3 (dashed vertical lines) for the volume-limited sample, we also eliminate region of the CMD with the highest fraction of edge-on galaxies. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 Y.-S. Loh et al. 60 the luminosity dependence like what we have done here would still leave residual clustering due to the difference in mass-to-light ratio of the respective colour-selected sample. A comprehensive analysis of the clustering as a function of star formation history with a dust-corrected CMD using the method of Johnson et al. (2006) is done in Heinis et al. (2009). Here, we adopt a geometric approach. Because dust lanes in galaxies usually appear when viewed edge-on, we can reduce the effect of dust on galaxy colour by restricting our analysis to galax- ies that are primarily face-on. The middle panel of Fig. 1 shows the ‘dust-corrected’ CMD obtained by including only galaxies with isophotal (minor-over-major) axis ratio b/a > 0.6. 4.4 Red, green, blue auto-correlation Figs 3 and 4 show the two-dimensional ACF ξ(π, rp) as a function of line-of-sight (π) and projected (rp) separation for the ﬂux- and volume-limited samples. The panels on the ﬁrst row in each ﬁgure are from the full distribution, while the second row is from the dust- corrected distribution. The panels are for red sequence (left), green valley (middle) and blue sequence (right) galaxies. We have binned π and rp linearly at 1 h−1 Mpc. For all panels, the grey-scale has the same range, and the contours are boxcar smoothed at 2 h−1 Mpc. The ξ(π, rp) contours indicate the constant probability of ﬁnding pairs at a given π and rp. The heavy solid line marks ξ(π, rp) = 1, and contours are spaced with increment (inner) and decrement (outer) by a factor of 2. The effect of redshift-space distortion is clearly seen in all panels, manifested by their departure from isotropy (concentric circles). At small rp, the contours are elongated along the line-of- sight (π) due to virial motions of galaxies in clusters. At large rp, the contours are compressed in the π direction due to the coherent large-scale streaming as galaxy infall into potential well. We recover the results of Zehavi et al. (2005): red sequence galaxies show the strongest ﬁnger-of-God effect and the larger correlation amplitude; and all three subpopulations (of all samples and distribution) show clear signs of large-scale compression. 4.3 Volume-limited sample While the resampled red, green and blue galaxies are matched in luminosity, they are not matched in volume and have different red- shift distributions. This makes the interpretation of the physical correlation function problematic. To this end, we select the largest possible volume within our catalogue, using a redshift cut of 0.03 < z < 0.12 and an additional luminosity cut at Mr < −20.3 to con- struct a volume-limited sample.5 The luminosity distributions of this sample are shown on the bottom row of Fig. 2. As before, the left-hand panel is for the full CMD, while the right-hand panel is for the dust-corrected (restricted) CMD. Similar to the ﬂux-limited case, the (original) blue dashed histograms are substantially dif- ferent from the red and the green, and are weighted more heavily towards the faint-end. We resample the histograms to match in lu- minosity. Note that in the case the procedure outlined in Section 4.2 essentially amounts to looking for an optimal function W(L) such that W(L)(L) gives the minimum luminosity function of the three subsamples. The new red, green and blue volume-limited subsam- ples each consists of 1971 (full) and 1226 (dust-corrected) galaxies with a common median luminosity of Mr ∼20.9, almost identical to the ﬂux-limited case. The right-hand panel of Fig. 1 shows the median ratio of edge-on (b/a < 0.4) to face-on (b/a > 0.8) as a function of colour and magnitude, with the same density contours from the middle panel. The region with the highest edge-on/face-on ratio falls in the green valley and towards the faint end of the luminosity distribution (Choi et al. 2007; Martin et al. 2007; Schiminovich et al. 2007). For the galaxies with the magnitude range with −23.5 < Mr < −19.0 used to construct our ﬂux-limited sample, the median b/a for red and blue sequence galaxies are 0.73 and 0.72, respectively, while for green valley galaxies it is 0.66. Choi et al. (2007) ﬁnd that edge-on galaxies are also statistically fainter due to the internal extinction, and it affects morphologically late-type galaxies (classiﬁed by eye) more than early-type galaxies. Hence, by limiting our analysis to face- on galaxies, we would reduce biases associated with differential dimming in addition to the biases from colour shifts. Following Choi et al. (2007), we restrict our analysis to galaxies with b/a > 0.6 as a proxy for a galaxy distribution derived from a dust-corrected CMD. 4 DEPENDENCE OF CLUSTERING ON UV–OPTICAL COLOUR To the extent that each of the subpopulations has the same intrinsic distribution of b/a, removing edge-on galaxies will give a correct mixture of galaxies that mimic the intrinsic colour–magnitude distribution. Compar- ing the dust-corrected CMD with the CMD on the left-hand panel (hereafter the full distribution), the density of green galaxies around Mr ∼−21 is reduced substantially, with the contours showing a more pronounced ‘valley’ separating the sequence of galaxies. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 4.2 Luminosity bias The luminosity distribution of our ﬂux-limited (top row) and volume-limited (bottom row) samples. The two panels on the left are for the full distribution, the right is for dust-corrected CMD, using only galaxies with b/a > 0.6. The dashed histograms show the original luminosity distribution for each of the subsamples of red, green and blue galaxies. We resampled the luminosity to match the number counts of the minimal of the three distribution at each magnitude bin, shown as solid histograms. Even for the volume-limited samples, the variations in the original luminosity distribution among the three subsamples are substantial. Blue galaxies are heavily weighted towards the less luminous end compared to red or green galaxies. 61 UV–optical clustering Downloaded from https://academic.oup.com/mnras/article/407/1/55/984101 by guest on 19 November 2021 https://academic.oup.com/mnras/article/407/1/55/984101 by guest on 19 November 2021 Figure 2. The luminosity distribution of our ﬂux-limited (top row) and volume-limited (bottom row) samples. The two panels on the left are for the full distribution, the right is for dust-corrected CMD, using only galaxies with b/a > 0.6. The dashed histograms show the original luminosity distribution for each of the subsamples of red, green and blue galaxies. We resampled the luminosity to match the number counts of the minimal of the three distribution at each magnitude bin, shown as solid histograms. Even for the volume-limited samples, the variations in the original luminosity distribution among the three subsamples are substantial. Blue galaxies are heavily weighted towards the less luminous end compared to red or green galaxies. to the optical blue cloud sample of Zehavi et al. (2005). We will consider the implications of this in the discussion below. The same conclusion can be drawn from the volume-limited sample shown in Fig. 4. The dust-corrected samples (bottom row) are substantially noisier. We also note that the contours of the blue sequence of the volume-limited full sample (top right panel) are roughly circular for ∼4 < π, rp < 10 h−1 Mpc where the ﬁnger-of-God elongation is balanced by compression due in infall. large scales (rp ≳1 h−1 Mpc), the ratio of projected ACF of the respective samples is constant as a function of scales, as expected from linear theory. All correlation functions appear to have a form that is well ﬁt by a broken power law with the dust-corrected distri- bution having a more pronounce convexity. It is noteworthy that the ﬂux-limited analysis (Fig. 4.2 Luminosity bias The clustering of galaxies is luminosity dependent. Norberg et al. (2002), Zehavi et al. (2005) and Li et al. (2006a) show that the am- plitude of the projected correlation function wp increases monoton- ically as a function of luminosity (or stellar mass) on scales ranging from 0.2 h−1 Mpc to 10 h−1 Mpc. In order to study the colour de- pendence on clustering, or to compare the clustering intrinsic to the membership of discrete subpopulations, it is vital to remove this known luminosity dependence. The top two panels of Fig. 2 show the luminosity distribution of the red, green and blue population of galaxies with Mr < −19 from our ﬂux-limited sample. The series of dashed histograms (in red, green and blue) show the original luminosity distributions. Blue galaxies are more numerous and less luminous, on average, compared to red and green galaxies, reﬂect- ing a steeper blue luminosity function at the faint-end (Wyder et al. 2007). To remove this luminosity dependence, we resample the lu- minosity histograms to match the number counts of the smallest of the three distributions at each magnitude bin. The result of this resampling is shown by the solid histograms. The left-hand panel shows histograms from the full distribution, while the right shows those from a dust-corrected distribution. There are 4177 (full) and 3148 (dust-corrected) galaxies in each of the resampled luminosity distribution of red, green and blue galaxies, with a common median luminosity of Mr ∼−21.0, about half a magnitude brighter than M∗, the typical luminosity (Blanton et al. 2003). Note that if the fundamental attribute that drive clustering is stellar mass, removing We see a clear ﬁnger-of-God effect for green valley galaxies, but not for blue sequence galaxies. In Fig. 3, if one compares the ﬁrst contour inwards from ξ = 1 (heavy line) for red galaxies to the ξ = 1 contour of green galaxies, green and red galaxies have identical kinematics, differing only by a scaling in the amplitude. The blue sequence appears to have a dynamical structure dominated by large- scale streaming distinct from both red and green galaxies, similar 5This is not strictly volume-limited since NUV is not complete at these redshifts. An additional weighting is applied using the luminosity function of Wyder et al. (2007). C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 UV–optical clustering 61 Figure 2. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 4.2 Luminosity bias 5) is more noisy than the volume-limited analysis (Fig. 6), despite having a factor of 2 greater numbers. We believe that this is due to the inclusion of uncorrelated (in redshift- space) galaxies, which dilute the clustering signal, especially for blue galaxies on small scales. From here onwards, we will restrict our analysis to the volume-limited sample. Figs 5 and 6 show the projected correlation function wp(rp) for the ﬂux-limited and volume-limited samples. The coloured (red, green, blue) points are the respective measured correlation func- tion. The panel on the left (right) is for the full (dust-corrected) distribution. As expected, in all four panels, the red sample has the largest amplitude, the blue the lowest and green in between. On When we ﬁt the projected correlation function of the full distri- bution with the standard two-parameter power law for scales from 1 < rp < 10 h−1 Mpc, red and green galaxies appear to have similar C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 62 Y.-S. Loh et al. Y.-S. Loh et al. 62 Figure 3. Contours and normalized counts of the two-dimensional correlation function ξ(π, rp) for red (left), green (middle) and blue (left) populations for the ﬂux-limited sample. The panels on the top row are for the full distribution, bottom row are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. Red galaxies have the strongest ﬁnger of God effect (extension in the π direction). The ﬁnger-of-God effect is seen in the green sample as well, while it is not present in the blue. Galaxies in the green valley appear to have clustering characteristics of the red sequence. Downloaded from https://academic.oup.com/mnras/article/407/1/55/984101 by guest on 19 November 2021 Figure 3. Contours and normalized counts of the two-dimensional correlation function ξ(π, rp) for red (left), green (middle) and blue (left) populations for the ﬂux-limited sample. The panels on the top row are for the full distribution, bottom row are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. Red galaxies have the strongest ﬁnger of God effect (extension in the π direction). 4.2 Luminosity bias The ﬁnger-of-God effect is seen in the green sample as well, while it is not present in the blue. Galaxies in the green valley appear to have clustering characteristics of the red sequence. slope with γ ∼1.93, while blue galaxies have a substantially shal- lower slope, with γ ∼1.75. The best-ﬁtting correlation lengths r0 are 7.5 h−1 Mpc (red), 5.3 h−1 Mpc (green) and 3.9 h−1 Mpc (blue). The result of the covariance analysis using 999 bootstrap samples is shown as conﬁdence interval contours – 68 per cent (inner) and 95 per cent (outer) in Fig. 7 (left-hand panel). These results are in excellent agreement with Heinis et al. (2009). The correlation function from the dust-corrected distribution is substantially nois- ier, in part due to the smaller number of galaxies. Applying the same power-law ﬁt, we obtain a larger uncertainty in γ , but comparable uncertainty in r0. This is shown on the right-hand panel of Fig. 7 and is tabulated in Table 1. deﬁned as bi b∗ (r) = ξi(r) ξ∗(r) ≃rγi/2 0,i rγ/2 0 r(γ −γi)/2 (11) (11) where ξi is the correlation function of interest and ξ∗is the ﬁducial correlation function that all correlation function is compared with. where ξi is the correlation function of interest and ξ∗is the ﬁducial correlation function that all correlation function is compared with. The approximate equality holds when the correlation function takes a power-law form. Zehavi et al. (2005) found that a typical L∗galax- ies in the local universe with −20 < Mr < −21 from the SDSS main spectroscopic sample have a ﬁducial power-law correlation function with ξ∗(r) = (r/5 h−1 Mpc)−1.8 on scale 1 < rp < 10 h−1 Mpc. By ﬁtting the correlation function using a constant γ = 1.8 on scales 1 < rp < 10 h−1 Mpc, we obtained the bias relative at rp = 5 h−1 Mpc to a ﬁducial L∗galaxies for each of our subpopulations: bred/b∗= 1.53±0.08, bgreen/b∗= 1.08±0.06 and bblue/b∗= 0.81±0.06 for the full-sample, and bred/b∗= 1.47±0.10, bgreen/b∗= 1.13±0.09 and bblue/b∗= 0.92±0.09 for the dust-corrected samples (Table 1). In Fig. 8, we plot bi/b∗(r = 5 h−1 Mpc) = (r0/5 h−1 Mpc)γ as a function of the comoving number density and compare our values with those obtained by Zehavi et al. (2005). C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 4.2 Luminosity bias Comoving number density of our sample is estimated using the 1/Vmax method and further corrected to match those obtained by Wyder et al. (2007). While all the samples by construction have almost identical comov- ing density, there is a range of relative bias, with the red having On the left-hand panel, the projected correlation function of green galaxies is evidently intermediate between the red and the blue on a range of scales (0.2 < rp < 6 h−1 Mpc), and runs faithfully parallel with the red correlation function, but with a lower amplitude. The picture for the dust-corrected distribution is slightly different. While the green still runs in between the red and blue for rp < 1 h−1 Mpc and mostly parallel to the red with a lower amplitude for broad range of scales, for larger scales (rp ≳2 h−1 Mpc), it coincides with the blue. This is a consequence of the more prominent two-halo excess on large scales seen in the blue correlation function. One way to compare the clustering between subpopulations of galaxies is through their relative bias (e.g. Norberg et al. 2002), C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 63 UV–optical clustering p g Figure 4. Contours and normalized counts of the two-dimensional correlation function ξ(π, rp) for red (left), green (middle) and blue (left) populations for the volume-limited sample. The panels on the top row are for the full distribution, bottom panels are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. Red galaxies have the strongest ﬁnger of God effect (extension in the π direction). Similar to Fig. 3, but with lower S/N, the ﬁnger-of-God effect is seen in the green sample as well, while it is not present in the blue. Figure 4. Contours and normalized counts of the two-dimensional correlation function ξ(π, rp) for red (left), green (middle) and blue (left) populations for the volume-limited sample. The panels on the top row are for the full distribution, bottom panels are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 4.2 Luminosity bias Red galaxies have the strongest ﬁnger of God effect (extension in the π direction). Similar to Fig. 3, but with lower S/N, the ﬁnger-of-God effect is seen in the green sample as well, while it is not present in the blue. Figure 5. The projected correlation function, wp(rp), of red sequence, green valley and blue sequence galaxies for the ﬂux-limited sample. The left (right) panel is from the full (dust-corrected) distribution. From red to blue, there is an increase in convexity, with the progressive shallowing of the slope on large scales, indicating an excess of two-halo signals at scale (rp ≳1 h−1 Mpc). Figure 5. The projected correlation function, wp(rp), of red sequence, green valley and blue sequence galaxies for the ﬂux-limited sample. The left (right) panel is from the full (dust-corrected) distribution. From red to blue, there is an increase in convexity, with the progressive shallowing of the slope on large scales, indicating an excess of two-halo signals at scale (rp ≳1 h−1 Mpc). C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 64 Y.-S. Loh et al. Figure 6. The projected correlation function, wp(rp), of red sequence, green valley and blue sequence galaxies for the volume-limited sample. Similar to Fig. 5, from red to blue, there is an increase in convexity, with the progressive shallowing of the slope on large scale, indicating an excess of two-halo signals, at scale (rp ≳1 h−1 Mpc). For the full distribution (left panel), the green wp(rp) is parallel to the red with a lower amplitude for scales 0.2 < rp < 10 h−1 Mpc. For the dust-corrected distribution (right panel), the blue wp(rp) approaches the green on large scales. Figure 6. The projected correlation function, wp(rp), of red sequence, green valley and blue sequence galaxies for the volume-limited sample. Similar to Fig. 5, from red to blue, there is an increase in convexity, with the progressive shallowing of the slope on large scale, indicating an excess of two-halo signals, at scale (rp ≳1 h−1 Mpc). For the full distribution (left panel), the green wp(rp) is parallel to the red with a lower amplitude for scales 0.2 < rp < 10 h−1 Mpc. For the dust-corrected distribution (right panel), the blue wp(rp) approaches the green on large scales. Figure 7. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 4.5 Cross-correlation functions Fig. 9 shows the two-dimensional ξ(rp, π) CCF for the three cross- pairs of galaxies in our volume-limited sample, with the panels on the top for the full distribution, and the bottom panels for the dust-corrected distribution. The large-scale infall effect is seen in all three panels suggesting that the galaxies, on average, trace a similar matter distribution as expected from linear theory. On small scales, the ﬁnger-of-God effect is strongest for the red–green CCF, intermediate for red–blue and green–blue. In Fig. 10, we plot the relative amplitude of ξ at the projected distance rp = 1 h−1 Mpc as a function of line-of-sight distance π. The circles are for green–red cross-correlation, squares for red–blue and triangles for green–blue. Green–red have largest amplitude for a wide range of π, while red– blue and green–blue have smaller amplitudes. The stronger ﬁnger- of-God component suggests that the dynamics of red and green are more strongly coupled compared with red–blue or green–blue pairs. Note that red–blue CCF have a very different ξ(π, rp) compared with the green ACF, the former has a weaker ﬁnger-of-God and stronger infall compression. As discussed in Section 3.1.2, one way to understand the rela- tionship between two populations is to compare the projected CCF between the two with their geometric mean. If the two populations are mixed evenly, the CCFs should trace the geometric mean. If they are spatial segregated (partially) beyond what was expected from their respective ACF, the CCF should be systematically be- low the geometric mean. The projected CCFs for the red and blue galaxies wr,b p (rp) are plotted as open circles in Fig. 11. Also plotted for comparison are the red and blue ACFs (solid lines), and the red–blue geometric mean wr,b p (rp) (dashed lines). On scales larger than ∼3 h−1 Mpc, the wr,g p (rp) approaches the geometric mean. For scales 0.2 < rp < 2 h−1 Mpc, wr,g p (rp) is systematically below the geometric mean for the full distribution (left-hand panel). For the dust-corrected distribution (right-hand panel), wr,b p (rp) starts to inch below the geometric mean only from rp ≲0.7 h−1 Mpc onwards. Our results are consistent with the partial morphology segrega- tion within galaxy clusters (Dressler 1980). 4.5 Cross-correlation functions At the one-halo regime (rp ≲1–3 h−1 Mpc), the relevant scales for galaxy clusters, red galaxies tend to occupy the cores of the clusters, while blue galax- ies tend to lie towards the periphery. The lower level of spatial mixing on these scales suppresses amplitude of the CCF. Note that the green ACF (green solid line) does trace the red–blue geometric mean on large scales, suggesting that blue and red galaxies do mix evenly on these scales. Figure 8. Bias relative to the typical L∗galaxy with a ξf id(r) = (r/r0)−1.8 at rp = 5 h−1 Mpc versus comoving number density. We also plotted points from SDSS (Zehavi et al. 2005). The solid circles are from their luminosity range analysis, i.e. galaxy with absolute magnitude between M1 and M2, while the open circles are from their luminosity threshold analysis, i.e. Mr < Mlim. While far from unique, the higher red bias suggests that on average red galaxies are more likely to be satellite galaxies than a typical galaxy with those number densities. If we assume the lower blue and green galaxies are all central galaxies in the halo they reside in, the lower observed number densities suggest that only a fraction of these haloes, as described by their bias, host a blue or green galaxy, as their expected number density is much higher. the highest bias and blue the lowest. The spread among the bias is much smaller among the dust-corrected samples. Red galaxies have clustering strength above the nominal strength (inferred from a typical SDSS galaxy) for a given number density. One plausible scenario suggests that red galaxies have a larger than average satel- lite fraction. On the other hand, blue and green galaxies have lower observed number density compared to their clustering. If we assume all blue and green galaxies are central galaxies in the halo they re- side in, the lower observed number densities (∼0.001 h3 Mpc−3) suggest that only a small fraction of these haloes, as described by their lower bias, host a blue or green galaxy, as their expected num- ber density (inferred from SDSS to be ∼0.01 h3 Mpc−3) is much higher. These fractions are increased if the average phases of green and blue are shorter than the lifetime of the haloes (Haiman & Hui 2001; Martini & Weinberg 2001). UV–optical clustering UV–optical clustering 4.2 Luminosity bias Conﬁdence contours of the power-law ﬁts to wp(rp) on scales 1 h−1 Mpc < rp < 10 h−1 Mpc for the full (left panel) and dust-corrected (right) distributions from the volume-limited sample. The three colours are for red, green and blue subsamples, with the inner (outer) contours encircling the 68 (95) per cent conﬁdence region in the r0–γ space. Figure 7. Conﬁdence contours of the power-law ﬁts to wp(rp) on scales 1 h−1 Mpc < rp < 10 h−1 Mpc for the full (left panel) and dust-corrected (right) distributions from the volume-limited sample. The three colours are for red, green and blue subsamples, with the inner (outer) contours encircling the 68 (95) per cent conﬁdence region in the r0–γ space. Table 1. Power-law ﬁt to the projected ACF of volume-limited samples (1 h−1 Mpc < rp < 10 h−1 Mpc). Full-sample N z Median Mr r0 γ Relative bias χ2/d.o.f . Red. . .. . .. . .. 1971 (0.03, 0.12) −20.9 7.5 ± 0.27 1.94 ± 0.07 1.53 ± 0.08 1.36 Green. . .. . ... 1971 (0.03, 0.12) −20.9 5.3 ± 0.19 1.93 ± 0.08 1.08 ± 0.06 0.40 Blue. . .. . .. . . 1971 (0.03, 0.12) −20.9 3.9 ± 0.25 1.73 ± 0.10 0.81 ± 0.06 1.54 Face-On Red. . .. . .. . .. 1226 (0.03, 0.12) −20.9 7.6 ± 0.38 1.81 ± 0.11 1.47 ± 0.10 2.47 Green. . .. . ... 1226 (0.03, 0.12) −20.9 5.5 ± 0.24 1.92 ± 0.15 1.13 ± 0.09 0.48 Blue. . .. . .. . . 1226 (0.03, 0.12) −20.9 4.6 ± 0.31 1.74 ± 0.17 0.92 ± 0.09 1.17 Table 1. Power-law ﬁt to the projected ACF of volume-limited samples (1 h−1 Mpc < rp < 10 h−1 Mpc) C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 65 6The halo mass that a typical L∗galaxy resides in. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 6The halo mass that a typical L∗galaxy resides in. 4.5 Cross-correlation functions In the case of the green galaxies, the transitional nature of these galaxies may be closely related to the AGN they host, or to minor mergers and starbursts, with triggering cycles corresponding to the duty cycles for these respective phe- nomena. We defer a more detail analysis of halo occupation and the life cycle of transition galaxies using SFR tracers of different lags to a future paper (Heinis et al. in preparation). Using a theoretical bias function of dark matter haloes (Seljak & Warren 2004) and normalizing to M∗= 1012 h−1 M⊙,6 red galaxies cluster similar to haloes with mass ∼1012.7 h−1 M⊙, green galaxies 1012.2 h−1 M⊙ and blue galaxies 1011.6 h−1 M⊙. Fig. 12 shows the projected CCF between the green and red wg,r p (rp), and green and blue wg,b p (rp). The solid green line is the ACF of the green sample (wggp(rp)), while the dashed lines are the geometric means: wg,r p (rp) and wg,b p (rp). For the full distribution (left-hand panel), wg,r p (rp) is systematically below wg,r p for a range of scales within errors. wg,b p (rp) are consistent, or slightly above wg,b p (rp) for rp ≳1 h−1 Mpc, and systematically below for 0.3 ≲ rp ≲1 h−1 Mpc. These inferences are shown more clearly in Fig. 13 (left for full, right for dust-corrected), where we plot the normalized cross clustering strength – the ratio wg,x p /wggp, where x is either red or blue – relative to the auto-correlation of the green galaxies. The additional solid lines are the normalized ACF of the red and blue. This suggests that red and green are consistent with being drawn from the same statistical sample on average for a large range of scales with a slight anti-bias on small scales. For the green and blue, there is a stronger anti-bias for r ≲1 h−1 Mpc as they avoid each other on these scales. Our results suggest that on scales typical C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 Y.-S. Loh et al. 66 Figure 9. Contours and normalized counts of the two-dimensional CCF ξ(π, rp) for red–blue (left), green–red (middle) and green–blue (left) for the volume- limited sample. The panels on the top row are for the full distribution, bottom panels are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. 4.5 Cross-correlation functions The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. The large-scale infall effect is seen in all three panels. On small scales, the ﬁnger-of-God effect is strongest for the red–green, intermediate for red–blue and weakest for green–blue, as expected. Note that red–blue CCF ξ(π, rp) has a weaker ﬁnger-of-God and stronger infall compression compare to the green valley ACF (Fig. 4, middle panels). Downloaded from https://academic.oup.com/mnras/article/407/1/55/984101 by guest on 19 November 2021 Figure 9. Contours and normalized counts of the two-dimensional CCF ξ(π, rp) for red–blue (left), green–red (middle) and green–blue (left) for the volume- limited sample. The panels on the top row are for the full distribution, bottom panels are restricted to face-on galaxies (b/a > 0.6). The contours obtained after 2 × 2 h−1 Mpc boxcar smoothing. The levels are 0.0 (dotted lines), 0.25, 0.5, 1.0 (heavy lines), 2.0, 4.0, 8.0 and 16.0. The large-scale infall effect is seen in all three panels. On small scales, the ﬁnger-of-God effect is strongest for the red–green, intermediate for red–blue and weakest for green–blue, as expected. Note that red–blue CCF ξ(π, rp) has a weaker ﬁnger-of-God and stronger infall compression compare to the green valley ACF (Fig. 4, middle panels). of dark matter haloes, galaxies drawn from the green and blue populations are less associated than would be predicted by their respective ACF. clearly in the wp(rp) analysis for the volume-limited sample (Fig. 6), where the green function has the form similar to that of the red function for scales 0.2 < rp < 15 h−1 Mpc, but is displaced to lower amplitude. It is noteworthy that the slope of the blue correlation function begins shallowing at r ∼1 h−1 Mpc, displaying the kind of one- and two-halo segregation expected from a correlation function dominated by central galaxies. In contrast to Coil et al. (2008), the green ACF converges to that of the blue at rp > 1 h−1 Mpc. We emphasize that the green galaxy sample of Coil et al. (2008) is deﬁned differently from ours. We divide the CMD into three disjoint parts to separate our galaxies into the subpopulations while in Coil et al., the green overlaps both the red and the blue. 5.1 Comparison with DEEP2 z ∼1 The GALEX NUV-selected sample used in our analysis is directly comparable to the high-redshift (z ≈1) galaxy sample of the DEEP2 survey (Coil et al. 2008) since their optical selection mim- ics the rest-frame NUV. In that paper, clustering analysis was done on green valley galaxies for the ﬁrst time. In their analysis, the green valley ξ(rp, π) appears to display kinematic structure in- termediate between red and blue galaxies, with an intermediate ﬁnger-of-God effect and an intermediate overall clustering ampli- tude. When redshift-space distortion is removed, the projected cor- relation function wp(rp) shows a scale dependence convergence. At rp > 1 h−1 Mpc the functions converge to those of red populations, while for rp < 1 h−1 Mpc it tends towards the clustering of the blue population. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 5.3 Blue (UV) versus blue (optical) In their studies using g −r colour, Zehavi et al. (2005) found that the correlation function of blue galaxies exhibits a lower amplitude and shallower correlation functions. By ﬁtting the wp(rp) with halo occupation distribution (HOD) models, they found that the majority (∼70–90 per cent) of blue galaxies are central galaxies in dark matter haloes, usually haloes with mass ≲1013 M⊙, in contrast to only the most luminous red galaxies being central objects of massive haloes, while the majority of (less luminous) red galaxies are satellites. This ﬁts well with the notion that blue galaxies are ﬁeld galaxies – the central objects of low-mass haloes; red galaxies, with the exception of the central galaxy in clusters and groups, are mostly satellites of massive haloes. Figure 10. This ﬁgure shows the relative amplitude of ξ at projected dis- tance rp = 1 h−1 Mpc as a function of the line-of-sight distance π. The circles are for green–red cross-correlation, squares for red–blue and trian- gles for green–blue. Green–red have largest amplitude for a wide range of π, while red–blue and green–blue have smaller amplitudes. These results suggest that the ﬁnger-of-God component of green–red CCF is stronger than those from red–blue or green–blue. One can infer a similar conclusion from the auto-correlation wp(rp) plot for the blue galaxies (Fig. 6). If we decompose the corre- lation function into two parts, one due to the one-halo term and the other due to the two-halo term, blue galaxies show strong two-halo excess on scales ≳1 h−1 Mpc, implying that on those such scales, the majority of galaxy pairs are from different haloes. This was seen in the optical analysis of Zehavi et al. (2005) but prominently in our blue sequence sample. For the dust-corrected volume-limited sample (right-hand panel of Fig. 6), the amplitude of the ACF of the blue sample beyond ∼3 h−1 Mpc actually rises to match the clustering strength of the green sample. green valley deﬁnition using the oblique colour cuts from Fig. 1, the AGN fraction is ∼50 per cent.7 In their study of AGN using SDSS, Constantin & Vogeley (2006) found that the redshift-space two-point correlation func- tion of Seyferts is less clustered than that of low-ionization nuclear emission-line regions survey (LINERS). However, Miller et al. (2003) and Li et al. (2006b) found that AGN as a whole cluster similarly as typical L∗galaxies, if one takes into account luminos- ity bias. 5.3 Blue (UV) versus blue (optical) As was discussed in Section 4.1, a substantial fraction of the galaxies in the green valley are dusty star-forming galaxies. Because dust content can modify the CMD we use to separate the galaxies into red, green and blue populations, this might potentially change 7This is a lower limit since the fraction of low-luminosity AGN and com- posite objects is unknown. 5.2 Green valley Many recent studies reveal that blue sequence mass has remained roughly constant since z ∼1 (Blanton 2006; Faber et al. 2007) because the average ongoing star formation over 0 < z ≲1 is balanced by mass ﬂux off the blue sequence, presumably towards the build-up of the red sequence since z ≈1 (Bell et al. 2004; Martin et al. 2007). Hence, green valley galaxies occupy a position where one expects to ﬁnd many transitional galaxies. Martin et al. also note that the AGN fraction peaks at the green valley. For our In contrast, we ﬁnd that our green valley ξ(rp, π) has a strong ﬁnger-of-God effect consistent with that measured for red sequence galaxies, differing only in their amplitude. This can be seen most C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 67 UV–optical clustering Figure 10. This ﬁgure shows the relative amplitude of ξ at projected dis- tance rp = 1 h−1 Mpc as a function of the line-of-sight distance π. The circles are for green–red cross-correlation, squares for red–blue and trian- gles for green–blue. Green–red have largest amplitude for a wide range of π, while red–blue and green–blue have smaller amplitudes. These results suggest that the ﬁnger-of-God component of green–red CCF is stronger than those from red–blue or green–blue. the behaviour of the correlation function. We argue here that to the extent that dust modiﬁes the number counts of green galaxies, it is to promote the migration from the blue sequence to the green valley (Choi et al. 2007). Our results from Fig. 4 (for the volume- limited sample) suggest that their inﬂuence is modest at best, and merely acts as additional Poisson noise to the two-dimensional ξ(π, rp) without altering the kinematics in the sample. The projected correlation functions of Fig. 6 show similar characteristics. 5.3 Blue (UV) versus blue (optical) Wang & Kauffmann (2008) argue that almost all galaxies in the local universe with stellar mass ≳10 h−1 M⊙have active nuclei, often LINERS with lines too weak to be detected spectro- scopically in SDSS. Our green galaxies, with luminosities peaked at Mr ∼−21, have stellar masses well above 10 h−1 M⊙, and could be dominated by LINERS (either detected or undetected). This would in part explain the clustering effect we see in Figs 3 and 4 (middle panels) where those bulge-dominated LINERS display kinematics similar to red sequence (primarily non-active) galaxies. The reason that AGN from r-band-selected survey (e.g. Miller et al. 2003) cluster on average much like typical L∗galaxies may merely be coincidental. Li et al. (2006a) found that the dependence of wp(rp) on optical g −r colour extends beyond 5 h−1 Mpc, suggesting that the con- ventional wisdom that clustering should converge at large scales may not occur until at a scale larger than 5 h−1 Mpc. Here, we argue that this is an effect due to the mixture of population between blue galaxies, and green and red, and that the optical g −r colour does not have sufﬁcient power to separate the green from the blue. Blue galaxies, by themselves, have a very pronounced two-halo excess and are dominated entirely by central galaxies, ﬂattening the corre- lation function substantially at large scales, compared to the red and green population. To the extent that one can eliminate or correct for the internal reddening due to dust, NUV −r colour is very efﬁcient in isolating a sequence of purely star-forming galaxies. The lack of such behaviour in the z ∼1 green valley (Coil et al. 2008) may be attributable to evolution in the AGN population. There may be fewer LINERS, or the red sequence galaxies may have been experiencing more gas infall, feeding their AGN. We note that with a relative bias ∼1.1, green galaxies cluster similarly to a typical galaxy with L∗−0.5 ≈−21, the median luminosity of our sample. The lack of such behaviour in the z ∼1 green valley (Coil et al. 2008) may be attributable to evolution in the AGN population. There may be fewer LINERS, or the red sequence galaxies may have been experiencing more gas infall, feeding their AGN. We note that with a relative bias ∼1.1, green galaxies cluster similarly to a typical galaxy with L∗−0.5 ≈−21, the median luminosity of our sample. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 6 SUMMARY AND CONCLUSIONS We have constructed a GALEX and SDSS-matched catalogue, where we have used the GR3 catalogue from GALEX and the SDSS DR5 main spectroscopic galaxy sample. We construct the galaxy distri- bution of NUV −r versus Mr CMD, and divide the distribution into populations of red sequence, green valley and blue sequence. Since our main goal is to study the colour dependence of clustering, we took substantial care in matching the luminosity distribution of each population. For each population, we measure the two-dimensional correlation function ξ(π, rp), and the one-dimensional projected correlation function wp. We also perform cross-correlation analy- ses between each of the subpopulations. As was discussed in Section 4.1, a substantial fraction of the galaxies in the green valley are dusty star-forming galaxies. Because dust content can modify the CMD we use to separate the galaxies into red, green and blue populations, this might potentially change C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 68 Y.-S. Loh et al. 68 Y. S. Loh et al. Figure 11. The projected CCFs between red and blue sequence galaxies wr,b p (rp) are shown as open circles. The solid lines are the ACF of the red and blue galaxies. The dashed line is the red–blue geometric mean wr,b p (rp). On scales larger than ∼3 h−1 Mpc, the cross-correlation approaches the geometric mean. For scales 0.2 h−1 Mpc < rp < 2 h−1 Mpc, the CCF is systematically below the geometric mean. This result suggests that on small scales, blue and red galaxies are spatially segregated beyond what was expected from their ACF. This is consistent with the morphology density relation (Dressler 1980). Figure 11. The projected CCFs between red and blue sequence galaxies wr,b p (rp) are shown as open circles. The solid lines are the ACF of the red and blue galaxies. The dashed line is the red–blue geometric mean wr,b p (rp). On scales larger than ∼3 h−1 Mpc, the cross-correlation approaches the geometric mean. For scales 0.2 h−1 Mpc < rp < 2 h−1 Mpc, the CCF is systematically below the geometric mean. This result suggests that on small scales, blue and red galaxies are spatially segregated beyond what was expected from their ACF. This is consistent with the morphology density relation (Dressler 1980). /academic.oup.com/mnras/article/407/1/55/984101 by guest on 19 November 2021 Figure 12. 6 SUMMARY AND CONCLUSIONS The projected CCF of green sample with blue wg,b p (rp) (blue open circles) and red wg,r p (rp) (red open circles) subsamples. The solid line shows the ACF of the green sample. The dashed lines are the geometric mean of the green–red (in red) and green–blue (in blue) auto-correlation. On the left (right) we show the analysis from the full (dust-corrected) distribution from the volume-limited sample (see Fig. 13 for more details). Figure 12. The projected CCF of green sample with blue wg,b p (rp) (blue open circles) and red wg,r p (rp) (red open circles) subsamples. The solid line shows the ACF of the green sample. The dashed lines are the geometric mean of the green–red (in red) and green–blue (in blue) auto-correlation. On the left (right) we show the analysis from the full (dust-corrected) distribution from the volume-limited sample (see Fig. 13 for more details). Our principal ﬁnding is that the red sequence and green valley appear to show similar clustering properties, as expressed in the ﬁnger-of-God effect in the ACF. The projected correlation function is consistent with red and green galaxies residing as satellites of massive haloes, while the blue sequence shows what appears to be a clear two-halo signature, hence primarily serving as central galaxies of less massive haloes. The CCF also shows that green and blue galaxies, on small scales, are not a mere statistical mix, but are spatially segregated from each other. massive galaxies that reside in massive haloes, and which cluster like the red sequence. We note that Martin et al. (2007), Wyder et al. (2007) and Salim et al. (2007) show that a large fraction of type II AGN are found in the green valley. Salim et al. show that in the plot of speciﬁc SFR versus stellar mass, the AGN tend to be found in massive (>3 × 1010 M⊙) galaxies. The AGN occupy a region in these plots that strongly resembles that of the reddest class of galaxies, the ‘no-Hα’ red sequence galaxies. Signiﬁcantly, the AGN are clearly offset from the locus of the blue sequence, in the plot of speciﬁc SFR versus mass. The signiﬁcance is that while a minority of AGN are found with properties that coincide with those The ﬁndings would appear to place the green valley population with the red sequence. The green valley would largely consist of C⃝2010 The Authors. 6 SUMMARY AND CONCLUSIONS Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 69 UV–optical clustering UV–optical clustering 69 Figure 13. The ratio of projected correlation function (wp(rp)) with the ACF (wggp(rp)) of the green subsample. The two ratios from the CCFs, wg,r p (rp) and wg,b p (rp), are indicated by coloured open circles. Dashed lines are for geometric means, as in Fig. 12. The additional solid lines are ratios from the ACF of red and blue, respectively. The fact that both wg,r p (rp) and wg,b p (rp) are below their respective geometric mean on small scales suggests that both red and blue galaxies are found in different environments compared to the green on these scales, albeit with large uncertainties. Figure 13. The ratio of projected correlation function (wp(rp)) with the ACF (wggp(rp)) of the green subsample. The two ratios from the CCFs, wg,r p (rp) and wg,b p (rp), are indicated by coloured open circles. Dashed lines are for geometric means, as in Fig. 12. The additional solid lines are ratios from the ACF of red and blue, respectively. The fact that both wg,r p (rp) and wg,b p (rp) are below their respective geometric mean on small scales suggests that both red and blue galaxies are found in different environments compared to the green on these scales, albeit with large uncertainties. star formation and move a blue sequence galaxy to the green valley. Any number of environmental effects (e.g. harassment, starvation) might speed the consumption of gas in a disc, again moving a galaxy to the green valley. A small sample of optically quiescent members of the green valley that nominally have an UV excess shows clear star formation signatures (spirals) when imaged in the UV using Hubble Space Telescope (Salim & Rich 2009). There is still the issue of the origin of the low-mass red sequence, and the evolution of blue sequence, into the green valley and ultimately the red sequence might have an important role in the growth of the lower mass portion of the red sequence. This was partially addressed by semi-analytical work of Benson et al. (2003) and Bower et al. (2006). of the more massive blue sequence galaxies, green valley galaxies – the subsample with the largest AGN fraction – exhibit properties similar to those of the red sequence, but showing mildly elevated star formation. 6 SUMMARY AND CONCLUSIONS In this study, we have shown that the green valley population clus- ters in ways that are characteristic of, but also less strongly than, the red sequence. One may suggest that these studies paint a picture in which both the properties of the green valley and the ‘demographics’ are different from those of the blue sequence, at the present epoch. These ﬁndings do not necessarily contradict the studies that ﬁnd an increase in the total mass of red sequence galaxies since z ∼1. They do suggest, however, that if blue sequence galaxies evolve by some process to the green valley, and ultimately to the red se- quence, such evolution must also be accompanied by a transition from the ﬁeld environment to a group/cluster environment. Such a change could conceivably occur if the blue population resides along ﬁlaments that infall into clusters over time. Our cross-correlation results show that green galaxies avoid both red and blue galaxies on small scales, which is consistent with the change in environ- ment hypothesis. We note that models like ram-pressure stripping (Gunn & Gott 1972), starvation of (cold) gas and the virial shock heating model of Dekel & Birnboim (2006) naturally incorporate environmental factors in their mechanism for colour transformation in galaxies. In contrast to the construction of CMDs for stellar populations, the environment, for galaxies, is a critical physical variable in their evolution. This is true in the sense of both their dark matter environ- ment and the presence of detectable companion stellar systems. In considering the major processes driving galaxy evolution, it would appear that evolution of both of these observables must be consid- ered as a function of look-back time. C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 8http://spectro.princeton.edu/idlutils ACKNOWLEDGMENTS YSL would like to thank C. Hirata, S. Salim, C. Park, J. Kormendy and Z. Zheng for helpful discussions. This work has made extensive use of IDLUTILS8 and Goddard IDL libraries. RMR acknowledges support from grant GO-11182 from the Space Telescope Science Institute. YSL would like to thank C. Hirata, S. Salim, C. Park, J. Kormendy and Z. Zheng for helpful discussions. This work has made extensive use of IDLUTILS8 and Goddard IDL libraries. RMR acknowledges support from grant GO-11182 from the Space Telescope Science Institute. It is also possible that the downsizing (Cowie et al. 1996) effect is so strong that most star-forming galaxies are evolving rapidly with redshift (e.g. Tinsley 1968). One must recall that star-forming activity at z ∼1 resides in considerably more massive galaxies, and that a colour-based population separation, as we have done, will refer to much higher masses; the rest-frame colours may be similar, but the fundamental nature of the galaxies would not. GALEX is a NASA Small Explorer, launched in 2003 April. We gratefully acknowledge NASA’s support for construction, opera- tion and science analysis for the GALEX mission, developed in One may speculate that the green valley is occupied by nominally red galaxies that experience the infall of a gas-rich system that either induces star formation and/or fuels the AGN, rendering it visible via its emission lines. However, the feedback of an AGN might inhibit C⃝2010 The Authors. Journal compilation C⃝2010 RAS, MNRAS 407, 55–70 Y.-S. 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https://openalex.org/W4366393926	https://escholarship.org/content/qt44x2k4ng/qt44x2k4ng.pdf?t=rxtu76	English	null	Impact of social determinants of health on perioperative opioid utilization in patients with lumbar degeneration	North American Spine Society journal	2,023	cc-by	5,995	Title Impact of social determinants of health on perioperative opioid utilization in patients with lumbar degeneration. Title Impact of social determinants of health on perioperative opioid utilization in patients with lumbar degeneration. Title Impact of social determinants of health on perioperative opioid utilization in patients with lumbar degeneration. a r t i c l e i n f o Keywords: Opioids Social determinants of health Lumbar Degenerative Keywords: Opioids Social determinants of health Lumbar Degenerative Background: Social determinants of health (SDOH), have been demonstrated to signiﬁcantly impact health out- comes in spine patients. There may be interaction between opioid use and these factors in spine surgical patients. We aimed to evaluate the social determinants of health (SDOH) which are associated with perioperative opioid use among lumbar spine patients. Methods: This retrospective cohort study included patients undergoing spine surgery for lumbar degeneration in 2019. Opioid use was determined based on prescription records from the electronic medical records. Preoperative opioid users (OU) were compared with opioid-naïve patients regarding SDOH including demographics like age and race, and clinical data such as activity and tobacco use. Demographics and surgical data, including age, comorbidities, surgical invasiveness, and other variables were also collected from the records. Multivariate logistic regression was used for analysis of these factors. Results: Ninety-eight patients were opioid-naïve and 90 used opioids preoperatively. All OU had ≥ 3 months of use, had more prior spine surgeries (1.07 vs. 0.44, p < .001) and more comorbidities including diabetes, hypertension, and depression (p = .021, 0.043, 0.017). Patients from lower community median income areas, unemployed, or with lower physical capacity (METS < 5) were more likely to use opioids preoperatively. Postoperative opioid use was strongly associated with preoperative opioid use, as well as alcohol use, and lower community median income. At one year postoperatively, OU had higher rates of opioid use [72.2% vs. 15.3%, p < .001]. Conclusions: Unemployment, low physical activity level, and lower community median income were associated with preoperative opioid use and longer-term opioid use postoperatively. Conclusions: Unemployment, low physical activity level, and lower community median income were associated with preoperative opioid use and longer-term opioid use postoperatively. Impact of social determinants of health on perioperative opioid utilization in patients with lumbar degeneration Aboubacar Wague, BA a , Jennifer M. O’Donnell, MD b , ∗ , Khuzaima Rangwalla, BS a , Ashraf N. El Naga, MD b , c , David Gendelberg, MD b , c , Sigurd Berven, MD b a University of California San Francisco School of Medicine, 505 Parnassus Ave MU 320W, San Francisco, CA 94143, USA b University of California San Francisco, Department of Orthopaedic Surgery, 505 Parnassus Ave, San Francisco, CA 94143, USA c Zuckerberg San Francisco General Hospital, 1001 Potrero Ave, San Francisco, CA 94110, USA FDA device/drug status: Not applicable. Author disclosures: AW: Nothing to disclose. JMO-D : Nothing to disclose. KR: Nothing to disclose. ANE-N: Nothing to disclose. DG: Nothing to disclose. SB: Royalties: Stryker (F); Stock Ownership: Green Sun Medical (75000 shares); Consulting: Medtronic (D), Globus (C), Accelus (C), Camber (B); Trips/Travel: AO Spine (B); Research Support (Investigator Salary): AO Spine (B). ∗ Corresponding author: University of California-San Francisco, 505 Parnassus Ave MU 320W, San Francisco, CA 94143, USA. Tel.: 908-907-2766. E-mail address: Jennifer.odonnell@ucsf.edu (J.M. O’Donnell) . Powered by the California Digital Library University of California eScholarship.org North American Spine Society Journal (NASSJ) 14 (2023) 100221 Contents lists available at ScienceDirect https://doi.org/10.1016/j.xnsj.2023.100221 Received 19 February 2023; Received in revised form 11 April 2023; Accepted 12 April 2023 Available online 19 April 2023 2666-5484/© 2023 The Authors. Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Available online 19 April 2023 2666-5484/© 2023 The Authors. Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Results Out of 521 patients that have underwent elective surgery for degen- erative lumbar pathology, 16 lacked medication data at the studies des- ignated time points and 317 patients had less than one year follow-up. A total of 188 patients were included in this cohort, with 98 included in the ON cohort and 90 in the OU cohort. The ON group had no preop- erative opioid use in the 3 months prior to surgery, and the OU group had a mean preoperative opioid use of 48.2 ± 4 MME ( Table 1 ). Patients were categorized into two groups, preoperative opioid users (OU) and ON according to whether they had been prescribed opioids preoperatively. Opioid use was measured in morphine milligram equiv- alents (MME). Demographic and baseline health status were collected including age, gender, body mass index, duration of symptoms, co- morbidities (including hypertension, hyperlipidemia, diabetes, coronary artery disease, depression/anxiety), length of hospital stay, duration of surgery, American Society of Anesthesiology (ASA) score, and prior sur- gical history. Additionally, intraoperative data including surgical inva- siveness index, estimated blood loss (EBL), and duration of operation were collected. Mean age was 59.7 years, and the cohort consisted of 46.8% women, was 82.4% white, and 11.7% Hispanic or Latinx. OU had more prior spinal surgeries (1.07 vs. 0.44, p < .001) and higher average BMI (30.2 vs. 27.8, p = .004). OU had higher levels of comorbidities, including dia- betes (20% vs. 8%, p = .019), hypertension (58% vs. 43%, p = .041), and anxiety/depression (48% vs. 31%, p = .016) ( Table 1 ). OU had higher ASA scores (2.40 vs. 2.09, p < .001) and longer length of hospital stay (3.44 days vs. 2.61 days, p = .014) ( Table 2 ). Univariate analysis demonstrates patients age 40 to 59 years had 3.2 times increased odds of preoperative opioid use (95% CI 1.10-10.4, p = .033). Black patients were 6.3 times more likely to use opioids before surgery (95% CI 1.01–166, p = .049). There were no increased odds based on gender or ethnicity. Those below the lower-income limit were 1.79 times more likely to have preoperative opioid use (95% CI 1.00–3.25, p = .051). Religious patients were 2.1 times more likely to use preop- eratively (95% CI 1.14–3.73, p = .016). Patients on Medicaid insurance were 4.3 times more likely to have preoperative opioid use (95% CI 1.46–14.6, p = .007). Introduction Opioid users had a higher number of prior spine surgeries, body mass index and frequency of diabetes, hypertension, coronary artery disease, and anxiety or depression compared to opioid naïve users. Table 1 Comparison of demographics between opioid naïve and opioid users. Opioid users had a higher number of prior spine surgeries, body mass index and frequency of diabetes, hypertension, coronary artery disease, and anxiety or depression compared to opioid naïve users. Table 1 Comparison of demographics between opioid naïve and opioid users. Opioid users had a higher number of prior spine surgeries, body mass index and frequency of diabetes, hypertension, coronary artery disease, and anxiety or depression compared to opioid naïve users. Comparison of demographics between opioid naïve and opioid users All Opioid naïve Opioid users p-value Age 59.7 (14.2) 59.9 (15.6) 59.4 (12.6) .778 Number of prior spine surgeries 0.74 (1.16) 0.44 (0.84) 1.07 (1.36) < .001 Body mass index 29.0 (5.56) 27.8 (4.90) 30.2 (6.00) .004 Duration of symptoms (years) 6.65 (9.50) 5.59 (7.57) 7.81 (11.2) .115 Diabetes 26 (13.8%) 8 (8.16%) 18 (20.0%) .019 Hypertension 94 (50%) 42 (42.9%) 52 (57.8%) .041 Hyperlipidemia 72 (38.3%) 38 (38.8%) 34 (37.8%) .888 Coronary artery disease 10 (5.32%) 8 (8.16%) 2 (2.22%) .070 Deep vein thrombosis 8 (4.26%) 2 (2.04%) 6 (6.67%) .117 Anxiety/depression 73 (38.8%) 30 (30.6%) 43 (47.8%) .016 Comparison of demographics between opioid naïve and opioid users (METS) as recorded in the preoperative anesthesia visit; METS are deter- mined based on the energy the patient is able to expend in daily tasks, such as walking 2 ﬂights of stairs [13] . (METS) as recorded in the preoperative anesthesia visit; METS are deter- mined based on the energy the patient is able to expend in daily tasks, such as walking 2 ﬂights of stairs [13] . Previous work in other ﬁelds have indicated that social conditions such as age, socioeconomic status and smoking status inﬂuence the like- lihood of chronic opioid use [ 9 , 10 ]. These factors, such as housing sta- tus, work status, income, religious belief, insurance status, and mari- tal status, are referred to as social determinants of health (SDoH). The Centers for Disease Control and Prevention (CDC) deﬁne SDoH as the “conditions in the places where people live, learn, work, and play that aﬀect a wide range of health and quality of life risks and outcome ”. Demographic and opioid utilization A retrospective cohort study was completed on a consecutive series of patients undergoing elective spine surgery for lumbar degenerative pathology from a single academic institution, from January 2019 to De- cember 2019. Patients who underwent 1- or 2-level lumbar spinal fusion surgery were included if they had greater than 3 months of preopera- tive data and at least 12 months of postoperative data. Exclusion criteria included missing prescription data from the electronic medical record (EMR), primary cancer diagnosis, and procedures for infection and de- bridement. The study was approved by the hospital’s Institutional Re- view Board. Results There were no increased odds related to marital status ( Table 3 ). Statistical analysis The primary outcome measures were odds ratios for SDoH and HRB factors after univariate and multivariate logistic regression among OU versus ON patients. All statistical analyses were performed using R soft- ware version 4.1.2 with statistical signiﬁcance deﬁned at p < .05 (R Foun- dation, Vienna, Austria). Descriptive statistics were collected with mean and standard deviation, or median and mode where applicable. ANOVA and t-test were used to obtain p-values for all linear variables, and chi- square test was performed for categorical variables. Odds ratios were de- termined with a 95% conﬁdence interval. Univariate analysis and mul- tivariate logistic regression of SDoH and HRB were conducted against opioid use status. Introduction of how to better understand and ultimately decrease opioid utilization in orthopedic patients. Opioid consumption in America is greater than any other country and opioid addiction is a nationwide crisis. In 2018, almost 70,000 Americans died of a drug overdose with two-thirds of these deaths at- tributed to opioid use [1] . Orthopedics is at the center of the opioid epidemic. Orthopedic surgeons prescribed an estimated 7.7% of opioid prescriptions in the United States in 2009, and accounted for the ﬁrst prescription of an estimated 8.8% of all opioid-naïve (ON) patients that develop chronic opioid dependence [ 2 , 3 ]. This calls for an assessment Lumbar decompression and fusion surgeries are among the most common spine surgeries. Chronic use of opioids in patients with lum- bar degenerative pathology has been correlated to longer hospital stays, increased risk of postoperative complications, and longer opioid use postoperatively [4–6] . Use of opioids has also been correlated to lower patient-reported outcome measurements [ 7 , 8 ]. To address these issues, it is imperative to identify risk factors that lead to preoperative opioid utilization. p 2666-5484/© 2023 The Authors. Published by Elsevier Ltd on behalf of North American Spine Society. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) North American Spine Society Journal (NASSJ) 14 (2023) 100221 A. Wague, J.M. O’Donnell, K. Rangwalla et al. Table 1 Comparison of demographics between opioid naïve and opioid users. Opioid users had a higher number of prior spine surgeries, body mass index and frequency of diabetes, hypertension, coronary artery disease, and anxiety or depression compared to opioid naïve users. Comparison of demographics between opioid naïve and opioid users All Opioid naïve Opioid users p-value Age 59.7 (14.2) 59.9 (15.6) 59.4 (12.6) .778 Number of prior spine surgeries 0.74 (1.16) 0.44 (0.84) 1.07 (1.36) < .001 Body mass index 29.0 (5.56) 27.8 (4.90) 30.2 (6.00) .004 Duration of symptoms (years) 6.65 (9.50) 5.59 (7.57) 7.81 (11.2) .115 Diabetes 26 (13.8%) 8 (8.16%) 18 (20.0%) .019 Hypertension 94 (50%) 42 (42.9%) 52 (57.8%) .041 Hyperlipidemia 72 (38.3%) 38 (38.8%) 34 (37.8%) .888 Coronary artery disease 10 (5.32%) 8 (8.16%) 2 (2.22%) .070 Deep vein thrombosis 8 (4.26%) 2 (2.04%) 6 (6.67%) .117 Anxiety/depression 73 (38.8%) 30 (30.6%) 43 (47.8%) .016 Table 1 Comparison of demographics between opioid naïve and opioid users. Introduction The impact of SDoH on opioid use in orthopedic spine patients has not yet been investigated. This study aims to evaluate the impact of SDoH and health-related behaviors (HRB) on preoperative and postoperative opioid utilization. Table 3 Table 3 Univariate comparison of SDoH and HRB between opioid naïve and opioid users. Univariate analysis revealed that several SDoH and HRB increased the odds of preoperative opioid use. Marital status, however, did not signiﬁcantly inﬂuence the odds of preoperative opioid use. Table 3 Univariate comparison of SDoH and HRB between opioid naïve and opioid users. Univariate analysis revealed that seve odds of preoperative opioid use. Marital status, however, did not signiﬁcantly inﬂuence the odds of preoperative opio Univariate comparison of SDoH and HRB between opioid naïve and opioid users. Univariate analysis revealed that several SDoH and HRB increased the odds of preoperative opioid use. Marital status, however, did not signiﬁcantly inﬂuence the odds of preoperative opioid use. Univariate comparison of SDoH and HRB between opioid naïve and opioid users. Univariate analysis revealed that several SDoH and HRB increased the odds of preoperative opioid use. Marital status, however, did not signiﬁcantly inﬂuence the odds of preoperative opioid use. Univariate comparison of social determinants of health and health related behaviors between opioid naïve and opioid users Opioid naïve Opioid users Odds ratio p-value Age range: 18–39 13 (13.3%) 6 (6.67%) Ref. Ref. 40–59 25 (25.5%) 38 (42.2%) 3.21 [1.10;10.4] .033 60–75 48 (49.0%) 36 (40.0%) 1.60 [0.57;5.02] .384 75 + 12 (12.2%) 10 (11.1%) 1.77 [0.49;6.82] .388 Race: White or Caucasian 84 (85.7%) 71 (79.8%) Ref Ref Asian 6 (6.12%) 1 (1.11%) 0.22 [0.01;1.39] .117 Black or African American 1 (1.02%) 6 (6.67%) 6.31 [1.01;166] .049 Other 7 (7.14%) 12 (13.3%) 2.00 [0.75;5.73] .165 Marital status: Married 71 (72.4%) 53 (58.9%) Ref Ref Single 20 (20.4%) 25 (27.8%) 1.67 [0.84;3.36] .146 Widowed 1 (1.02%) 2 (2.22%) 2.50 [0.20;80.1] .478 Divorced 6 (6.12%) 10 (11.1%) 2.20 [0.76;6.96] .149 Work status: Full time 44 (44.9%) 19 (21.1%) Ref Ref Not employed 19 (19.4%) 45 (50.0%) 5.38 [2.55;11.8] < .001 Retired 35 (35.7%) 26 (28.9%) 1.71 [0.82;3.64] .156 Median income: 100,000 + 45 (45.9%) 25 (27.8%) Ref. Ref. 30,000–59,999 17 (17.3%) 22 (24.4%) 2.31 [1.04;5.23] .04 60,000–79,999 12 (12.2%) 21 (23.3%) 3.10 [1.32;7.57] .009 80,000–99,999 24 (24.5%) 22 (24.4%) 1.64 [0.77;3.54] .202 Median income dichotomous: Above low-income limit 64 (65.3%) 46 (51.1%) Ref. Ref. Below low-income limit 34 (34.7%) 44 (48.9%) 1.79 [1.00;3.25] .051 Religion: Nonreligious 51 (52.0%) 31 (34.4%) Ref. Ref. Religious 47 (48.0%) 59 (65.6%) 2.05 [1.14;3.73] .016 Insurance: Private 44 (44.9%) 30 (33.3%) Ref. Ref. Social determinants of health Comparison between OU and ON groups were based on ten SDoH fac- tors available in the EMR: age, gender, race, preferred language, ethnic- ity, work status, community median income, religious belief, insurance status, and marital status. Age was divided into quartile groups. Com- munity median income was determined using 2019 American Commu- nity Survey data and was divided into below and above the low-income limit, as determined by the US Department of Housing and Urban De- velopment [ 11 , 12 ]. The groups were also compared based on four HRB: tobacco, alcohol, and drug use, and physical activity capacity. Physi- cal activity capacity was assessed using metabolic equivalence of task Among HRB, patients who smoked were 2.2 times more likely to have preoperative opioid use (95% CI 1.25–4.06, p = .007). Those with 2 North American Spine Society Journal (NASSJ) 14 (2023) 100221 A. Wague, J.M. O’Donnell, K. Rangwalla et al. Table 2 Comparison intra and postoperative data between opioid naïve and opioid users. Opioid users had signiﬁcantly higher ASA scores and longer length of hospital stay. Comparison of surgical factors and outcomes between opioid naïve and opioid users All Opioid naïve Opioid users p-value Surgical invasiveness index 6.99 (6.24) 6.33 (5.45) 7.71 (6.95) .133 American Society of Anesthesiology Score 2.24 (0.57) 2.09 (0.50) 2.40 (0.60) < .001 Estimated blood loss (mL) 281 (470) 233 (385) 333 (545) .154 Total operation time (mins) 226 (133) 218 (139) 234 (125) .416 Length of hospital stay (days) 3.01 (2.32) 2.61 (2.08) 3.44 (2.50) .014 a e Comparison intra and postoperative data between opioid naïve and opioid users. Opioid users had signiﬁcantly higher ASA scores and longer length of hospital stay. Table 3 Medicaid 5 (5.10%) 15 (17.4%) 4.26 [1.46;14.6] .0067 Medicare 49 (50.0%) 42 (48.8%) 1.25 [0.67;2.35] .475 Other public 0 (0.00%) 3 (3.33%) - - Smoking history: Negative 62 (63.3%) 39 (43.3%) Ref. Ref. Positive 36 (36.7%) 51 (56.7%) 2.24 [1.25;4.06] .007 METS range: > 5 83 (84.7%) 57 (63.3%) Ref. Ref. < 5 15 (15.3%) 33 (36.7%) 3.17 [1.60;6.54] .001 Univariate comparison of social determinants of health and health related behaviors between opioid naïve and opioid users Opioid naïve Opioid users Odds ratio low physical activity capacity (METS < 5) were 3.2 times more likely to have preoperative opioid use (95% CI 1.60–6.54, p = .001). No associa- tion was found among alcohol use or drug use ( Table 3 ). Table 4 Multivariate comparison of social determinants of health and health related be- haviors between opioid naïve and opioid users. Odds ratio p-value Median Income B‘60,000 – 79,999 3.85 [1.27;11.7] .001 Not Employed 5.14 [1.95;14.4] .001 METS range < 5 3.76 [1.52;9.33] .005 Discussion The current study evaluates preoperative opioid use among spine surgery patient who underwent elective lumbar degenerative surgery, in the context of the social factors which can aﬀect patients’ health. Im- portantly, those with lower income, unemployed, or with lower physi- cal capacity (METS < 5) were more likely to use opioids preoperatively. Postoperative opioid use was strongly associated with preoperative opi- oid use, as well as alcohol use, and lower community median income. Furthermore, those who use opioids preoperatively used opioids post- operatively at higher rates, at higher doses, and for a longer period of time. Lower community median income (below the US HUD low-income limit) was also associated with preoperative opioid use, with 3.9 times increased odds. There are many historical and socioeconomic factors that can contribute to this, and the association between lower income and opioid use is well-established in the literature. Correlation has been found between low socioeconomic status and higher rates of opioid pre- scription renewal, higher preoperative opioid consumption in neighbor- hoods of lower socioeconomic status, and lower socioeconomic status and postoperative or chronic opioid use [32–35] . There are hidden con- founders within this variable, such as race or access to medical care [36–37] . Access is especially diﬃcult for subspecialty and surgical care. Additionally, when patients present at later timepoints or with more ad- vanced disease, it is understandable that they may be in more pain or require more opioids. Late presentation is also a multifactorial problem, which is seen at higher rates among those in marginalized groups. The opioid epidemic of the 21st century was unprecedented and had a sweeping eﬀect on Americans, and on how doctors must handle opi- oids [14–16] . The eﬀects of the opioid epidemic are especially pertinent for patients undergoing treatment for spine pathology [17–19] . Our results are consistent with previous work in this area. Previ- ous studies have demonstrated similar associations between preopera- tive and postoperative opioid utilization. An analysis of the Humana database by Jain et al. observed that 87.6% of preoperative OU con- tinued opioid use 1 year after a 1- or 2-level posterior lumbar fusion, and this has been corroborated in later studies as well [20–22] . Yerneni et al. [22] showed that outcomes were poor for those with preoperative opioid use. Spine surgeons see a high percentage of patients who are using opi- oids when presenting to their clinic. Table 4 Kaplan-Meier curve of opioid use among patients undergo- ing spine surgery for lumbar degenerative disease. Figure 1. Kaplan-Meier curve of opioid use among patients undergo- ing spine surgery for lumbar degenerative disease. use as the single most important risk factor for long-term opioid use [ 20 , 26 , 27 ]. Multivariate analysis of predictive factors for postop- erative opioid use. In order to better understand who is at risk for long-term opioid use or misuse, we must understand what risk factors contribute to their use before presentation or surgery. Studies have examined opioid use in spine clinics and patient populations but not through a social deter- minants lens. SDoH have shown to be reliable predictors of health out- comes in spine surgery. They have been linked to poorer outcomes in spine patients, such as postoperative complications and increased length of stay [ 28 , 29 ]. Regarding postoperative opioid use, multivariate analysis showed that preoperative opioid use, alcohol use, and community median in- come below the lower-income limit were independent risk factors (p < .001, .030, and .042 respectively). Those with preoperative opioid use were 13.2 times more likely to have postoperative opioid use (95% CI 5.90–31.9, p < .001) ( Table 5 ). In this study, we have identiﬁed several social factors that are as- sociated with preoperative opioid use. Lower physical capacity, lower community median income, and unemployment were all associated with preoperative opioid use in multivariate analysis. Lower physical capacity, marked by METS less than 5 in patients’ preoperative anesthesia visit, was associated with preoperative opioid use with 3.8 times increased odds of opioid use. A study on stress testing indicated a METS of less than 5 as poor exercise capacity [30] . Previous studies have shown that prehabilitation can be an early intervention to decrease postoperative opioid use, and this may be an appropriate inter- vention for patients scored in this lower METS group [31] . Given that this risk factor is modiﬁable, it may be a good target for preoperative optimization in spine surgery patients. There is caution, however, of pos- sible confounding with this metric; patients who are in more pain and therefore using more opioids may be limited in their physical capacity secondary to pain level. Table 4 Table 4 Multivariate comparison of social determinants of health and health related be- haviors between opioid naïve and opioid users. Odds ratio p-value Median Income B‘60,000 – 79,999 3.85 [1.27;11.7] .001 Not Employed 5.14 [1.95;14.4] .001 METS range < 5 3.76 [1.52;9.33] .005 operatively at higher rates, at higher doses, and for a longer period of time ( Figure ). Analysis comparing preoperative and postoperative MME in chronic OU indicated no diﬀerence (p = .170). Multivariate comparison of social determinants of health and health related be- haviors between opioid naïve and opioid users. Odds ratio p-value Median Income B‘60,000 – 79,999 3.85 [1.27;11.7] .001 Not Employed 5.14 [1.95;14.4] .001 METS range < 5 3.76 [1.52;9.33] .005 Multivariate analysis demonstrated unemployment (OR 5.15, 95%CI 1.95–14.4, p < .001), lower capacity for physical activity marked by METS < 5 (OR 3.76, 95%CI 1.52–9.33, p = .005), and those with com- munity median income below the lower-income limit (OR 3.85, 95%CI 1.27–11.7, p < .001) as independent risk factors for preoperative opioid use ( Table 4 ). The OU cohort had a greater proportion of patients still using opioids at 3 months (83% vs. 22%, p < .001) and 1 year postoperatively (72% vs. 15%, p < .001). The OU group used more daily opioids postoperatively (57.2 MME vs. 10.0, p < .001) ( Table 6 ). The OU group used opioids post- operatively at higher rates, at higher doses, and for a longer period of time ( Figure ). Analysis comparing preoperative and postoperative MME in chronic OU indicated no diﬀerence (p = .170). 3 A. Wague, J.M. O’Donnell, K. Rangwalla et al. North American Spine Society Journal (NASSJ) 14 (2023) 100221 Figure 1. Kaplan-Meier curve of opioid use among patients undergo- ing spine surgery for lumbar degenerative disease. A. Wague, J.M. O’Donnell, K. Rangwalla et al. North Figure 1. Kaplan ing spine surgery Table 5 use as the single most i A. Wague, J.M. O’Donnell, K. Rangwalla et al. North American Spine Society Journal (NASSJ) 14 (2023) 100221 Figure 1. Kaplan-Meier curve of opioid use among patients undergo- ing spine surgery for lumbar degenerative disease. North American Spine Society Journal (NASSJ) 14 (2023) 100221 North American Spine Society Journal (NASSJ) 14 (2023) 100221 A. Wague, J.M. O’Donnell, K. Rangwalla et al. A. Wague, J.M. O’Donnell, K. Rangwalla et al. Figure 1. References [1] Centers for Disease Control and Prevention. (2021). America’s drug overdose epi- demic: Putting data to action . Centers for Disease Control and Prevention. Re- trieved February 3, 2023, from https://www.cdc.gov/injury/features/prescription- drug-overdose/index.html . [2] Volkow ND, McLellan TA, Cotto JH, Karithanom M, Weiss SR. Charac- teristics of opioid prescriptions in 2009. JAMA 2011;305(13):1299–301. doi: 10.1001/jama.2011.401 . The main limitation of the current study is the data available, which is limited by what is collected in the EMR. As a retrospective chart re- view, further data that encompass other SDOH could not be obtained. Some factors which were not available include information about phys- ical environment (transportation and housing), education (education level, language, literacy), and the patient’s experience with the health- care system. Additionally, proxies such as community median income (via zipcode) were used for socioeconomic status. Actual opioid usage was not tracked directly (such as via drug testing). [3] Schoenfeld AJ, Jiang W, Chaudhary MA, Scully RE, Koehlmoos T, Haider AH. Sustained prescription opioid use among previously opioid-naive patients insured through TRICARE (2006-2014). JAMA Surg 2017;152(12):1175–6. doi: 10.1001/ja- masurg.2017.2628 . g [4] Kha ST, Scheman J, Davin S, Benzel EC. The impact of preoperative chronic opioid therapy in patients undergoing decompression laminectomy of the lumbar spine. Spine (Phila Pa 1976) 2020;45(7):438–43. doi: 10.1097/BRS.0000000000003297 . [5] Kalakoti P, Hendrickson NR, Bedard NA, Pugely AJ. Opioid utilization following lumbar arthrodesis: trends and factors associated with long-term use. Spine (Phila Pa 1976) 2018;43(17):1208–16. doi: 10.1097/BRS.0000000000002734 . [6] Jain N, Sharma M, Wang D, Ugiliweneza B, Drazin D, Boakye M. Burden of preopera- tive opioid use and its impact on healthcare utilization after primary single level lum- bar discectomy. Spine J 2021;21(10):1700–10. doi: 10.1016/j.spinee.2021.04.013 . Future directions should include preoperative optimization of all fac- tors which are modiﬁable. This includes prehabilitation for improve- ment of physical capacity, reduction of HRBs such as alcohol and smok- ing, and reducing preoperative opioid intake. We must also continue to focus on improving healthcare access and delivery of equitable care (care that does not vary with gender, ethnicity, or socioeconomic status); this can have an impact on patients who are unemployed, of lower so- cioeconomic status, or otherwise disadvantaged and presenting to spine surgeons on higher doses of opioids. The spine surgery community must continue to deliver equitable care which includes further studies of bar- riers to access, disparity in care, and diﬀerences in surgical outcomes. Conclusions To summarize, in adults treated with surgery for lumbar degenera- tive pathology, low physical activity capacity, low community median income, and unemployment were associated with preoperative opioid use. Preoperative opioid use is a strong predictor of postoperative opi- oid utilization at every follow-up timepoint up to 1 year after surgery. Minimizing preoperative opioid use may represent a strategy for reduc- ing postoperative opioid use. [13] Jetté M, Sidney K, Blümchen G. Metabolic equivalents (METS) in exercise test- ing, exercise prescription, and evaluation of functional capacity. Clin Cardiol 1990;13(8):555–65 . [14] Florence CS, Zhou C, Luo F, Xu L. The economic burden of prescription opi- oid overdose, abuse, and dependence in the United States, 2013. Med Care 2016;54(10):901–6 . [15] Brummett CM, Waljee JF, Goesling J, et al. New persistent opioid use after minor and major surgical procedures in US adults. JAMA Surg 2017;152(6):e170504 Epub 2017 Jun 21 . [16] Chaudhary MA, Schoenfeld AJ, Harlow AF, et al. Incidence and predictors of opioid prescription at discharge after traumatic injury. JAMA Surg 2017;152(10):930–6 . Declarations of Competing Interests [17] Armaghani SJ, Lee DS, Bible JE, et al. Preoperative opioid use and its associa- tion with perioperative opioid demand and postoperative opioid independence in patients undergoing spine surgery. Spine (Phila Pa 1976). 2014;39(25):E1524–30. doi: 10.1097/BRS.0000000000000622 . The authors have no copyrighted materials included in the attached submission. The local Institutional Review Board approved this work. [18] 3rd Connolly J, Raji JZ, Chan W, Kuo YF, Baillargeon J. Predictors of longterm opioid use following lumbar fusion surgery. Spine (Phila Pa 1976) 2017;42(18):1405–11 . [19] Faour M, Anderson JT, Haas AR, et al. Preoperative opioid use: a risk factor for poor return to work status after single-level cervicalfusion for radiculopathy in a workers’ compensation setting. Clin Spine Surg 2018;31(1):19–24 . References Minimizing disparity is paramount to delivering excellent, equitable care. [7] Kim S, Ozpinar A, Agarwal N, et al. Relationship between preoperative opioid use and postoperative pain in patients undergoing minimally invasive stand-alone lat- eral lumbar interbody fusion. Neurosurgery 2020;87(6):1167–73. doi: 10.1093/neu- ros/nyaa207 . [8] Weiner JA, Snavely JE, Johnson DJ, Hsu WK, Patel AA. Impact of preoperative opioid use on postoperative patient-reported outcomes in lumbar spine surgery patients. Clin Spine Surg 2021;34(3):E154–9. doi: 10.1097/BSD.0000000000001067 . [9] Ghertner R, Groves L. The opioid crisis and economic opportunity: geographic and economic trends. ASPE Research Brief 2018;11:1–22 . [10] Zale EL, Dorfman ML, Hooten WM, Warner DO, Zvolensky MJ, Ditre JW. Tobacco smoking, nicotine dependence, and patterns of prescription opioid misuse: results from a nationally representative sample. Nicotine Tob Res 2015;17(9):1096–103. doi: 10.1093/ntr/ntu227 . [11] U.S. Census Bureau (2021). Factﬁnder for the nation. The Census Bureau’s American Community Survey (ACS) Public Use Microdata Sample (PUMS). Retrieved February 3, 2023, from https://www.census.gov/library/publications/time-series/cﬀ.html . [12] US Department of Health and Human Services (HHS). (2023). FY 2023 income limits documentation system. FY 2023 Income Limits Documentation System – Summary. Retrieved February 3, 2023, from https://www.huduser.gov/portal/datasets/il/ il2022/2022summary.odn . ests (as provided in the authors’ ICMJE forms) do not raise any concerns and represent a low risk for bias in the present study. or their unemployed work status has an eﬀect on their opioid intake [38] . Those who used opioids preoperatively had more than 13 times odds of using opioids postoperatively. This underlines the importance of the preoperative opioid use risk factors, as preoperative use has direct im- plications for postoperative. Additional associated risk factors for post- operative opioid use were alcohol use and community median income below the lower income limit. Lower community median income re- mained a risk factor from preoperative to postoperative, independent from preoperative opioid use itself. The impact of socioeconomic status on opioid use cannot be understated, and is likely multifactorial. Discussion Patients undergoing spine surgery have a high incidence of preoperative opioid use, ranging from 20% to 70% [23–25] . Studies continue to characterize preoperative opioid Similarly, in this study we found that unemployed patients had more than 5 times odds of using opioids preoperatively. Again, it is diﬃcult to discern whether those patients are unemployed as a result of their pain, 4 North American Spine Society Journal (NASSJ) 14 (2023) 100221 A. Wague, J.M. O’Donnell, K. Rangwalla et al. Table 6 Table 6 Comparison of postoperative opioid use between opioid naïve and opioid users. Opioid users had signiﬁcantly greater opioid use at both 3 months and 1 year postoperatively compared to opioid naïve users. Opioid naïve Opioid users p-value Preoperative morphine milligram equivalents - 48.2 (44.8) - Immediately prior to surgery morphine milligram equivalents - 48.7 (43.7) - Postoperative opioid use after 3 mo 22 (22.4%) 75 (83.3%) < .001 Postoperative opioid use after 1 y 15 (15.3%) 65 (72.2%) < .001 Postoperative morphine milligram equivalents 10.0 (27.1) 57.2 (65.7) < .001 ests (as provided in the authors’ ICMJE forms) do not raise any concerns and represent a low risk for bias in the present study. Funding [20] Jain N, Phillips FM, Weaver T, Khan SN. Preoperative chronic opioid therapy: a risk factor for complications, readmission, continued opioid use and increased costs after one- and two-level posterior lumbar fusion. Spine (Phila Pa 1976). 2018;43(19):1331–8. doi: 10.1097/BRS.0000000000002609 . All authors certify that no funding was provided for the current study. After study-speciﬁc appraisal for potential conﬂict of interest- associated biases, the authors conclude that any ﬁnancial or other inter- 5 A. Wague, J.M. O’Donnell, K. Rangwalla et al. North American Spine Society Journal (NASSJ) 14 (2023) 100221 [30] Kharabsheh SM, Al-Sugair A, Al-Buraiki J, Al-Farhan J. Overview of exercise stress testing. Ann Saudi Med 2006;26(1):1–6. doi: 10.5144/0256-4947.2006.1 . [21] Jr Schoenfeld AJ, Belmont PJ, Blucher JA, et al. Sustained preoperative opioid use is a predictor of continued use following spine surgery. J Bone Joint Surg 2018;100(11):914–21. doi: 10.2106/JBJS.17.00862 . [31] Lee A, Shelton E, Bidwell S, et al. Association of prehabilitation with postopera- tive opioid use in colorectal surgery: an Observational Cohort Study. J Surg Res 2022;273:226–32. doi: 10.1016/j.jss.2021.12.023 . 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https://openalex.org/W2777755454	https://bmcnephrol.biomedcentral.com/track/pdf/10.1186/s12882-017-0785-1	English	null	On-line hemodiafiltration did not induce an overproduction of oxidative stress and inflammatory cytokines in intensive care unit-acute kidney injury	BMC nephrology	2,017	cc-by	6,570	* Correspondence: k-klouche@chu-montpellier.fr 1Intensive Care Medicine Department, University of Montpellier Lapeyronie Hospital, 371, Av Doyen Gaston Giraud, 34295 Montpellier, France 4Lapeyronie University Hospital. PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, 34295 Montpellier cedex 5, France Full list of author information is available at the end of the article On-line hemodiafiltration did not induce an overproduction of oxidative stress and inflammatory cytokines in intensive care unit-acute kidney injury Kada Klouche1,4* , Laurent Amigues1, Marion Morena2,4, Vincent Brunot1, Anne Marie Dupuy2, Audrey Jaussent3, Marie Christine Picot3, Noémie Besnard1, Delphine Daubin1 and Jean Paul Cristol2,4 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Klouche et al. BMC Nephrology (2017) 18:371 DOI 10.1186/s12882-017-0785-1 Klouche et al. BMC Nephrology (2017) 18:371 DOI 10.1186/s12882-017-0785-1 Patients Since 2004, we exclusively used OL-HDF besides con- tinuous therapies to treat our critically ill patients. During one-year period, we consecutively enrolled all patients ad- mitted to the ICU with AKI requiring RRT support and who underwent OL-IHDF. Exclusion criteria included preg- nancy, age < 18 years old, previous chronic renal failure, and severe neutropenia. Epidemiological data and severity of patients assessed by the Simplified Acute Physiologic (SAPS) II [18] and Sepsis-related Organ Failure Assessment (SOFA) scores [19] were collected. Decisions regarding the initiation, management, and discontinuation of RRT were made by the referring physician according to the KDIGO recommendations [20]. The choice of RRT modality was depending on patient hemodynamic stability and was daily re-evaluated. Patients with hemodynamic instability or severe fluid overload were preferentially treated with con- tinuous venovenous hemodiafiltration and with OL-IHDF when they had or recovered hemodynamic stability. Only OL-IHDF sessions were investigated. Outcome was assessed at ICU discharge. g In intensive care units (ICUs), around 5% of patients suffering from acute kidney injury (AKI) require renal replacement therapy (RRT) [1]. Intermittent conven- tional hemodialysis (IHD) and continuous venovenous hemo(dia)filtration are the 2 principal modalities used for RRT. However, the ideal renal replacement method for intensive care patients remains under scrutiny [2, 3]. It should combine the advantages of continuous RRT (CRRT) with those of IHD, be simple to implement and induce minimal work with limited cost. On-line dialysis fluids preparation may fit these conditions since it offers favorable technical possibilities and highly flexible proce- dures to apply various forms of cost-effective high effi- ciency hemodiafiltration (HDF) modalities in intermittent or sustained mode [4–7]. On-line HDF (OL-HDF) is a RRT based on cold sterilization of dialysis fluid to prepare the infusate which is readily administered into the extracorporeal bloodstream. It necessitates a fully microbiological integ- rity of on-line produced dialysis fluids. In chronic dialy- sis facilities, this technique became a routine and safe modality of RRT and now represents the most effective dialysis therapy [8–10]. A few reports exist about its use in ICUs and its routine application is mainly restricted to ICU facilities working with a trained nephrological team [4–7, 11–17]. Patients The limited use of this technique by intensivists is largely related to the technical complexity with water treatment and HDF machines and to the po- tential infectious risk of on-line produced fluids infusion especially in septic patients who represent the majority of admissions in ICUs. We have previously reported our experience of OL-HDF use in ICU [11]. A regular bac- teriological control of dialysis fluids showed that this technique is safe and well tolerated [11]. Whether OL-HDF modulates plasma cytokine concentration and oxidative stress production is still not investi- gated. The combined use of synthetic biocompatible membrane and ultrapure dialysis fluid may limit the additional inflammatory risk induced by OL-HDF. However, this potential acute inflammatory risk in- duced by OL intermittent HDF (OL-IHDF) which re- mains possible in ICU-AKI has never been evaluated. We designed therefore a study to determine whether OL-IHDF would induce an overproduction of oxida- tive stress, cytokines and growth factors in critically ill patients. On-line intermittent hemodiafiltration: Clinical tolerance OL-IHDF clinical tolerance was investigated by collect- ing the following intradialytic parameters at baseline and every 30 min: pulse, temperature, mean arterial pressure (MAP). A pyrogenic reaction was defined as the onset of objective chills and an increase in temperature of more than 1 °C in a patient who had no recorded signs or symptoms of infection before RRT [23]. An intradialytic Abstract BMC Nephrology (2017) 18:371 Page 2 of 8 Page 2 of 8 Page 2 of 8 On-line intermittent hemodiafiltration On-line intermittent hemodiafiltration: Description, disinfection procedures p OL-IHDF was performed using a RRT generator (Fresenius 5008, Fresenius Medical Care, Bad Homburg, Germany) with a standard 1.8 m2 Polysulfone hemodiafilter HF80 (Fresenius Medical Care, Bad Homburg, Germany). Countercurrent dialysate flow (QD) was routinely set at 500 mL/min, on-line infusate flow in pre-dilution mode (Qi) at 100 mL/min and blood flow (QB) at 300 mL/min. Dialysate and infusate temperature were adjusted to 36 °C and the sodium dialysate concentration at 145 mmol/L. The net-ultrafiltration rate was adapted to the hemo- dynamic parameters and extracellular volume status of each patient. Our ICU water production and distribution system was identical to that routinely used in chronic dialysis facilities performing on-line therapies [21] as pre- viously described [11]. Dialysate and infusate purity has been also validated previously [9, 11, 22] and was ensured by regular endotoxin and microbiological testing. Vascular access was obtained through double lumen jugular catheter, with unfractionated heparin for anticoagulation whenever needed. Abstract Background: Though on-line intermittent hemodiafiltration (OL-IHDF) is a routine therapy for chronic dialysis patients, it is not yet widespread used in critically ill patients. This study was undergone to evaluate efficiency and tolerance of OL-IHDF and to appreciate inflammatory consequences of its use in intensive care unit (ICU)-acute kidney injury (AKI) patients. Methods: In this prospective cohort study conducted in a medical academic ICU in France, 30 AKI patients who underwent OL-IHDF were included. OL-HDF used an ultrapure water production: AQ 1250 line with double reverse osmosis, a generator 5008 with a 1.8m2 dialyzer with Polysulfone membrane (Fresenius Medical Care). Tolerance and efficiency of OL-IHDF were evaluated as well as its inflammatory risk by the measurement of plasma concentrations of proinflammatory (Interleukin 6, IL1β, IL8, Interferon γ) and anti-inflammatory (IL4, IL10) cytokines, Epidermal growth factor (EGF), Vascular Endothelial growth factor (VEGF) and Macrophage Chemoattractive Protein-1 (MCP-1) before and after sessions. Results: Intradialytic hypotensive events were observed during 27/203 OL-IHDF sessions accounting for a mal-tolerated session’s rate at 13.3%. Mean delivered urea Kt/V per session was 1.12 ± 0.27 with a percentage of reduction for urea, creatinine, β2-microglobulin and cystatine C at 61.6 ± 8.8%, 55.3 ± 6.7%, 51.5 ± 8.7% and 44.5 ± 9.8% respectively. Production of superoxide anion by leukocytes, mean levels of pro- and anti-inflammatory cytokines and plasmatic concentrations of EGF, VEGF and MCP-1 did not differ before and after OL-IHDF sessions. We observed however a significant decrease of mean TNFα plasmatic concentrations from 8.2 ± 5.8 to 4.8 ± 3.5 pg/ml at the end of OL-IHDF. Conclusions: OL-IHDF was not associated with an increase in pro and anti-inflammatory cytokines, oxidative stress or EGF, VEGF and MCP-1 in AKI patients and seems therefore a secure and feasible modality in ICUs. Keywords: Acute kidney injury, On-line Hemodiafiltration, Oxidative stress, Inflammatory cytokines, Anti-inflammatory cytokines, Egf, Vegf, Mcp-1 © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Klouche et al. Methods This observational prospective study was carried out at the Medical ICU of Lapeyronie University Hospital at Montpellier and was approved by the Ethics Research Committee of our hospital; PHRC régional: N° 2006-A00510–51. Page 3 of 8 Klouche et al. BMC Nephrology (2017) 18:371 hypotensive event was defined by a 20% reduction of MAP or by an initiation or/and increase in vasocon- strictive agents’ dose. production measure were as follows: intra-assay CV = 3.5% (basal O2°- production) and 3.9% (PMA-stimulated O2°- production); interassay CV = 5.0% (basal O2°- production) and 9.7% (PMA-stimulated O2°- production). Statistical analysis Statistical analysis was performed using SAS Entreprise Guide version 4.1. We first performed a descriptive analysis by computing frequencies and percents for cat- egorial data, means, standard deviations, quartiles and extreme values for continuous data. For every patient in- cluded, 2 or more OL-IHDF sessions were investigated. The session that induced the highest post treatment cytokine increase was solely analyzed per patient. We checked for normality of continuous data distribution (O2 °- anion production, cytokines and proinflammatory mediators measurements), using Shapiro-Wilk’s tests. To analyze differences between before and after treatment measurements, univariate analysis was performed using two-tailed Student t-test, or two-tailed Mann-Whitney- Wilcoxon’s test (signed Rank Statistic) when appropriate. Kinetic of oxidative stress and cytokine release before and after OL-IHDF was also investigated in all included sessions using a linear mixed model. A value of p < 0.05 was considered significant. Determination of superoxide (O2 °-) anion production by whole blood O2 °- anion production was measured in blood samples before and after OL-IHDF sessions. It was determinated in 200 μL of fresh whole blood (treated immediately after collection) diluted in 820 μl of DMEM medium and 200 μL of lucigenin (1.5.10−4 mol/L) (Sigma Chemical, Saint Quentin Fallavier, France) [28]. After a 20-min incubation at 37 °C under gentle agitation, whole blood was stimulated by using Phorbol 12-Myristate 13-Acetate (PMA) (10−7 M) and the luminescence was immediately recorded at 37 °C by means of a Victor Wallac luminometer (Perkin Elmer, Turku, Finland). Lumines- cence intensity was normalized to leukocyte count. Re- sponse of PMA-free whole blood (basal O2 °- production) incubated simultaneously was used as control and consid- ered as equal to 100%. To rule out autoproduction of O2 °- by lucigenin or by plasma compounds, O2 °- production was determined in whole blood, de-leukocyted blood, plasma and culture medium. Imprecision studies of O2°- Determination of plasmatic cytokines, growth factors, and advanced oxidation protein products, measurements Determination of plasmatic cytokines, growth factors, and advanced oxidation protein products, measurements Pre-and post-OL-IHDF sessions blood samples were im- mediately centrifuged at 1000 g for 10 min at 4 °C and stored at −80 °C until use. A panel of cytokines was de- termined on frozen plasma using a proteomic approach on an Evidence Investigator® biochip system (Randox, Mauguio, France). This proteomic method allows the simultaneous determination of IL1β, IL4, IL6, IL8, IL10, interferonγ, Epidermal growth factor (EGF), Vascular Endothelial growth factor (VEGF), Tumor Necrosis factor α (TNFα) and Macrophage Chemoattractive Protein-1 (MCP-1) levels. After addition of a sample (100 μl) to the biochip, the degree of binding of each analyte to its specific ligand is determined using a chemiluminescence light source and quantified using a super-cooled charge- coupled camera and an image-processing software [29]. Biological plasma parameters Blood solutes including urea, creatinine, β2-microglobulin (β2-M) and cystatine C (CyC) were routinely monitored at the beginning and the end of each OL-IHDF session. Blood samples were collected at the end of the treatment by standard stop- flow technique [24] and after the first hour of treatment, simultaneously at the arterial and venous ports after a temporary net ultrafiltration cessation. Urea, creatinine, β2-M and CyC removals per session were evaluated by the percentage of solute reduction ratios according to: RR = [(Cpre–Cpost)/Cpre]100 where Cpre and Cpost are respectively pre-treatment (baseline) and post-treatment concentrations [25]. Kt/V were determined by using Daugirdas second generation, single pool urea kinetic model equation: Kt/V = −ln (T-0.008 time duration ses- sion in minutes) + (4–3.5T)(UF/weightpostsession) where T represents plasma ureapostsession/ureapresession [26]. Instantaneous whole blood (KW.B) and plasma water solutes clearances (KP.W.) were estimated as follows: KW.B = QB[(Cart-Cven)/Cart] where QB is effective blood flow, Cart and Cven are solute concentration in arterial and venous blood line; KP.W. = KW.B (1– 0.00107Tp)[(SPC*Ht) + (1 - Ht)] where Ht is the pa- tient’s predialysis hematocrit level, Tp the average of total protein level in arterial and venous blood line (Tp = [Tpart + Tpven]/2) and solute partition coefficient: 0.86 for urea, 0.73 for creatinine, and 0 for β2-M and CyC [27]. Plasma Advanced Oxidation Protein Products (AOPP) levels (μM/l) were measured in pre- and post-OL-IHDF sessions blood samples by spectrophotometry [30, 31]. Patient demographic data During the study period, 34 of 51 patients admitted to our ICU for severe AKI treated by RRT were included in the study. Main reasons for non-inclusion were: contra- indication (8 patients for aplasia, 2 for non-consent, and 2 for life expectancy less than 48 h), and 5 patients Klouche et al. BMC Nephrology (2017) 18:371 Page 4 of 8 Table 2 On-line IHDF sessions with hypotensive events OL-IHDF sessions, n = 203 n (%) Table 2 On-line IHDF sessions with hypotensive events Table 2 On-line IHDF sessions with hypotensive events OL-IHDF sessions, n = 203 n (%) Sessions with hypotension 27 (13.3) Requiring only UF cessation 23 (11.3) Requiring a vasopressor support 5 (2.5) Requiring a fluid challenge 4 (2) Requiring a dialysis cessation 0 (0) One or more therapeutic interventions may be used treated only with continuous RRT. In addition, 4 pa- tients were excluded because of missing data. Thus, 30 patients were enrolled in the study and completely ana- lyzed. Age, gender, cause of AKI, severity scores are listed in Table 1. The cause of AKI was septic in ap- proximately ¾ of the cases. All patients were anuric, treated by vasoactive agents and most of them venti- lated. The ICU mortality rate was 26.7%. One or more therapeutic interventions may be used Parameters, clinical and tolerance evaluation of on- line intermittent hemodiafiltration sessions. (choosing the session with the highest cytokine increase after treatment according to Statistical analysis). Mean delivered urea Kt/V session was 1.12 ± 0.27. RR for urea, creatinine, β2-M and CyC were respectively 61.6 ± 8.8%, 55.3 ± 6.7%, 51.5 ± 8.7% and 44.5 ± 9.8%. KW.B. and KP.W. were 239.2 ± 22.3 and 213.4 ± 20.7 mL/min for urea, 197 ± 22 and 168.6 ± 20.5 mL/min for creatinine, 58.9 ± 17.3 and 38.5 ± 10.9 mL/min for CyC, 77.8 ± 29.6 and 50.3 ± 17.4 mL/min for β2-M. Albuminemia increased from 27.5 ± 4.0 g/L to 28.5 ± 4.3 g/L (p < 0.05). Ol-IHDF duration time ranged from 4 to 6 h with a median time at 4.8 h. Sessions parameters were as fol- lows: blood flow: 285(326–185) ml/mn, dialysate flow: 468 (442–489) ml/mn, predilution infusate flow: 89 (110–85) ml/mn with convection volume at 28 (20–36) l per session. Venous recirculation was less than 5% in all sessions. Patient demographic data Among the 203 OL-IHDF sessions (>3/patient) evaluated, mean arterial pressure (MAP) increased from 86.2 ± 16 to 90 ± 16 mmHg after treatment (p < 0.05) with a significant increase of MAP after 119/203 (58%) sessions. An intradialytic hypotensive event was ob- served during 27 sessions accounting for a mal-tolerated session’s rate at 13.3% (Table 2). Cessation of ultrafiltra- tion was sufficient to restore hemodynamic stability in 23 sessions while a fluid challenge or a vasopressor support was necessary during the remaining sessions (Table 2). No pyrogenic reactions occurred among all OL-IHDF sessions performed. On-line intermittent hemodiafiltration and inflammatory mediators Mean basal and PMA-stimulated production of O2°- anion by leukocytes did not differ before and after OL-IHDF ses- sions (Fig. 1a). The activation rate of PMA-stimulated O2°- production reached 294% ± 273 at the initiation and 372% ± 415 at the end of OL-IHDF, but differences were not statistically significant (Fig. 1b). However, OL-IHDF sessions provided a slight but significant decrease in AOPP (p = 0.008) (Fig. 1c). On-line intermittent hemodiafiltration efficiency and solutes control Cytokines plasmatic measurements showed a significant heterogeneity between individuals before initiation of OL- IHDF but their variations after OL-IHDF sessions remained totally similar. Thus, mean levels of pro- (IL6, IL10, IL8, interferon γ) and anti- (IL4, IL10) inflammatory cytokines were not significantly different before and after OL-IHDF sessions (Figs. 2 and 3). We observed however a significant decrease of mean TNFα plasmatic concentrations from 8.2 ± 5.8 to 4.8 ± 3.5 pg/mL after OL-IHDF sessions, a reduction ratio at 41.4% (Fig. 3). Analysis of plasmatic concentrations of EGF, VEGF and MCP-1, before and after OL-IHDF sessions showed no significant differences (Fig. 4). Evaluation for efficiency and inflammatory consequences of OL-IHDF was achieved for 76 out of 203 (37.4%) OL-IHDF sessions accounting for at least one session per patient Table 1 Epidemiological data of patients Patient characteristics n = 30 Age, years 61.1 ± 15.3 Male, n (%) 25 (83.3) SAPS II 58.4 ± 20.8 APACHE II 29.8 ± 6.6 SOFA 11.6 ± 3.8 Mechanical ventilation, n (%) 19 (63.3) Vasoactive support, n (%) 28 (93.3) Causes of AKI, n (%) Septic 21 (70) Ischemic 11 (36.6) Toxic 12 (40) Miscellaneous 4 (13.3) ICU mortality, n (%) 8 (26.7) All parameters, otherwise specified, are presented as mean ± standard deviation Table 1 Epidemiological data of patients Patient characteristics n = 30 Age, years 61.1 ± 15.3 Male, n (%) 25 (83.3) SAPS II 58.4 ± 20.8 APACHE II 29.8 ± 6.6 SOFA 11.6 ± 3.8 Mechanical ventilation, n (%) 19 (63.3) Vasoactive support, n (%) 28 (93.3) Causes of AKI, n (%) Septic 21 (70) Ischemic 11 (36.6) Toxic 12 (40) Miscellaneous 4 (13.3) ICU mortality, n (%) 8 (26.7) All parameters, otherwise specified, are presented as mean ± standard deviation Discussion The present study demonstrated that OL-IHDF achieved an adequate dialysis dose with a fair hemodynamic toler- ance in critically ill patients, and was not associated with an increased inflammatory risk. It did not induce an overproduction of oxidative stress and of pro- and anti- inflammatory cytokines but a significant decrease of TNFα and AOPP plasmatic levels. On-line substitution fluid preparation is a standard feature of modern dialysis devices for chronic treatments Klouche et al. BMC Nephrology (2017) 18:371 Page 5 of 8 a b c Fig. 1 Anion superoxide production (a), superoxide activation rate (b), and advanced oxidation protein products plasmatic levels (c) before and after OL-IHDF sessions. Values are shown as mean and standard deviation. Phorbol 12-myristate 13-acetate (PMA), On-line intermittent hemodiafiltration (OL-IHDF) b b Fig. 1 Anion superoxide production (a), superoxide activation rate (b), and advanced oxidation protein products plasmatic levels (c) before and after OL-IHDF sessions. Values are shown as mean and standard deviation. Phorbol 12-myristate 13-acetate (PMA), On-line intermittent hemodiafiltration (OL-IHDF) [32]. In ICU settings, on-line RRT modalities are how- ever rarely used and their routine application is mainly restricted to ICU facilities working with a trained nephrological team [4–7, 9, 11–17]. The carefulness of intensivist to use OL-IHDF is related to the potential in- fectious risk and deleterious effects of on-line produced fluid infusion especially in septic patients who represent the majority of those admitted to ICUs. On-line prepar- ation is not accompanied by on-line control of the microbiological quality and inadequate infusate would be administered directly without prior product check and release. In a previous work, we have evaluated, over a 7 year-period, the purity of on-line produced ultrapure water and dialysis fluids by a weekly bacterial control monitoring in ICU settings: more than 90% of samples showed negative bacterial growth and undetectable levels of endotoxin indicating an overall compliance rate of 99% [11]. However, water and dialysis fluids may still contained cytokines-inducing substances other than en- dotoxins like breakdown products of microorganisms, peptidoglycans and β-glycans [33]. They have the poten- tial capability of penetrating ultrafiltration and dialysis membranes with subsequent blood exposure and intra- dialytic cytokine and oxidative stress induction [34]. We re and after OL-IHDF sessions Values are shown as mean and standard Fig. 2 Mean plasmatic concentrations of pro-inflammatory cytokines before and after OL-IHDF sessions. Values are shown as mean and standard deviation. Discussion On-line intermittent hemodiafiltration (OL-IHDF), Interleukin 6 (IL6), Interleukin 8 (IL8), Interferon γ (infγ), Interleukin 1β (IL1β) Fig. 2 Mean plasmatic concentrations of pro-inflammatory cytokines before and after OL-IHDF sessions. Values are shown as mean and standard deviation. On-line intermittent hemodiafiltration (OL-IHDF), Interleukin 6 (IL6), Interleukin 8 (IL8), Interferon γ (infγ), Interleukin 1β (IL1β) Klouche et al. BMC Nephrology (2017) 18:371 Page 6 of 8 Fig. 3 Mean plasmatic concentrations of anti-inflammatory cytokines and TNFα before and after OL-IHDF sessions. Values are shown as mean and standard deviation. Interleukin 4 (IL4), Interleukin 10 (IL10), Tumor Necrosis Factor alpha (TNFα) Fig. 3 Mean plasmatic concentrations of anti-inflammatory cytokines and TNFα before and after OL-IHDF sessions. Values are shown as mean and standard deviation. Interleukin 4 (IL4), Interleukin 10 (IL10), Tumor Necrosis Factor alpha (TNFα) contribute to the progression of renal failure and that higher AOPP levels are associated with poor renal recovery [40]. Cytokine induction has been also con- sidered as the trigger of the inflammatory response and a critical parameter of dialysis biocompatibility during RRT [41]. Moreover, high levels of pro-inflammatory cytokines have been associated with increased mortality in AKI [35, 42]. We found that both pro- and anti- inflammatory cytokines plasma levels did not increase after OL-IHDF suggesting that it might not alter the balance of cytokines production. Other studies reported, like us, the lack of cytokines reduction by hemodiafiltration [43]. Sub- stance clearance is dependent on its molecular size but also on ultrafiltration rate and on whether the substitution fluid is administered before and after the filter. In our study, we used a predilution modality of HDF diluting the blood be- fore filter passage and convection volumes were less than 30 l per session explaining, at least for a part, the observed non significative reduction of plasmatic cytokines. aimed therefore to assess the inflammatory risk for crit- ically ill patients receiving OL-HDF by the quantification of cytokines, oxidative stress and growth factors poten- tially produced during OL-IHDF. y p g Critically ill patients with AKI have higher circulating plasma concentration of inflammatory biomarkers impli- cated in RRT dependence and mortality than those with- out AKI [35, 36]. This inflammatory process is partly due to the generation of oxidative stress which is mainly of multifactorial origin including sepsis, and accumula- tion of uremic toxins in case of AKI but may be also re- lated to RRT modalities. Abbreviations AKI: Acute kidney injury; Cpost: Post-treatment concentration; Cpre: Pre-treatment concentration; CRRT: Continuous renal replacement therapy; CyC: Cystatine C; EGF: Epidermal growth factor; HDF: Hemodiafiltration; ICU: Intensive care unit; IHD: Intermittent conventional hemodialysis; KP.W.: Plasma water solutes clearances; Kt/V: Clearance adjusted for total body volume; KW.B.: Instantaneous whole blood solutes clearances; MCP-1: Macrophage Chemoattractive Protein-1; O2°-: Superoxide anion; OL-HDF: On-line hemodiafiltration; OL-IHDF: On-line intermittent hemodiafiltration; PMA: Phorbol 12-myristate 13-acetate; QB: Blood flow; QD: Dialysate flow; Qi: Infusate flow; RR: reduction ratio; RRT: Renal replacement therapy; SAPS II: Simplified Acute Physiologic II score; SOFA: Sepsis-related Organ Failure Assessment score; TNF α: Tumor Necrosis factor alpha; VEGF: Vascular Endothelial growth factor; β2-M: β2-microglobulin Competing interests h h d l h Competing interests h h ’ d l h Competing interests The authors’ declare that they have no competing interest. Competing interests The authors’ declare that they have no competing interest. Received: 1 March 2017 Accepted: 8 December 2017 In conclusion, our study shows that OL-IHDF does not in- duce additional inflammatory risks in critically ill patients with AKI and may be used securely in these settings. On- line production of ultrapure water seems to be very useful for ICU acute renal failure as it gives possibilities for a large scale of dialysate and infusate rate prescription. Further studies should however investigate its effect on all-cause mortality in comparison to other RRT modalities. Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Furthermore, on-line IHDF was best tolerated and achieved an adequate urea reduction rate. We did not observe any pyrogenic reactions whereas data on chronic on-line HDF reported an incidence at 0.04% [47]. A low incidence of intradialytic hypotensive events was also repertoried (13.3%) lower than recently reported (18.7%) (47). However, we may not state that OL-IHDF lead to a better hemodynamic tolerance than other modalities especially continuous therapies since this study was only observational. Discussion Indeed, online therapy itself can exacerbate oxidative stress production through leuko- cytes activation induced by dialysis and substitution fluids. Herein, we found that OL-IHDF did not alter superoxide anion production by leukocytes either basal or after stimulation by PMA. Our AKI patients treated by RRT have an increased levels of AOPPs [37–39], varying from 35 to 120 μmol/L as previously reported by Du et al. [39] but lower than those observed by Lentini et al. [38]. We observed that OL-IHDF provided a slight but still significant decrease in AOPP plasma concentra- tions. Of note, it has been suggested earlier that AOPPs Last, OL-IHDF sessions were not associated with an increased production of VGEF, EGF and MCP-1 in our patients. Enhanced production of growth factors after endothelial activation has been reported in critical Fig. 4 Mean plasmatic concentrations of Endothelial Growth factor (EGF), Vascular Endothelial Growth factor (VEGF), Macrophage Chemoattractive protein 1 (MCP-1) before and after OL-IHDF sessions. Values are shown as mean and standard deviation Fig. 4 Mean plasmatic concentrations of Endothelial Growth factor (EGF), Vascular Endothelial Growth factor (VEGF), Macrophage Chemoattractive protein 1 (MCP-1) before and after OL-IHDF sessions. Values are shown as mean and standard deviation Klouche et al. BMC Nephrology (2017) 18:371 Page 7 of 8 Page 7 of 8 conditions especially sepsis-related AKI leading to in- creased capillary permeability, systemic vasodilatation and multi-organ failure [44, 45]. Chancharoenthana W [46] reported however that OL-HDF provided a signifi- cant removal of VEGF and was associated with better renal outcome as compared to high-flux hemodialysis. Acknowledgements None. Acknowledgements None. Author details 1 1Intensive Care Medicine Department, University of Montpellier Lapeyronie Hospital, 371, Av Doyen Gaston Giraud, 34295 Montpellier, France. 2Departments of Biochemistry, University of Montpellier, INSERM U1046, CNRS UMR 9214, 34295 Montpellier cedex 5, France. 3Departments of Medical statistics, University of Montpellier, INSERM U1046, CNRS UMR 9214, 34295 Montpellier cedex 5, France. 4Lapeyronie University Hospital. PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, 34295 Montpellier cedex 5, France. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, et al. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA. 2005;294:813–8. 2. Vinsonneau C, Camus C, Combes A, Costa de Beauregard MA, Klouche K, Boulain T, et al. Continuous venovenous haemodiafiltration versus intermittent haemodialysis for acute renal failure in patients with multiple-organ dysfunction syndrome: a multicentre randomised trial. Lancet. 2006;368:379–85. 3. Mehta RL, McDonald B, Gabbai FB, Pahl M, Pascual MT, Farkas A, et al. A randomized clinical trial of continuous versus intermittent dialysis for acute renal failure. Kidney Int. 2001;60:1154–63. Authors’ contributions KK, LA and JPC designed the study. KK, LA, AMD, MM, VB, NB, DD, JPC analyzed and interpreted the data. AMD, MM and JPC performed the biological sample analysis and were contributor in writing the manuscript. AJ and MCP made the statistical analysis. All authors participated in drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript. KK, LA and JPC designed the study. KK, LA, AMD, MM, VB, NB, DD, JPC analyzed and interpreted the data. AMD, MM and JPC performed the biological sample analysis and were contributor in writing the manuscript. AJ and MCP made the statistical analysis. All authors participated in drafting the article or revising it critically for important intellectual content. All authors read and approved the final manuscript. Ethics approval and consent to participate pp p p This observational prospective study was carried out at the Medical ICU of Lapeyronie University Hospital at Montpellier and was approved by the Ethics Research Committee of our hospital; PHRC régional: N° 2006-A00510–51. A written informed consent was obtained from the patients. If the patient has not the capability to give consent, a written informed consent was obtained from its closest relative. All the patients confirmed their consent by writing later on. We must acknowledge some limitations to the study. First, our work shares the limitations of single-center stud- ies. Our unit is indeed familiar with online therapies which are not the general rule in ICU settings. Second, the num- ber of patients included may be considered small but a high number of OL-HDF sessions were analyzed and se- cure our observations. Third, our RRT patients exhibited a cellular reactivity as reflected by inflammatory and oxida- tive stress parameters with a medium intensity thanks to the biocompatibility membranes. An evaluation of more sensitive biomarkers like isoprostanes may evaluate more thoroughly oxidative stress. Nevertheless, most of the tested parameters were not modified during the on-line sessions performed, a result that represents our primary hypothesis in this work. Last, outcome was not studied in this study since our concern was focused on feasibility and potential risk of this technique in critically ill patients. Consent for publication Not applicable. Consent for publication Not applicable. J . 005; 9 :8 3 8. 2. Vinsonneau C, Camus C, Combes A, Costa de Beauregard MA, Klouche K, Boulain T, et al. Continuous venovenous haemodiafiltration versus intermittent haemodialysis for acute renal failure in patients with multiple-organ dysfunction syndrome: a multicentre randomised trial. Lancet. 2006;368:379–85. 3. Mehta RL, McDonald B, Gabbai FB, Pahl M, Pascual MT, Farkas A, et al. A randomized clinical trial of continuous versus intermittent dialysis for acute renal failure. Kidney Int. 2001;60:1154–63. 4. Saudan P, Niederberger M, De Seigneux S, Romand J, Pugin J, Perneger T, et al. Adding a dialysis dose to continuous hemofiltration increases survival in patients with acute renal failure. Kidney Int. 2006;70:1312–7. 5. Marshall MR, Ma T, Galler D, Rankin APN, Williams AB. Sustained low-efficiency daily diafiltration (SLEDD-f) for critically ill patients requiring renal replacement therapy: towards an adequate therapy. Nephrol Dial Transplant. 2004;19:877–84. 6. Abe M, Okada K, Suzuki M, Nagura C, Ishihara Y, Fujii Y, et al. Comparison of sustained hemodiafiltration with continuous venovenous hemodiafiltration for the treatment of critically ill patients with acute kidney injury. Artif Organs. 2010;34:331–8. Conclusions Received: 1 March 2017 Accepted: 8 December 2017 Received: 1 March 2017 Accepted: 8 December 2017 Abbreviations AKI A kid 4. Saudan P, Niederberger M, De Seigneux S, Romand J, Pugin J, Perneger T, et al. Adding a dialysis dose to continuous hemofiltration increases survival in patients with acute renal failure. Kidney Int. 2006;70:1312–7. 5. Marshall MR, Ma T, Galler D, Rankin APN, Williams AB. Sustained low-efficiency daily diafiltration (SLEDD-f) for critically ill patients requiring renal replacement therapy: towards an adequate therapy. Nephrol Dial Transplant. 2004;19:877–84. 6. Abe M, Okada K, Suzuki M, Nagura C, Ishihara Y, Fujii Y, et al. Comparison of sustained hemodiafiltration with continuous venovenous hemodiafiltration for the treatment of critically ill patients with acute kidney injury. Artif Organs. 2010;34:331–8. Page 8 of 8 Page 8 of 8 Klouche et al. BMC Nephrology (2017) 18:371 7. Kron J, Kron S, Wenkel R, Schuhmacher H-U, Thieme U, Leimbach T, et al. Extended daily on-line high-volume haemodiafiltration in septic multiple organ failure: a well-tolerated and feasible procedure. Nephrol Dial Transplant. 2012;27:146–52. 30. Witko-Sarsat V, Friedlander M, Capeillère-Blandin C, Nguyen-Khoa T, Nguyen AT, Zingraff J, et al. Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int. 1996;49:1304–13. 31. Delample D, Durand F, Severac A, Belghith M, Mas E, Michel F, et al. Implication of xanthine oxidase in muscle oxidative stress in COPD patients. Free Radic Res. 2008;42:807–14. 8. Nistor I, Palmer SC, Craig JC, Saglimbene V, Vecchio M, Covic A, Strippoli GF. Convective versus diffusive dialysis therapies for chronic kidney failure: an updated systematic revierw of randomized controlled trials. Am J Kidney Dis. 2014;63:954–67. 32. Canaud B, Barbieri C, Marcelli D, Bellocchio F, Bowry S, Mari F, et al. Optimal convection volume for improving patient outcomes in an international incident dialysis cohort treated with online hemodiafiltration. Kidney Int. 2015;88:1108–16. 9. Nistor I, Palmer SC, Craig JC, Saglimbene V, Vecchio M, Covic A, Strippoli GF. Haemodiafiltration, haemofiltration and haemodialysis for end-stage kidney disease. Cochrane Database Syst Rev. 2015;5:CD006258. 33. Tsuchida K, Takemoto Y, Yamagami S, Edney H, Niwa M, Tsuchiya M, et al. Detection of peptidoglycan and endotoxin in dialysate, using silkworm larvae plasma and limulus amebocyte lysate methods. Nephron. 1997;75:438–43. 10. Morena M, Jaussent A, Chalabi L, Leray-Moragues H, Chenine L, Debure A, Thibaudin D, et al. Treatment tolerance and patient-reported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly. Kidney Int. 2017;17:30040–6. 34. Schindler R, Krautzig S, Lufft V, Lonnemann G, Mahiout A, Marra MN, et al. Abbreviations AKI A kid 2007;18:528–38. 18. Le Gall JR, Lemeshow S, Saulnier F. A new simplified acute physiology score (SAPS II) based on a European/north American multicenter study. JAMA. 1993;270:2957–63. 41. Vaslaki L, Weber C, Mitteregger R, Falkenhagen D. Cytokine induction in patients undergoing regular online hemodiafiltration treatment. Artif Organs. 2000;24:514–8. 19. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonça A, Bruining H, et al. 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Nephrol Dial Transplant. 1998;13(Suppl 5):3–11. 21. Canaud B, Bosc JY, Leray H, Stec F, Argiles A, Leblanc M, et al. On-line haemodiafiltration: state of the art. Nephrol Dial Transplant. 1998;13(Suppl 5):3–11. 44. van der Flier M, van Leeuwen HJ, van Kessel KP, Kimpen JL, Hoepelman AI, Geelen SP. Plasma vascular endothelial growth factor in severe sepsis. Shock. 2005;23:35–8. 22. Canaud B, Bosc JY, Leray H, Stec F. Microbiological purity of dialysate for on-line substitution fluid preparation. Nephrol Dial Transplant. 2000;15(Suppl 2):21–30. 45. Pickkers P, Sprong T, van Eijk L, van der Hoeven H, Smits P, van Deuren M. Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock. 2005;24:508–12. 23. Pegues DA, Oettinger CW, Bland LA, Oliver JC, Arduino MJ, Aguero SM, et al. A prospective study of pyrogenic reactions in hemodialysis patients using bicarbonate dialysis fluids filtered to remove bacteria and endotoxin. J Am Soc Nephrol. 1992;3:1002–7. 46. Chancharoenthana W, Tiranathanagul K, Srisawat N, Susantitaphong P, Leelahavanichkul A, Praditpornsilpa K, et al. Abbreviations AKI A kid Induction of interleukin-1 and interleukin-1 receptor antagonist during contaminated in-vitro dialysis with whole blood. Nephrol Dial Transplant. 1996;11:101–8. 11. Klouche K, Amigues L, Serveaux-Delous M, Machado S, Delabre J-P, Laydet E, et al. Implementing on-line hemodiafiltration as a renal replacement therapy for ICU acute renal failure: a single-center report of feasibility, safety and hemodynamic tolerance over a seven-year period. Blood Purif. 2012;34:10–7. 35. Simmons EM, Himmelfarb J, Sezer MT, Chertow GM, Mehta RL, Paganini EP, et al. Plasma cytokine levels predict mortality in patients with acute renal failure. Kidney Int. 2004;65:1357–65. 12. Pettilä V, Tiula E. Intermittent hemodiafiltration in acute renal failure in critically ill patients. Clin Nephrol. 2001;56:324–31. 36. Chawla LS, Seneff MG, Nelson DR, Williams M, Levy H, Kimmel PL, et al. Elevated plasma concentrations of IL-6 and elevated APACHE II score predict acute kidney injury in patients with severe sepsis. Clin J Am Soc Nephrol. 2007;2:22–30. 13. Laurent I, Adrie C, Vinsonneau C, Cariou A, Chiche J-D, Ohanessian A, et al. High-volume hemofiltration after out-of-hospital cardiac arrest: a randomized study. J Am Coll Cardiol. 2005;46:432–7. 37. Selmeci L, Seres L, Antal M, Lukács J, Regöly-Mérei A, Acsády G. Advanced oxidation protein products (AOPP) for monitoring oxidative stress in critically ill patients: a simple, fast and inexpensive automated technique. Clin Chem Lab Med. 2005;43:294–7. 14. Kawanishi H. On-line hemodiafiltration in critical care. Ther Apher. 2002;6:199–203. 15. Kawanishi H. On-line continuous hemodiafiltration in sepsis. Transfus Apher Sci. 2006;35:265–9. 38. Lentini P, de Cal M, Cruz D, Chronopoulos A, Soni S, Nalesso F, et al. The role of advanced oxidation protein products in intensive care unit patients with acute kidney injury. J Crit Care. 2010;25:605–9. 16. Kumar VA, Yeun JY, Depner TA, Don BR. Extended daily dialysis vs. continuous hemodialysis for ICU patients with acute renal failure: a two-year single center report. Int J Artif Organs. 2004;27:371–9. 39. Du S, Zeng X, Tian J, Ai J, Wan J, He J. Advanced oxidation protein products in predicting acute kidney injury following cardiac surgery. Biomarkers. 2015;20:206–11. 17. Kielstein JT, Kretschmer U, Ernst T, Hafer C, Bahr MJ, Haller H, et al. Efficacy and cardiovascular tolerability of extended dialysis in critically ill patients: a randomized controlled study. Am J Kidney Dis. 2004;43:342–9. 40. Li HY, Hou FF, Zhang X, Chen PY, Liu SX, Feng JX, et al. Advanced oxidation protein products accelerate renal fibrosis in a remnant kidney model. J Am Soc Nephrol. 47. The VA/NIH Acute Renal Failure Trial Network. Intensity of renal support in critically ill patients with acute kidney injury. N Engl J Med. 2008;359:7–20. Abbreviations AKI A kid Enhanced vascular endothelial growth factor and inflammatory cytokine removal with online hemodiafiltration over high-flux hemodialysis in sepsis-related acute kidney injury patients. Ther Apher Dial. 2013;17:557–63. 24. NKF-DOQI clinical practice guidelines for hemodialysis adequacy. Am J Kidney Dis. 1997;30:suppl2 S38-S42. 24. NKF-DOQI clinical practice guidelines for hemodialysis adequacy. Am J Kidney Dis. 1997;30:suppl2 S38-S42. 25. Bergström J, Wehle B. No change in corrected beta 2-microglobulin concentration after cuprophane haemodialysis. Lancet. 1987;1:628–9. 25. Bergström J, Wehle B. No change in corrected beta 2-microglobulin concentration after cuprophane haemodialysis. Lancet. 1987;1:628–9. 25. Bergström J, Wehle B. No change in corrected beta 2-microglobulin concentration after cuprophane haemodialysis. Lancet. 1987;1:628–9. 26. Daugirdas JT. Second generation logarithmic estimates of single-pool variable volume Kt/V: an analysis of error. J Am Soc Nephrol. 1993;4:1205–13. 47. The VA/NIH Acute Renal Failure Trial Network. Intensity of renal support in critically ill patients with acute kidney injury. N Engl J Med. 2008;359:7–20. 27. Badiou S, Dupuy AM, Descomps B, Cristolead JP. Comparison between the enzymatic vitros assay for creatinine determination and three other methods adapted on the Olympus analyzer. J Clin Lab Anal. 2003;17:235–40. 28. Ventura E, Durant R, Jaussent A, Picot M-C, Morena M, Badiou S, et al. Homocysteine and inflammation as main determinants of oxidative stress in the elderly. Free Radic Biol Med. 2009;46:737–44. 29. Dupuy AM, Lehmann S, Cristol JP. Protein biochip systems for the clinical laboratory. Clin Chem Lab Med. 2005;43:1291–302.
https://openalex.org/W4220990705	https://www.frontiersin.org/articles/10.3389/fpsyt.2022.843984/pdf	English	null	Prevalence and Associated Factors of Common Mental Disorders Among Pregnant Mothers in Rural Eastern Ethiopia	Frontiers in psychiatry	2,022	cc-by	8,568	Prevalence and Associated Factors of Common Mental Disorders Among Pregnant Mothers in Rural Eastern Ethiopia Dawit Tamiru1, Tadesse Misgana2, Mandaras Tariku2, Dejene Tesfaye2, Daniel Alemu2, Adisu Birhanu Weldesenbet3, Berhe Gebremichael3 and Merga Dheresa4 1 Department of Midwifery, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia, 2 Department of Psychiatry, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia, 3 School of Public Health, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia, 4 School of Nursing and Midwifery, College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia Background: Antenatal common mental disorder is a signiﬁcant public health issue, especially in low- and middle-income countries with an extensive treatment gap. Common mental disorders have multifaceted implications on maternal and fetal health outcomes during pregnancy with long-running economic and social sequels. This study aimed to determine the prevalence of common mental disorder and associated factors among pregnant mothers in eastern Ethiopia, Kersa and Haramaya Health, and Demographic surveillance sites. ORIGINAL RESEARCH published: 28 March 2022 doi: 10.3389/fpsyt.2022.843984 Keywords: common mental disorder, pregnancy, antenatal care, gestational age, rural Ethiopia Edited by: Suraj Bahadur Thapa, University of Oslo, Norway Edited by: Suraj Bahadur Thapa, University of Oslo, Norway Edited by: Suraj Bahadur Thapa, University of Oslo, Norway Suraj Bahadur Thapa, University of Oslo, Norway Reviewed by: Eero Kajantie, National Institute for Health and Welfare, Finland Saeeda Paruk, University of KwaZulu-Natal, South Africa Methods: A community-based cross-sectional study was conducted in Kersa and Haramaya health and demographic surveillance sites from January 30 to April 30, 2021. World Health Organization Self-Reporting Questionnaire (SRQ-20) was used to measure common mental disorder among 1,015 randomly selected pregnant women. Data were collected face-to-face using open data kit software. Logistic regression was ﬁtted to identify factors associated with common mental disorders. Correspondence: Dawit Tamiru dawittamru5@gmail.com Results: The overall prevalence of common mental disorders (SRQ > 6) among pregnant women was 37.5% (95% CI: 34.5, 40.5). Current substance use (AOR = 1.99, 95% CI 1.37, 2.88), intimate partner violence (AOR = 2.67, 95% CI 2.02, 3.53), null parity (AOR = 3.10, 95% CI 1.65, 5.84), gestational age [ﬁrst trimester (AOR = 2.22, 95% CI 1.01, 4.93) and third trimester (AOR = 1.74, 95% CI 1.31, 2.31)], history of abortion (AOR = 2.03, 95% CI 1.27, 3.24), and absence of antenatal care follow-up (AOR = 1.43, 95% CI 1.08, 1.89) were signiﬁcantly associated with common mental disorder during pregnancy. Specialty section: This article was submitted to Anxiety and Stress Disorders, a section of the journal Frontiers in Psychiatry Received: 27 December 2021 Accepted: 01 March 2022 Received: 27 December 2021 Accepted: 01 March 2022 Published: 28 March 2022 Keywords: common mental disorder, pregnancy, antenatal care, gestational age, rural Ethiopia INTRODUCTION include lack of social support, history of violence, pressure to have a child, history of mental illness, unplanned pregnancy, and adverse life events, complications in past or index pregnancy, and pregnancy loss (13, 19, 20). Globally more than one billion people are aﬀected by mental or addictive disorders, causing 19% of all years lived with disability (YLD) (1). Common mental disorders (CMDs) refer to a range of non-psychotic mental health conditions such as anxiety, depressive and somatoform disorders (1–3). The conditions are rampant in the population hence why they are deemed common and impact the mood or feelings of aﬀected person. It is also common people experiencing more of these conditions simultaneously (3, 4). In LMICs, the treatment gap for mental health conditions is estimated to be over 80% (21). The high mental health burden among pregnant women accentuates the need for mental health services within the maternal health care system in low-income settings (22). The World Health Organization (23) endorsed maternal mental health care be integrated into primary health care for improved access (24, 25). Integration of CMD screening with antenatal care has also been mentioned as an eﬀective strategy to realize WHO’s Mental Health Gap Action Program (MhGAP), which will lead to better maternal and child health outcomes (23, 26). Although Ethiopia has recently integrated mental health services into the primary healthcare system, the implementation faces many barriers (27, 28). The number of individuals with a CMD is increasing globally, mainly in lower-income countries, because of the increment in population and more people living to the age when depression and anxiety are common (2, 5). Globally, it is estimated that 3.6% (264 million) and 4.4% (322 million) and of the world’s population suﬀer from anxiety and depressive disorders, respectively. Estimates vary across regions, from a low of 2.6% in males in the western Paciﬁc region to 7.7% among females in the Americas. In Ethiopia, 3.3% of the population suﬀers from anxiety disorders, while 4.7% face depression causing 3.5 and 10.1% of total YLD, respectively (2). The disorders are more common among females than males, particularly in pregnant women (2, 5, 6). Ethiopia is galvanizing eﬀorts to achieve continuum Growth and Transformation Plan II (GTP II) and Sustainable Development Goals (SDG). The need for interdisciplinary and inter-sectorial solutions to reach maternal and child health, as well as mental health goals, is highlighted (29). Abbreviations: ANC, antenatal care; AOR, adjusted odds ratio; ASSIST, Alcohol, Smoking, and Substance Involvement Screening Test; CI, conﬁdence interval; CMD, common mental disorder; COR, crude odds ratio; GTP, Growth and Transformation Plan; HDSS, health and demographic surveillance sites; IPV, intimate partner violence; MhGAP, Mental Health Gap Action Program; ODK, open data kit; SDG, sustainable development goals; SRQ-20, Self-Reporting Questionnaire; VIF, variance inﬂation factor; YLD, years lived with disability. INTRODUCTION Attitudes and professional treatment-seeking behavior for mental health problems in Ethiopia are generally low; doubt of discrimination for having a mental disorder, depending in non- formal aid source, and negative beliefs toward mental health services were the most commonly cited barriers (30, 31). Despite eﬀective treatments are available, studies reported that many aﬀected people do not seek professional help. It was indicated that less than 35% of adults with identiﬁable mental problems looked for professional help, in which more than 80% visited informal help sources (31, 32). Modern mental health services centers are concentrated in urban settings, which are not accessible to the majority of the population. Mental health in pregnant women is a signiﬁcant public health matter, especially in low- and middle-income countries (LMICs) (7, 8). Although pregnancy is generally considered a period of contentment, the state increases vulnerability to psychiatric conditions like common mental disorders (9). Even though symptom scores for depression in studies are elevated in antepartum compared to after childbirth, postnatal depression has been a focus of concern, while depression during pregnancy has been relatively neglected (10). The prevalence of CMDs among pregnant women ranges from 1 to 37%, with an increasing threshold, mainly in low and middle-income countries (11–13). However, common mental disorders during pregnancy are often left under diagnosed and untreated because conditions are usually ascribed to typical experiences of pregnancy (14). Describing the correlates of common mental disorders in pregnancy and childbirth is a crucial step in preventing and treating of the associated complications in lower-income countries. With a largely rural population, low mental health care service, and high maternal, infant, and neonatal mortality rates, Ethiopia lacks reliable data on the burden and eﬀects of mental disorders (33, 34). Demographic and health surveys in the country generally have not included maternal mental health status as expected health outcomes and national data are rarely available; thus, the exact extent of the broader impact remains unclear and likely under-estimated (34, 35). Untreated CMD during pregnancy can lead to the negative child, mother, and family conditions, including poor antenatal clinic follow-up, poor nutrition and self-care, higher substance abuse, suicidal ideation, and thoughts of hurting the fetus, development of depression after childbirth, neglect of the child, and family breakdown (15, 16). INTRODUCTION CMDs during the antenatal period have also been associated with adverse pregnancy and child well-being eﬀects, including lower birth weight, prematurity, neonatal mortality, and deﬁcient child nutrition status, behavioral, emotional, and cognitive development (17, 18). When resources are available, data are predominantly from health facility surveys, whereas a high proportion of pregnant women remain at home without seeking maternal health care service (34, 36). Therefore, this study aimed to determine the magnitude and determinants of common mental disorders during pregnancy in the eastern region of Ethiopia. Multiple constituents and psychosocial determinants have been distinguished as contributing to common mental disorders during pregnancy. The most common ascertained circumstances Citation: Tamiru D, Misgana T, Tariku M, Tesfaye D, Alemu D, Weldesenbet AB, Gebremichael B and Dheresa M (2022) Prevalence and Associated Factors of Common Mental Disorders Among Pregnant Mothers in Rural Eastern Ethiopia. Front. Psychiatry 13:843984. doi: 10.3389/fpsyt.2022.843984 Tamiru D, Misgana T, Tariku M, Tesfaye D, Alemu D, Weldesenbet AB, Gebremichael B and Dheresa M (2022) Prevalence and Associated Factors of Common Mental Disorders Among Pregnant Mothers in Rural Eastern Ethiopia. Front. Psychiatry 13:843984. doi: 10.3389/fpsyt.2022.843984 Conclusion: Common mental disorders are prevalent among pregnant women in the study area with signiﬁcant correlates. Administration of regular screening programs for maternal mental health conditions in rural, low-income communities, integrating into primary health care settings is imperative to reduce the risk. March 2022 \| Volume 13 \| Article 843984 1 Frontiers in Psychiatry \| www.frontiersin.org Common Mental Disorder Pregnant Ethiopia Tamiru et al. Frontiers in Psychiatry \| www.frontiersin.org MATERIALS AND METHODS Study Setting, Design, and Period A community-based cross-sectional study was conducted in Kersa and Haramaya Health and Demographic March 2022 \| Volume 13 \| Article 843984 Frontiers in Psychiatry \| www.frontiersin.org 2 Common Mental Disorder Pregnant Ethiopia Tamiru et al. Surveillance Sites (HDSS) from January 30 to April 30, 2021. a tool adapted and modiﬁed from the Ethiopian Demographic and Health Survey (EDHS) of 2016 (40). Alcohol, Smoking, and Substance Involvement Screening Test (41) comprising eight questions or items covering ten substances, was used assessing substance use behavior of pregnant women. Khat was included under the stimulant category, and shisha was incorporated under tobacco. The experience of intimate partner violence (IPV) was assessed using the WHO multi-country study questionnaire constituting psychological, physical, and sexually violent acts, often accompanied by controlling behavior where a single positive answer indicated the presence of violence (42). Kersa and Haramaya Health and Demographic Surveillance Site is one of the seven HDSS sites in the Ethiopian universities intended to reﬂect the countries’ health and demography. Kersa and Haramaya Demographic Surveillance and Health Research Center (KDS-HRC) was established in 2007. Kersa HDSS is located in Kersa District, Oromia Regional State, eastern Ethiopia. There are 35 rural sub-districts (Kebeles) and three small-town kebeles. The Kersa HDSS covers 24 of the 38 kebeles. Most inhabitants are farmers, with a small numbers working in trade, public service, or casual laborers. Haramaya Woreda (district) is located in the East Hararghe Zone of Oromia Regional State. Haramaya Woreda has thirty- three rural and two urban kebeles (sub-districts). Haramaya HDSS covers 12 rural kebeles. Data Quality Control To maintain the consistency of the data collection tool, the questionnaire was prepared in English language and translated to local languages, Afaan Oromo and Amharic languages, and vice versa. To evaluate the acceptability and applicability of the procedures and tools, pre-testing was done on 5% of the sample size 1 week before the actual data collection in the kebeles that are not included in the samples from each site. To keep the completeness and consistency of the questionnaire, data collectors were closely supervised during the data collection process by the supervisors and investigators. Data Collectors and Data Collection Procedure Twenty data collectors who have at least a B.Sc. degree in health sciences and working in Kersa and Haramaya Demographic Surveillance and Health Research Center (KDS-HRC) collected the data for this study. Besides, they were supervised by six M.Sc. holders. In addition, an intensive 3 days training was provided to data collectors and supervisors. Rural eastern Ethiopia is where the majority of Khat (Catha edulis), psychoactive substances, in the country produced and exported abroad (37). Study Population All pregnant women living in Kersa and Haramaya Health and Demographic Surveillance Sites (HDSS) were the source population. All registered pregnant women were enrolled in the study irrespective of their age and pregnancy trimester. Pregnant women were located from Health and Demographic surveillance sites registration server and interviewed in their homes. Pregnant women who could not communicate due to serious medical and psychiatric illnesses were excluded from the study. The data were collected by face-to-face interview using structured and semi-structured questionnaires prepared in ODK collect form, completed data were sent directly to the server. Pregnant women were interviewed in separate rooms in their home environment or inside their compounds during working hours 7 days of the week. Sample Size Determination The minimum sample size required for this study was determined using a single population proportion formula with assumptions of conﬁdence level at 95% = 1.96, a margin of error (d) = 0.04, and a reasonable proportion of common mental disorder (P = 0.267) (38). After incorporating a design eﬀect of 2 and a 10% non- response rate, the maximum sample size calculated for this study was 1,034. The calculated sample size was proportionally allocated based on the conﬁrmed number of pregnant women in selected Kebeles (clusters); the study participants were then determined using simple random sampling. Frontiers in Psychiatry \| www.frontiersin.org Study Variables and Measurements g y After data ﬁles were downloaded from the server, the data set were exported to Stata version 14.0 for cleaning, coding, and analysis. Categorical variables were described using simple frequencies and percentages. Continuous variables were deﬁned using mean or median and inter-quartile ranges depending on their distribution and standard deviation. Bivariate analysis was done using binary logistic regression to see the association between each independent variable and common mental disorder. The ﬁnal multivariate analysis model included all variables from the bivariate analysis with p ≤0.2 to control all possible confounders. The direction and strength of statistical association were measured by odds ratio with 95% CI. Finally, a P-value less than 0.05 were considered to declare the presence of a statistically signiﬁcant association. The Hosmer–Lemeshow statistic and Omnibus test tested the model goodness of ﬁt. The y Data was collected using Open Data Kit (ODK) tools. Observations were uploaded to the ODK aggregate server. The data collection instrument included socio-demographic and economic characteristics, reproductive, obstetric and gynecologic conditions, pre-existing medical and psychiatric conditions, substance use, intimate partner violence, and common mental disorder. Self-Reporting Questionnaire (SRQ-20) was used to measure common antenatal mental disorders. The SRQ-20 is composed of twenty yes/no items asking about depressive, anxiety, panic, and somatic symptoms during the preceding 30 days. For this study, the total score was dichotomized (SRQ-20 < 6 versus SRQ ≥6), with high scores indicating a high level of CMD (39). Data on household economic status were collected using March 2022 \| Volume 13 \| Article 843984 Frontiers in Psychiatry \| www.frontiersin.org 3 Common Mental Disorder Pregnant Ethiopia Tamiru et al. Psychosocial and Substance Use Behavior of Participants multi co-linearity test was also carried out to see the correlation between independent variables using VIF, standard error (SE), and tolerance tests. Based on the assessment made on substance abuse using ASSIST, about 207 (20.39%) and 158 (15.57%) of the participants reported as they were used any type of substance at least once in their lifetime and the last 3 months, respectively (Figure 1). Concerning psycho-social factors, 493 (48.57%) of women reported that they experienced any act of physical, sexual, or emotional (psychological) violence or any combination of the three by an intimate partner during their current pregnancy. Socio-Demographic Characteristics Socio Demographic Characteristics From a total of 1,034 participants selected for the study, 1,015 consented to participate in the study yielding a response rate of 98.2%. Among the participants, 676 (66.6%) were between the age of 20–35 years with a mean of 30.1 years (SD = 8.5). The majority of the study participants, 982 (96.75%) were married in their current relationship, 1,012 (99.7%) were Muslim followers, and 1,010 (99.51%) were Oromo in ethnicity. About 772 (76.06%) of the participants neither read nor write and 900 (88.67) were housewives. About 388 (38.2%) of the participants were on a lesser quintile of wealth index (Table 1). TABLE 1 \| Socio-demographic and economic characteristics of pregnant women. Variables Category Frequency Percentage (%) Age <20 94 9.26 20–35 676 66.60 >35 245 24.14 Marital status Married 982 96.75 Othersa 33 3.25 Religion Muslim 1,012 99.70 Orthodox 3 0.30 Ethnicity Oromo 1,010 99.51 Amhara 5 0.49 Educational status High school and above 218 21.48 Can read and write 25 2.46 Neither read nor write 772 76.06 Occupation Farmer 47 4.63 Housewife 900 88.67 Othersb 68 6.70 Wealth index First quintile 388 38.23 Second quintile 375 36.95 Third quintile 252 24.83 aSingle, divorced, and widowed. bPetty trader, student, retired, and unemployed. TABLE 1 \| Socio-demographic and economic characteristics of pregnant women. RESULTS On bivariate analysis, age, wealth index, history of mental illness, history of chronic medical illness, current substance use, intimate partner violence, parity, gestational age, history of abortion, ANC follow up, history of the gynecological problem and gynecological operation had p-value score of less than 0.2 and entered into the multivariable analysis. In the multivariable logistic regression analysis model, however, only current substance Ethics Approval and Consent to Participate Ethical approval was obtained from Haramaya University, College of Health and Medical Sciences, Institutional Health Research Ethics Review Committee (IHRERC) and submitted to the respective health bureau of each study site. The study was conducted under the Declaration of Helsinki and, participants were informed that participation in the study is voluntary; had the right to withdraw or refuse to participate in the study at any time. Throughout the study period, the conﬁdentiality of the data was strictly followed; participants were interviewed in separate rooms in their home environment. Before starting data collection, information sheets are read, and written and signed consent was obtained from each participant. Pregnant women who screened positive for the common mental disorder were linked to health facilities for assessment and interventions. Prevalence of Common Mental Disorders The overall prevalence of common mental disorder (SRQ > 6) among pregnant mother was found to be 37.5% (95% CI: 34.5, 40.5). About 180 (17.73%) of the women had no CMD symptoms and 454 (44.73%) had low CMD symptoms. Regarding the distribution of the symptoms, poor appetite (n = 350) was the most complained symptom followed by sleep disturbance (n = 285). The median SRQ-20 score was 2 (25th centile 1, 75th centile 8). Frontiers in Psychiatry \| www.frontiersin.org Clinical, Reproductive, Obstetric, and Gynecological Characteristics Among the study participants, 26 (2.56%) had a previous history of mental illness with a mean duration of 2.7 years (SD = 1.37). Of them, 10 (38.46%) were on the treatment. Twenty-three (2.27%) had a chronic medical illness, the majority (43.5%) being hypertension. The majority of the study participants were grand multipara, woman who has had ≥5 births (live or stillborn) ≥28 weeks of gestation, (46.50%). Over half, 52.12% of the participants were in the third trimester and the mean gestational age of pregnant mothers was 28.91 (SD = 6.63) weeks. About 98 (9.66%) of the participants had a previous history of abortion while 48.08% have Antenatal Care (ANC) follow-up. About 70 (6.9%) of the participants had a history of a gynecological problem, of this tumor (41.43%) was the most prevalent one. The median age of pregnancy interval and marriage was 1.7 years (IQR = 0.56) and 17.7 years (IQR = 2.12), respectively (Table 2). March 2022 \| Volume 13 \| Article 843984 Frontiers in Psychiatry \| www.frontiersin.org 4 Common Mental Disorder Pregnant Ethiopia Tamiru et al. use, intimate partner violence, parity, gestational age, history of abortion, and ANC follow-up were signiﬁcantly associated with common mental disorders among pregnant women at a p-value of <0.05. pregnant on the second trimester, respectively. Women with a history of abortion are two times (AOR = 2.03, 95% CI 1.27, 3.24) more likely to experience antenatal CMD. Pregnant women who didn’t have an ANC follow-up for their current pregnancy were 1.43 times (AOR = 1.43, 95% CI 1.08, 1.89) more likely to develop CMD as compared to those who had an ANC visit (Table 3). Pregnant women who use any type of substance in the past 3 months were two times (AOR = 1.99, 95% CI 1.37, 2.88) more likely to have CMD as compared to those women who didn’t use any types of substance. Those pregnant women who reported that they experienced intimate partner violence were 2.67 times (AOR = 2.67, 95% CI 2.02, 3.53) more likely to have CMD as compared to those women who didn’t report any act of physical, sexual, or psychological violence or any combination of the three by an intimate partner during the current pregnancy. DISCUSSION Almost two in ﬁve (37.5%) pregnant women had a common mental disorder in this study. In addition, current substance use, intimate partner violence, parity, gestational age, history of abortion, and lack of ANC attendance increased the odds of CMD. The odds of developing CMD were three times (AOR = 3.10, 95% CI 1.65, 5.84) higher among nulliparous women as compared with a multiparous woman. Pregnant women who had been in the ﬁrst trimester (AOR = 2.22, 95% CI 1.01, 4.93) and third trimester (AOR = 1.74, 95% CI 1.31, 2.31) were 2.2 times and 1.74 times more likely to have CMD as compared to those The prevalence of 37.5% (95% CI: 34.5, 40.5) for common mental disorder among pregnant women in this study is consistent with other ﬁndings found in Bale, Ethiopia (35.8%) (19), Tanzania (39.5%) (43), South Africa (39%) (44), and Vietnam (37.4%) (45). On the other hand, this result was higher than studies conducted in various regions of Ethiopia and other countries; Butajira (12%) (46), Gondar (23%) (47), and Addis Ababa (24.94%) (48), Brazil (20.2%) (49), and Nigeria (24.5%) (49). Likewise, in one community-based cross-sectional survey done in 20 sites across four countries, the prevalence of CMD was 21% in Vietnam, 33% in Ethiopia, 30% in India, and 30% in Peru (50). This diﬀerence might be due to the high magnitude of substance use, particularly Khat, in the current study setting, which signiﬁcantly contributed to the development of mental illness (51). Additionally, poor utilization of ANC follow-up in the study setting might contribute to high maternal CMD. TABLE 2 \| Medical, reproductive, obstetric, and gynecological characteristics of pregnant women. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION FIGURE 1 \| Description of the types of substance used by pregnant mothers. FIGURE 1 \| Description of the types of substance used by pregnant mothers. of developing antenatal common mental illnesses (59, 78, 79), while others did not ﬁnd a strong association between parity and perinatal mental disorder (80, 81). Khat, as the women beneﬁted from stimulant eﬀects of Khat, including elation and alleviation of their symptoms (64). Pregnant women, who reported intimate partner violence, were more likely to have CMD than those who did not report any acts of physical, sexual, or psychological violence or any combination of the three by an intimate partner during the current pregnancy. A similar ﬁnding was reported from the studies conducted in other areas of Ethiopia, Bale (19) and Butajira (65), Tanzania (66), and Vietnam (67). This might be because IPV can directly aﬀect antenatal mental illnesses (68). First, having experienced partner violence, pregnant women may develop a stress response proﬁle which heightens the neuroendocrine responses (69, 70). The collective eﬀect of these responses can lead to mental illnesses. Secondly, women who had perinatal mental illness are more likely to be exposed to IPV during pregnancy than those who do not (71, 72). Finally, psychiatric disorders and IPV can happen along with structural stressors, such as low socioeconomic status or poor social support (73). Another factor associated with antenatal CMD is gestational age. Pregnant women were more likely to experience CMD during the ﬁrst and third trimester compared to those in their second trimester. Previous longitudinal studies conducted in England (82), China (83), and Lithuania (84) found a similar trend. Thus, the increased prevalence of CMD in the third trimester might be linked with the proximity of giving birth. Additionally, these results might be induced by hormonal changes in the ﬁrst trimester (85). Furthermore, in our study, women with past pregnancy complications or abortion had a higher odds of antenatal CMD. This is similar to the ﬁndings from various other studies (86–88). This might be because women who had a previous abortion or pregnancy complications may develop diﬀerent psychosocial problems, worrying about possible complications in the index pregnancy. This may induce fear, uncertainty, worry, and anxiety, further enhancing the risk of depression during the subsequent pregnancies (89, 90). Nulliparous women were more likely to have CMD as compared to multiparous women. Frontiers in Psychiatry \| www.frontiersin.org DISCUSSION Variables Category Frequency Percentage (%) History of mental illness Yes 26 2.56 No 989 97.44 History of chronic medical illness Yes 23 2.27 No 992 97.73 Parity Nulliparous 52 5.12 Multipara (has had two or more births ≥28 weeks of gestation) 491 48.37 Grand multipara (has had ≥5 births ≥28 weeks of gestation) 472 46.50 Gestational age (in a week) First trimester 31 3.05 Second trimester 455 44.83 Third trimester 529 52.12 History of abortion Yes 98 9.66 No 917 90.34 ANC follow up Yes 488 48.08 No 527 51.92 Interval between last delivery and LNMP <24 months 754 92.29 24–59 55 6.73 >59 8 0.98 History of gynecological problem Yes 70 6.90 No 945 93.10 History of gynecological operation Yes 46 4.53 No 969 95.47 TABLE 2 \| Medical, reproductive, obstetric, and gynecological characteristics of pregnant women. The result is, however, lower than studies done in Jamaica (56%) (52), Turkish women (47.6%) (53), Nicaragua (57%) (54), Pakistan (49%) (55), and India (65%) (56). These variations might be due to disparities in measurement tools, and cut-oﬀ points used, Jamaican study used Zung self-rating depression scale (SDS > 50), Edinburgh postnatal depression scale (EDS) and the state and trait anxiety scale (STAI) were used in Turkey, India, and Nicaragua studies while Anxiety and Depression Scale (HADS, 11–21) were used in Pakistan. Conjointly, socio-cultural and economic variations, and geographical settings might also explain the observed diﬀerence. p In congruent with population-based cohort studies conducted in Ethiopia (57), India (58), Jordan (59), and Netherlands (60), pregnant women who use any type of substance were more likely to have CMD compared to those women who did not use any substance in the past 3 months. Mental health and substance use issues have a bidirectional cause-and-eﬀect relationship. A pregnant woman using a substance often has complex social and mental health problems (51, 61). On the other hand, antenatal anxiety and depression could have a signiﬁcant role in the causal mechanisms of substance use and prenatal stress, leading individuals to poor stress management and behavioral problems, both of which increase the risk of substance use disorder (62, 63). Similarly, a recent cross-sectional study found pregnant women with antenatal distress were more likely to chew March 2022 \| Volume 13 \| Article 843984 5 Tamiru et al. Common Mental Disorder Pregnant Ethiopia FIGURE 1 \| Description of the types of substance used by pregnant mothers. DISCUSSION This ﬁnding was in agreement with studies done in Ethiopia (74), Pakistan (75), Vietnam (76), and Finland (77). This might be because primigravida women do not have any previous delivery experience, may have uncertainty and fear of childbirth, and they don’t have the knowledge and skills to take care of a child, which may heighten the incidence of depression and increase vulnerability to other psychological morbidities. There have been contradictory ﬁndings in studies as some have found multiparous women are at increased risk Consistent with previous studies conducted in Gondar (47) and Debre Markos (74), Ethiopia, pregnant women who did not have ANC follow-up for their current pregnancy were more likely to have CMDs than those who had an ANC visit. The most likely explanation for this association is that antenatal clinic attendance may construct maternal self-esteem and resiliency, increase chance to obtain information about pregnancy preparedness and minimize risk factors, and increase March 2022 \| Volume 13 \| Article 843984 6 Common Mental Disorder Pregnant Ethiopia Tamiru et al. TABLE 3 \| Results of bivariate and multivariate analysis for factors associated with antenatal common mental disorders. DISCUSSION Common Mental Disorder Variables Yes, n (%) No, n (%) Crude OR (95% CI) Adjusted OR (95% CI) Age <20 31 (33.0) 63 (67.0) 1.00 1.00 20–35 251 (37.1) 425 (62.9) 1.20 (0.76–1.89) 1.21 (0.74–1.99) >35 99 (40.4) 146 (59.6) 1.38 (0.84–2.27) 1.20 (0.69–2.10) Wealth index First quantile 154 (39.7) 234 (60.3) 1.34 (0.96–1.87) 1.26 (0.88–1.81) Second quantile 144 (38.4) 231 (61.6) 1.27 (0.91–1.77) 1.27 (0.89–1.82) Third quantile 83 (32.9) 169 (67.1) 1.00 1.00 History of mental illness Yes 14 (53.8) 12 (46.2) 1.98 (0.90–4.32) 1.63 (0.68–3.86) No 367 (37.1) 622 (62.9) 1.00 1.00 History of chronic medical illness Yes 9 (39.1) 14 (60.9) 1.07 (0.46–2.49) 0.74 (0.28–1.91) No 372 (37.5) 620 (62.5) 1.00 1.00 Current use of substance Yes 81 (51.3) 77 (48.7) 1.95 (1.39–2.75) 1.99 (1.37–2.88)* No 300 (35.0) 557 (65.0) 1.00 1.00 Intimate partner violence Yes 244 (49.5) 249 (50.5) 2.75 (2.15–3.58) 2.67 (2.02–3.53)* No 137 (26.5) 385 (73.5) 1.00 1.00 Parity Nulliparous 29 (55.8) 23 (44.2) 2.54 (1.42–4.52) 3.10 (1.65–5.84)*** Multiparous 163 (33.2) 328 (66.8) 1.00 1.00 Grand multiparous 189 (40.0) 283 (60.0) 1.34 (1.03–1.75) 1.21 (0.90–1.62) Gestational age (in week) First trimester 16 (51.6) 15 (48.4) 2.35 (1.12–4.84) 2.22 (1.01–4.93)* Second trimester 143 (31.4) 312 (68.6) 1.00 1.00 Third trimester 222 (42.0) 307 (58.0) 1.58 (1.21–2.05) 1.74 (1.31–2.31)* History of abortion Yes 50 (51.0) 48 (49.0) 1.84 (1.21–2.80) 2.03 (1.27–3.24) No 331 (36.1) 586 (63.9) 1.00 1.00 ANC follow up Yes 154 (31.6) 334 (68.4) 1.00 1.00 No 227 (43.1) 300 (56.9) 1.64 (1.27–2.12) 1.43 (1.08–1.89)** History of gynecological problem Yes 36 (51.4) 34 (48.6) 1.84 (1.13–2.99) 1.69 (0.99–2.89) No 345 (36.5) 600 (63.5) 1.00 1.00 History of gynecological operation Yes 23 (50.0) 23 (50.0) 1.71 (0.94–3.09) 1.57 (0.81–3.01) No 358 (36.9) 611 (63.1) 1.00 1.00 P-value < 0.05; P-value < 0.01; *P-value < 0.001. TABLE 3 \| Results of bivariate and multivariate analysis for factors associated with antenatal common mental disorders. P-value < 0.05; P-value < 0.01; *P-value < 0.001. knowledge about complications during pregnancy, including mental illnesses (91, 92). follow-up were signiﬁcantly associated with antenatal CMD. Administration of regular screening programs for maternal mental health conditions in rural, low-income communities, integrating into primary health care settings is imperative to reduce the risk. This study also underscores the importance of routine maternal mental health assessment and intervention when pregnant women present to maternity services. DISCUSSION Pregnant women in this setting should be informed that substance use in pregnancy should be avoided or reduced. CONCLUSION Pregnancy is a time of increased susceptibility for the occurrence of common mental disorder. Almost two in every ﬁve pregnant women suﬀered from antenatal CMD in the study area. Currently, using a substance in early and late pregnancy, experiencing intimate partner violence, null parity, having a history of abortion or pregnancy complications, and no ANC Limitations of the Study This study has some limitations that should be kept in mind when interpreting the results. SRQ-20 is a screening but not diagnostic tool. Beside, this report did not evaluate biological determinants of common mental disorders, such as thyroid dysfunction and immunity. Additionally, the study did not diﬀerentiate women with new-onset mental disorders from those with a pre-existing conditions. Lastly, since we recruited multiple data collectors, there may be interviewer bias. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Frontiers in Psychiatry \| www.frontiersin.org REFERENCES 16. Pagel MD, Smilkstein G, Regen H, Montano D. 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Yitbarek K, Birhanu Z, Tucho GT, Anand S, Agenagnew L, Ahmed G, et al. Barriers and facilitators for implementing mental health services into the Ethiopian health extension program: a qualitative study. Risk Manag Health Policy. (2021) 14:1199–210. doi: 10.2147/RMHP.S298190 14. Whitton A, Warner R, Appleby L. The pathway to care in post-natal depression: women’s attitudes to post-natal depression and its treatment. Br J Gen Pract. (1996) 46:427–8. 28. Sudhakar M, Woldie M, Yitbarek K, Abamecha F, Tamirat A. Primary care systems proﬁles and performance (PRIMASYS): Ethiopian case study. Alliance Heal Policy Syst Res. ACKNOWLEDGMENTS All authors contributed to the conception of the study, organized the data collection process, equally contributed to data analysis, drafting or revising the article, gave ﬁnal approval of the version to be published, and agreed to be accountable for all aspects of the work. We would like to thank Kersa and Haramaya HDSS for providing opportunity and technical support throughout the study. We are grateful to all mothers who participated in this research. We would also like to acknowledge the Haramaya University for funding and facilitating the research, and the data collectors. FUNDING and Medical Sciences, Institutional Health Research Ethics Review Committee (IHRERC). The patients/participants provided their written informed consent to participate in this study. This study was funded by the Haramaya University. The funding body has no role in the design of the study and collection, analysis, and interpretation of data, and writing the manuscript. ETHICS STATEMENT The studies involving human participants were reviewed and approved by the Haramaya University, College of Health Frontiers in Psychiatry \| www.frontiersin.org March 2022 \| Volume 13 \| Article 843984 7 Common Mental Disorder Pregnant Ethiopia Tamiru et al. 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Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 80. Abujilban SK, Abuidhail J, Al-Modallal H, Hamaideh S, Mosemli O. Predictors of antenatal depression among Jordanian pregnant women in their third trimester. Health Care Women Int. (2014) 35:200–15. doi: 10.1080/07399332. 2013.817411 Copyright © 2022 Tamiru, Misgana, Tariku, Tesfaye, Alemu, Weldesenbet, Gebremichael and Dheresa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 81. Dibaba Y, Fantahun M, Hindin MJ. 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https://openalex.org/W4212837238	https://journals.pnu.edu.ua/index.php/pcss/article/download/5390/6047	Russian	null	Strong correlation effects in vanadium oxide films	Fìzika ì hìmìâ tverdogo tìla	2,022	cc-by	3,066	Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty Strong correlation effects in vanadium oxide films Ternopil Ivan Puluj National Technical University, Ternopil, Ukraine, skorenkyy@tntu.edu.ua Ternopil Ivan Puluj National Technical University, Ternopil, Ukraine, skorenkyy@tntu.edu.ua In the present work the metal-insulator transition in doubly orbitally degenerated model of quasi-two- dimensional material based on V2O3 film, in which a crucial role is played by on-site Coulomb interaction and correlated hopping of electrons, has been investigated. With use of a projection procedure in the Green function method the energy spectrum of electrons has been calculated to model variations of the material properties at the temperature changes, the external pressure application and doping. The obtained expressions for thermodynamic potential and the energy gap widths allow analyzing the possible phase transitions in a system, the dependency of characteristics on the external actions for this strongly correlated material. Keywords: Metal-Insulator Transition, Strong Electron Correlations, Energy Spectrum. Received 25 November 2021; Accepted 24 January 2022. energy levels is required to describe MIT in this compound. Such a model has been studied in [12] within the LDA + DMFT (QMC) approximation. In this work, realistic critical values of the intra-atomic Coulomb interaction U were obtained and the calculated spectrum reproduced the experimental data, in contrast to the simpler LDA method. It could be assumed that the theory of MIT in V2O3 has been completed however, recently intensive research of nanosystems began for the design of novel electronic devices. New results obtained for thin films of transition metals oxides have significantly raised the interest to vanadium oxides, primarily to V2O3 and VO2. In 2010, the inhomogeneity of the metallic phase at submicron level was discovered [13]. At temperature decrease in Cr-doped V2O3 the appearance of metallic domains in the dielectric environment has been observed, and the application of the external pressure was shown to stabilize the homogeneous metallic phase. The effect of structural disorder in V2O3 and VO2 has been studied in [14] by irradiating samples with oxygen ions. At a slight irradiation of V2O3, the insulating phase quickly turns into a metallic one. On the contrary, in VO2 much more intensive (by an order of magnitude) irradiation and the corresponding increase in lattice defects number weakly affect the gradual decrease in the resistance of the insulating sample. This indicates that the electronic ISSN 1729-4428 ISSN 1729-4428 Introduction We should single out the work [17] in which by the method of controlled epitaxial deformation the intermediate electronic and optical properties of thin films of pure and chromium-doped V2O3 have been stabilized in intermediate states between metallic and insulating phases (which is impossible to realize in bulk samples). In-plane lattice constant in this study has been tuned to control the properties of the material. These results open new prospects for the creation of electronic devices based on Mott-Hubbard narrow band systems. Introduction Despite more than half a century of studies of the metal-insulator transition (MIT) and magnetic phase transitions in pure and doped vanadium oxides (see [1] for a review), these compounds attracted much attention recently. After the first experimental research [2], where phase diagram of doped V2O3 was explored, theorists proposed various models and mechanisms to explain the electrical and magnetic properties of these materials [3- 7]. Most of these models took into account the Mott- Hubbard mechanism. Until 2000, the filling of 3d V- orbitals (vanadium) for different phases of vanadium oxides had never been measured by direct methods, although these values were the starting assumptions for the formulation of different theoretical models. MIT temperatures in V2O3 and VO2 are 150 K and 200-300 K respectively, which values are 40 - 20 times lower than the energy gaps, 0.66 eV and 0.3-0.5 eV [8-11]. Recently, completely new properties of these well studied compounds have been discovered and these findings suggest new applications of these systems [3], in particular, as thin film devices with controlled phase transitions. New experiments have shown that the spin of vanadium atoms in V2O3 is equal to one [11] and a model which takes into account the orbital degeneracy of 62 Strong correlation effects in vanadium oxide films correlations in V2O3 are long-range (magnetic ordering) and in VO2 these correlations are local (Mott-Hubbard ones). Note that MIT in VO2 has never been attributed to the Mott-Hubbard mechanism because of the simultaneous structural phase transition. In an earlier experimental work [15] it was shown that in VO2 during MIT a metallic phase appears first and only then a structural phase transition occurs. This can be considered a confirmation of the Mott-Hubbard character of MIT in VO2 and an example proving that ultrafast spectroscopy allows new insights into the complex processes associated with MIT. Along with the experimental work, there were many theoretical studies attempting explanations of the new experimental features of MIT in vanadium oxides. For example, in [16] a modified degenerate Hubbard model is considered, which allows the study of MIT induced by an electric field. The paper shows the possibility of the existence of an intermediate metastable metal phase. I. The model Hamiltonian for vanadium oxide film We start from the partial case of the doubly orbitally degenerate model [18, 19] suitable for description of vanadium d-electron subsystem, which takes into account the essential peculiarities of narrow energy bands 𝐻= (𝐸𝑑−𝜇) ∑ 𝑐𝑖𝛾𝜎 + 𝑐𝑖𝛾𝜎 𝑖𝛾𝜎 + 𝑈∑ 𝑛𝑖𝛾↑𝑛𝑖𝛾↓ 𝑖𝛾 + 𝑈′ ∑ 𝑛𝑖𝛼𝜎𝑛𝑖𝛽𝜎 𝑖𝜎 + (𝑈′ −𝐽𝐻) ∑ 𝑛𝑖𝛼𝜎𝑛𝑖𝛽𝜎 𝑖𝜎 + + ∑ (𝑡𝑖𝑗(𝑛, 𝑢)+𝑡𝑖𝑗 𝑎(𝑛𝑖𝛾𝜎+𝑛𝑗𝛾𝜎)+𝑡𝑖𝑗 𝑏(𝑛𝑖𝛾𝜎+𝑛𝑗𝛾𝜎)) 𝑐𝑖𝛾𝜎 + 𝑐𝑖𝛾𝜎 𝑖𝑗𝛾𝜎 + ∑ ℏ𝜔𝑓(𝑞⃗)𝑏𝑞⃗⃗𝑓 + 𝑏𝑞⃗⃗𝑓 𝑞⃗⃗𝑓 + + 1 2 𝑁𝑉0𝐶𝑢2. (1) (1) V0 is the unit cell volume. Electron-phonon interactions play an important role in dynamics of strongly correlated electron systems, as revealed by studies of Hubbard- Holstein model. The system energy is to be minimized with respect to lattice parameters, thus the lattice elastic energy is to be taken into account as well (the last term in Hamiltonian). The model energy parameters and the corresponding processes are illustrated by fig. 1. Oxygen atoms are not shown, however we note that electron hoppings between vanadium ions in V2O3 lattice are mediated by the oxygen sites, so the hopping parameters implicitly include the overlapping of wave functions on V and O sites and can be affected by the structural disorder substantially. We use the standard notations of the second quantization approximation [20], with c-operators of creation and annihilation of an electron with spin σ at orbital γ (α or β) of a site, electron number operator 𝑛𝑖𝛾𝜎= 𝑐+ 𝑖𝛾𝜎𝑐𝑖𝛾𝜎 and b-operators for creation and destruction of phonons. For the doubly degenerated band, three different electron correlation parameters are to be introduced [18], namely the intra-atomic Hubbard repulsion energy U of two electrons of different spins in the same orbital state, U’ of two electrons of different spins in the different orbital states and the lowest in energy Hund’s doublon with two electrons of same spin in the different orbitals having energy U’-JH, where JH is Hund’s rule coupling. Band hopping integral is concentration-dependent in consequence of taking into account the correlated hopping of electrons and two additional mechanisms of correlated hopping which are peculiar to doubly degenerated systems [18]. In distinction from the previously used models of electronic subsystem with orbital degeneracy, here we couple the electronic subsystem to the phonon one with strain- dependent hopping amplitude Fig. 1. Two-dimensional lattice of V atoms with possible electronic configurations of a site and different hopping amplitudes, dependent on the site occupation. Fig. 1. 2.1. Energy spectra for AFI and PM phases 2.1. Energy spectra for AFI and PM phases For calculation of the single electron Green function we use the standard approach of equation of motion For calculation of the single electron Green function we use the standard approach of equation of motion 𝐸⟨⟨𝑐𝑝𝛾′↑ + ⟩⟩= 𝛿𝑝𝑝′ 2𝜋+ ⟨⟨𝑐𝑝′↑ + ⟩⟩ (7) (7) in which a variant of the projection method [20] is used to obtain a closed system of equation [𝑐𝑝𝛾↑; ∑ 𝑡𝑖𝑗 𝑒𝑓𝑐𝑖𝛾𝜎 + 𝑐𝑖𝛾𝜎 𝑖𝑗𝛾𝜎 ] = ∑ 𝜀𝑝𝑙𝑐𝑙𝛾↑ 𝑙 + ∑ 𝜉𝑝𝑟𝑐𝑟𝛾↑ 𝑙 (8) 1 𝑁∑(⟨𝑐𝑖𝛾↑ + 𝑐𝑖𝛾↑⟩+ ⟨𝑐𝑖𝛾↓ + 𝑐𝑖𝛾↓⟩) 𝑖𝛾 = 𝑛. (10) (10) and the hopping terms are transformed in the mean-field ideology Finally, the quasiparticle energy spectrum below the transition temperature is is 𝑡𝑖𝑗 𝑒𝑓= 𝑡𝑖𝑗(𝑛, 𝑢) + 2𝑡𝑖𝑗 𝑎𝑛𝛾𝜎+ 2𝑡𝑖𝑗 𝑏𝑛𝛾𝜎 (9) 𝑡𝑖𝑗 𝑒𝑓= 𝑡𝑖𝑗(𝑛, 𝑢) + 2𝑡𝑖𝑗 𝑎𝑛𝛾𝜎+ 2𝑡𝑖𝑗 𝑏𝑛𝛾𝜎 (9) 𝐸1,2 𝛾𝜎= −𝜇+ 1 2 (𝑈′ −𝐽𝐻)𝑛𝛾𝜎+ 1 2 𝜀𝑘⃗⃗+ 1 2 𝜉𝑘⃗⃗∓ 1 2 √((𝑈′ −𝐽𝐻)𝑛𝛾¯𝜎+𝜀𝑘⃗⃗+𝜉𝑘⃗⃗) 2 + 4𝜀𝑘⃗⃗𝜉𝑘⃗⃗. (11) (11) When under an external action the energy gap closes, for the metallic paramagnet the band spectrum becomes conducting characteristics and the magnetic nature of the compound. For numerical calculation, the model rectangular electronic density of states has been used to speed up calculations, though more realistic DOS’s are available for such quasi-two-dimensional systems. This restriction, however, does not undermine the soundness of our conclusions as the gap criterion is used to detect the transition point, which is quite insensitive to the DOS form [23]. 𝐸𝛾𝜎= −𝜇+ 1 2 (𝑈′ −𝐽𝐻)𝑛𝛾𝜎+𝑡𝑘⃗⃗ 𝑒𝑓(𝑛, 𝑢) . (12) (12) In the metal-insulator transition point the energy spectrum, sublattice magnetizations, magnetic susceptibilities, conductivities all change, however in this study we just check the viability of the approach and investigate the conditions for the energy gap closure, leaving more detailed discussion for the forthcoming researches. Lattice strain u is found to depend weakly on the variation of the applied pressure and exponentially increase close to the transition point. Nevertheless, even a small variation of the lattice constant may trigger the transition and completely rebuild the single electron energy spectrum. I. The model Hamiltonian for vanadium oxide film Two-dimensional lattice of V atoms with possible electronic configurations of a site and different hopping amplitudes, dependent on the site occupation. The averaged value of the lattice strain u is to be calculated in a self-consistent way from the minimum of the Gibbs thermodynamic potential 𝐺= 𝐹+ 𝑃𝑉= 𝐹+ 𝑁𝑃𝑉0(1 + 𝑢), (3) (3) where F denotes free energy. From the relation dF/du=<dH/du>, one has 𝑡𝑖𝑗(𝑛, 𝑢) = 𝑡𝑖𝑗(1 + 𝐵𝑉0 2𝑤𝑢) (2) ⟨ 𝜕𝐻 𝜕𝑢⟩+𝑁𝑝𝑉0 = 0. (4) ⟨ 𝜕𝐻 𝜕𝑢⟩+𝑁𝑝𝑉0 = 0. (4) (2) (2) (4) Taking into account, that 𝛼𝑢= 𝜏𝑝𝑉0, the equilibrium lattice strain is derived as Taking into account, that 𝛼𝑢= 𝜏𝑝𝑉0, the equilibrium lattice strain is derived as where 2w is the unperturbed band width (w=z\|tk(n)\|), renormalized by correlated hopping processes [20], B is the characteristic constant for a particular compound and 63 Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty 𝑢= − 1 𝑉0 ( 𝑆 𝑁∑ ⟨𝑐𝑘⃗⃗𝛾𝜎 + 𝑐𝑘⃗⃗𝜎⟩ 𝑘⃗⃗𝛾𝜎 + 𝐵𝑉0 2𝑤 1 𝑁∑ 𝑡𝑘⃗⃗⟨𝑐𝑘⃗⃗𝛾𝜎 + 𝑐𝑘⃗⃗𝛾𝜎⟩ 𝑘⃗⃗𝜎 ) − 𝑃𝑉0 𝐶𝑉0, (5) (5) where averages (6) are related to the single electron Green function spectral intensity J(E). To study the strain-induced MIT we have to calculate Green functions and energy spectra for both paramagnetic metal (PM) state and antiferromagnetic insulator (AFI). Below, we restrict our consideration to the low-energy sector of the model, thus the higher- energy states of the lattice sites will be effectively excluded. Here 𝑡𝑖𝑗 𝑒𝑓is the effective concentration- and strain- dependent hopping amplitude introduced to enable the projection coefficients 𝜀𝑝𝑙, 𝜉𝑝𝑟 calculation by the method of work [20]. The reason for applying the above projection procedure is the following. In V2O3 the transition from the high-temperature PM phase to the low-temperature AFI phase is accompanied by onset of alternating sublattices with opposite majority spin projections (their sites are shown as open or shaded circles in Fig. 1). The inverse transition can be induced by either the external pressure application or the chemical doping. Therefore, the physically distinct processes of electron hoppings from one of the two possible orbital states (shown as arrows of different colors in Fig. 1) to the corresponding orbital of the nearest neighboring site in a V2O3 film (with the opposite majority spin orientation) or to the next-nearest-neighbor (with the parallel majority spin orientation) are to be represented by different projection coefficients [21, 22]. To calculate the chemical potential we fix the electron concentration Strong correlation effects in vanadium oxide films Strong correlation effects in vanadium oxide films Strong correlation effects in vanadium oxide films Fig. 2. Lattice strain as a function of the applied external pressure. The upper curve (dashed black one) corresponds to pV0/w=2 and the lower one (shown by the solid red line) is built for pV0/w=2.1. Fig. 3. The temperature dependences of the energy gap in spectrum. The upper curve corresponds to pV0/w=2, the middle one corresponds to pV0/w=2.05 and the lower one to pV0/w=2 1 Fig. 4. Experimental phase diagram from work [24]. Fig. 5. AFI/PM phase boundaries calculated within the considered model. Conclusions Fig. 2. Lattice strain as a function of the applied external pressure. The upper curve (dashed black one) corresponds to pV0/w=2 and the lower one (shown by the solid red line) is built for pV0/w=2.1. Fig. 4. Experimental phase diagram from work [24]. Fig. 5. AFI/PM phase boundaries calculated within the considered model. Fig. 3. The temperature dependences of the energy gap in spectrum. The upper curve corresponds to pV0/w=2, the middle one corresponds to pV0/w=2.05 and the lower one – to pV0/w=2.1. Fig. 5. AFI/PM phase boundaries calculated within the considered model. Fig. 3. The temperature dependences of the energy gap in spectrum. The upper curve corresponds to pV0/w=2, the middle one corresponds to pV0/w=2.05 and the lower one – to pV0/w=2.1. 2.2. Lattice strain and the metal-insulator transition Lattice contraction under the external pressure application or at chemical doping appears to be essential to switch the transition, which changes both the 64 Conclusions In the doubly degenerated model of strongly correlated electron system the projection procedure allows to describe metal-insulator transitions as well as stabilization of magnetic orderings in the generalized Hubbard model (with electron-hole asymmetry). There is an important distinction of the present model from the non-degenerated one, namely, the lowest in energy doublon configuration is the Hund’s spin triplet, which considerably reduces the energy benefit of having the alternate antiferromagnetic lattices if the hopping is intensified at increasing temperatures. The temperature dependences of the energy gap in the spectrum are shown in Figure 3 for close values of the external pressure. One can see that our approach allows us to reproduce three different regimes. The upper curve corresponds to the antiferromagnetic state with an energy gap; the middle one corresponds to the transition and the lower curve (with negative values which mean that no gap in the spectrum is present) corresponds to the metallic paramagnet state. In Fig.5, the constructed phase diagram of the antiferromagnetic insulator (AFI) to the paramagnetic metal (PM) transition is shown, with wide boundaries of the transition region due to the uncertainty of the experimental findings for the model parameters we have used. For comparison, the experimental phase diagram of work [24] is given in Fig.4. One can see the qualitative agreement of PM/AFI phase boundaries on those diagrams. Our results support the findings of paper [18] which open new prospects for use of strain engineering to design novel electronic devices. The phase diagram allows the qualitative explanation of the transition, therefore the proposed model may serve as a basis for more detailed studies. 63 65 Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty Skorenkyy Yuriy - Head of Physics Department at Ternopil Ivan Puluj National Technical University, 56 Ruska St, Ternopil, 46001, Ukraine; Kramar Oleksandr - Associate professor of Physics Department, Ternopil Ivan Puluj National Technical University; Dovhopyaty Yuriy – Lecturer of Physics Department, Ternopil Ivan Puluj National Technical University. Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty Skorenkyy Yuriy - Head of Physics Department at Ternopil Ivan Puluj National Technical University, 56 Ruska St, Ternopil, 46001, Ukraine; Kramar Oleksandr - Associate professor of Physics Department, Ternopil Ivan Puluj National Technical University; Dovhopyaty Yuriy – Lecturer of Physics Department, Ternopil Ivan Puluj National Technical University. Conclusions Skorenkyy Yuriy - Head of Physics Department at Ternopil Ivan Puluj National Technical University, 56 Ruska St, Ternopil, 46001, Ukraine; Kramar Oleksandr - Associate professor of Physics Department, Ternopil Ivan Puluj National Technical University; Dovhopyaty Yuriy – Lecturer of Physics Department, Ternopil Ivan Puluj National Technical University. [1] P.A. Metcalf et al, Thin Solid Films 515(7-8), 3421 (2007); https://doi.org/10.1016/j.tsf.2006.10.003. [ ] , y , ( ); p g y [9] H. Kuwamoto, J. M. Honig, J. Appel. Phys. Rev. B 22, 2626 (1980); https://doi.org/10.1103/PhysRevB.22.2626. y p g y [11] J.-H. Park et al, Phys. Rev. B 61, 11506 (2000); https://doi.org/10.1103/PhysRevB.61.11506. [11] J.-H. Park et al, Phys. Rev. B 61, 11506 (2000); https://doi.org/10.1103/Ph [13] [S. Lupi et al, Nat Commun 1, 105 (2010); https://doi.org/10.1038/ncomms1109. p p g [14] J.G. Ramirez et al, Phys. Rev. B 91, 205123 (2015); https://doi.org/10.1103/PhysRevB.91.205123. [16] G. Mazza, A. Amaricci, M. Capone, M. Fabrizio, Phys. Rev. Lett. 117, 176401 (2016); https://doi.org/10.1103/PhysRevLett.117.176401. [16] G. Mazza, A. Amaricci, M. Capone, M. Fabrizio, Phys. Rev. Lett. 117, 176401 (2016); https://doi.org/10.1103/PhysRevLett.117.176401. [17] P. Homm et al, APL Materials. 9, 021116 (2021); https://doi.org/10.1063/5.0035865 [19] Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty, Condens. Matter Phys. 23, 43714 (2020); https://doi.org/10.5488/CMP.23.43714. [19] Yu. Skorenkyy, O. Kramar, Yu. Dovhopyaty, Condens. Matter Phys. 23, 43714 (2020); https://doi.org/10.5488/CMP.23.43714. p g [20] L. Didukh, Yu. Skorenkyy, O. Kramar, Condens. Matter Phys. 11(3), 443 (2008); https://doi.org/10.5488/CMP.11.3.443. p g [21] Yu. Skorenkyy, O. Kramar, Mol. Cryst. Liq. Cryst. 639(1), 24 (2016) https://doi.org/10.1080/15421406.2016.1254507. p g [21] Yu. Skorenkyy, O. Kramar, Mol. Cryst. Liq. Cryst. 639(1), 24 (2016) https://doi.org/10.1080/15421406.2016.1254507. yy y p g [23] L. Didukh, O. Kramar, Fizika Nizkikh Temperatur (Kharkov), 28(1), 42 (2002) https://doi.org/10.1063/1.1449182. p g [24] G. Keller et al, Phys. Rev. B 70, 205116 (2004); https://doi.org/10.1103/PhysRevB.70.205116. Ефекти сильних електронних кореляцій в плівках оксидів ванадію Тернопільський національний технічний університет імені Івана Пулюя, вул. Руська 56, м. Тернопіль, 46001 Україна, skorenkyy@tntu.edu.ua Тернопільський національний технічний університет імені Івана Пулюя, вул. Руська 56, м. Тернопіль, 46001 Україна, skorenkyy@tntu.edu.ua Досліджено перехід метал-діелектрик в двократно орбітально виродженій моделі квазідвовимірного матеріалу на основі плівки V2O3, в якому ключову роль відіграє внутрішньоатомна кулонівська взаємодія та корельований перенос електронів, що зумовлює електрон-діркову асиметрію властивостей. З застосуванням процедури проєктування в методі функцій Гріна розраховано енергетичний спектр електронів, що дозволило змоделювати зміни властивостей матеріалу під дією зовнішніх впливів - тиску, легування та змін температури. Отримані вирази для термодинамічного потенціалу та ширини енергетичної щілини дозволяють аналізувати можливі фазові переходи в системі, залежність її характеристик від зовнішніх впливів для цього класу матеріалів з сильними електронними кореляціями. Ключові слова: перехід метал-діелектрик, сильні електронні кореляції, енергетичний спектр. 64 66 64 66
https://openalex.org/W4231935085	https://archive.org/download/southafricaunion00thea/southafricaunion00thea.pdf	English	null	South Africa	null	1,917	public-domain	141,535	THE STORY OF THE NATIONS I. Rome. By AKTHUK (in. man, M.A. 36. Spain. By H. E. Watts. I. Rome. By AKTHUK (in. man, M.A. 2. The Jews.' By Prof J. K. Hosmkk. 36. Spain. By H. E. Watts. 37- Japan. By David Murray, Ph.D. 36. Spain. By H. E. Watts. 37- Japan. By David Murray, Ph.D. 2. The Jews.' By Prof J. K. Hosmkk. 5. Germany. By Rev. S. BARIXG- GOULD M.A. 37- Japan. By David Murray, Ph.D. 38. South Africa. By GEORGE M. Theal. GOULD M.A. 4. Carthage. By Prof. ALFRED \|. Church. 39. Venice. Bv ALETHEA WlEL. 40. The Ciusades. By T, A. AR 40. The Ciusades. By T, A. ARCHER and C. L. KlNGSFORD. Church. 5. Alexander's Empire. By Prof. J. P Mahakky. 41. Vedic India. By Z. A. RAGOZIN. J. P Mahakky. 6. The Moors in Spain, Bv STANLEY Lane-Poole. 41. Vedic India. By Z. A. RAGOZIN. 42. The West Indies and the Spanish Main. By Fames Rodway. Lane-Poole. 7. Ancient Egypt. By Prof. GEORGE Rawlinson. Main. By Fames Rodway. 43. B ohemia." By C. Edmund MAURICE. Rawlinson. S. Hungary By Prof. Aumimis Vambery. 43. B ohemia." By C. Edmund MAURICE. 44. The Balkans. By W. Miller, M.A. 45. Canada. By Sir J. G. BOURINOT, LL.D. Vambery. <). The Saracens. By ARTHUR OIL- MAN, M.A. LL.D. 46. British India, By R. \V. FRAZER, LL.B. MAN, M.A. 10. Ireland. By the Hon. EMILY Lawless. LL.B. 47. Modern France. By ANDRE Le Bon. Lawless. 11. Chalriea. By Zfkaide A Ragozix. 11. Chalriea. By Zfkaide A Ragozix. 12. The Goths." Hv HENRY BRADLEY. Bon. 48. The Franks, By LEWIS SER- GEANT. 12. The Goths." Hv HENRY BRADLEY. 13. Assyria. By ZENAfDK A. Ragozix. 13. Assyria. By ZENAfDK A. Ragozix. 14. Turkey. By Stanley Lane-Poole. GEANT. 49. Austria. By Sidney Whitman. 14. Turkey. By Stanley Lane-Poole. 15. Holland. By Prof. J. E. THOROLD ROGKRS. 49. Austria. By Sidney Whitman. 50. Modern England. Before the Re- form Bill. By Justin McCarthy. ROGKRS. 16. Mediaeval France. By Gustaye Masson. form Bill. By Justin McCarthy. 51. China. By Prof. U.K. Douglas. Masson. 17. Persia. By S. G. W. BENJAMIN. 51. China. By Prof. U.K. Douglas. 52. Modern England. From the Reform BUI to the Present Time. By Justin McCarthy. 17. Persia. By S. G. W. BENJAMIN. 18. Phoenicia. By Prof. (1. Rawlin-ON. 18. Phoenicia. By Prof. (1. Rawlin-ON. 10. Media. By ZenaIDE A.' Rac.ozi.v. Justin McCarthy. 53. SOUTH AFRICA SOUTH AFRICA SOUTH AFRICA THE STORY OF THE NATIONS Modern Spain. Bv MARTIN A. S. HUME 10. Media. By ZenaIDE A.' Rac.ozi.v. 20. The Hansa Towns. By HELEN ZlMMERN. HUME 54. Modern Italy. Bv PlETRO Orsi. ZlMMERN. z\. Early Britain. By Prof. ALFRED \|. Chlrcm. 54. Modern Italy. Bv PlETRO Orsi. 55. Norway. Bv H. H. HOYESEN. \|. Chlrcm. 22. The Barbary Corsairs. BvSTANLKY Lank-Poolk. 55. Norway. Bv H. H. HOYESEN. 56. Wales. Bv 0. M. EDWARDS. ^7. Mediseval Rome. Bv W. MILLER, 56. Wales. Bv 0. M. EDWARDS. ^7. Mediseval Rome. Bv W. MILLER, M A. Lank-Poolk. 2.;. Russia. By W. R. MORFILL, M.A. M A. ^s. The Papal Monarchy. Bv William Barry, d.d. 2.;. Russia. By W. R. MORFILL, M.A. 24. The Jews under the Romans. By \v. I). Morrison. Barry, d.d. 59. Mediaeval India under Mohamme- dan Rule. Bv Stanley Lane- Poole. \v. I). Morrison. 2^. Scotland. By John Mackintosh, LLD. LLD. 26. Switzerland. By Mrs. LlNA HUG and R. STEAD. Poole. 60. Buddhist India. By Prof. T. W. Rhys-Davids. and R. STEAD. 27. Mexico. Hv Sis\n Hale Rhys-Davids. 61. Parliamentary England. Bv Ed WARD fENKS. M.A. 27. Mexico. Hv Sis\n Hale 2^. Portugal. By H. Morse Stephens. 2^. Portugal. By H. Morse Stephens. 29 The Normans. By SARAH ORME [EWET1 . WARD fENKS. M.A. 62. Mediaeval England. Bv MARY B vi ESON. [EWET1 . 30. The Byzantine Empire. By C. W. <-'. ()\\| ,w. 63. The Coming of Parliament. Bv L Cecil Jane. <-'. ()\\| ,w. 31. Sicily: Phoenician, Greek and Roman. By, the Prof. E. A. FREEMAN. Cecil Jane. 04. The Story of Greece. From the Earliest Times to A.D. 14. Bv E. S. SHUCKpURGH. FREEMAN. 32. The Tuscan Republics. By BELLA Duffy. E. S. SHUCKpURGH. 65. The Story of the Roman Empire. (B.C. 29 to a.d. 476.) By H. Stuart Joxkf. Duffy. 33. Poland. By W. R. MORFILL M.A. 33. Poland. By W. R. MORFILL M.A. 34. Parthia. By Prof. GEORGE RAW- l.ixsox. 33. Poland. By W. R. MORFILL M.A. 34. Parthia. By Prof. GEORGE RAW- l.ixsox. Stuart Joxkf. 66. Denmark and Sweden, with Ice- land and Finland. By fox STEFANSSON, Ph.D. 34. Parthia. By Prof. GEORGE RAW- l.ixsox. l.ixsox. 33. The Australian Commonwealth. By Greville TREGARTHEN. 73/ 7"37 19/7 I London ■ T. FISHER UNWI x, LTD., 1 Adelphi Terrace GATEWAY 01- THE CASTLE 01 GOOD HOPE. GATEWAY 01- THE CASTLE 01 GOOD HOPE. COPYRIGHT BY T. FlSHER UNWIN, 1894 (For Great Britain) copyright by G. ?. Putnam's, 1894 (For the United States of America) 19/7 I SOUTH AFRICA (THE UNION OF SOUTH AFRICA, RHODESIA, AND ALL OTHER TERRITORIES SOUTH OF THE ZAMBESI) By GEORGE M'CALL THEAL, Litt.D., LL.D. EICHTH EDITION' (eleventh impression) BROUGHT DOWN TO JUNE 1916 LONDON T. FISHER UNWIN LTD. ADELPHI TERRACE AUG z b 1985 By GEORGE M'CALL THEAL, Litt.D., LL.D. EICHTH EDITION' (eleventh impression) BROUGHT DOWN TO JUNE 1916 AUG z b 1985 First Edition . . . i$qj Second Edition . . i8<J5 Third Impression . . iSq6 Third Edition . . . i8qj {Fourth Impression) fifth Impression . . i8gg Fourth Edition . . i8gg (Sixth Impression) Fifth Edition . . . njoo (Seventh Impression) Sixth Edition . . . 10.00 (Eighth Impression) Ninth Impression . . 10,01 Seventh Edition . . 1Q10 (Tenth Impression) Eighth Edition . . 1917 (Eleventh Impression) COPYRIGHT BY T. FlSHER UNWIN, 1894 (For Great Britain) copyright by G. ?. Putnam's, 1894 (For the United States of America) PREFACE TO EIGHTH EDITION PREFACE TO EIGHTH EDITION The first edition of this book was published in 1894, and with each succeeding issue either the concluding chapters were enlarged or a supple- mentary chapter was added. To continue this method of keeping the volume up to the date of issue is not advisable, because it breaks the line of continuity too frequently, and I have therefore recast a number of chapters so that they appear now as if written for the first time. I have also rearranged these chapters in order to bring those relating to the same province of the present Union in consecutive order, which I think will be found an improvement. improvement. It has been my aim to give as faithful an account of the past of South Africa as diligent research during half a century and constant effort to avoid bias of any kind could make it. The narrative, of events preceding 1873 has been condensed from my detailed " History of South Africa," in eight large volumes, and of those since 1872 is derived from personal observation. GEO, M'CALL THEAL. GEO, M'CALL THEAL. YVyxberg, Cape Province, June 1916. FORMATION of a Refre Stati in Table Valley by the Dutch East India Company 2 FORMAT a Re St in Ta Valley by the Dutch East India Company 25-31 Trade with Hottentots — The first cattle raid — Extension to Rondebosch. Trade with Hottentots — The first cattle raid — Extension to Rondebosch. i i PAGE Ancient Inhabitants of South Africa . . 1-7 Bushmen — Hottentots — Bantu — Constant warfare. Discovery of the South African Coast by the Portuguese . . . . .8-16 Discoveries of the Portuguese — First ships in Table Bay — Portuguese maps. Events that fed to the Occupation of Table Valley by the Dutch East India Company 1 Events that fed to the Occupation of Table Valley by the Dutch East India Company 17-24 Rise of the Dutch Republic — The Dutch East India Com- pany— The Eastern trade route — Wreck of the Haarlem — Advantages of Table Valley — Loss of life by scurvy — Mr. Van Riebeek. t IX The End of the East India Company's Rule in South Africa ..... 96-1 The End of the East India Company's Rule in South Africa ..... 96-1 11 in 96-1 11 Reckless expenditure — Second Kaffir war — Churches in the colony — Affairs in Europe — Arrival of a British force — Fruitless negotiations — Feeble defence of the colony — Review of the Company's rule. Progress ok the Cape Colony from 1700 to Life of the early settlers YVilhem Adrian van der Stel — First outbreak of small-pox — System of administration — Effort to improve Table Bay — Growth of the settlement. VI PADS The Second Hottentot War and its Conse- quences ...... 45-59 Origin of the cattle fanners — Extension of the settlement — Arrival of Huguenots— Form of government — System of taxation— Exploration. VII Progress ok the Cape Colony from 1700 to 1750 60 Life of the early settlers YVilhem Adrian van der Stel — First outbreak of small-pox — System of administration — Effort to improve Table Bay — Growth of the settlement. Course ok Events in the Cape Colony from 1750 to 1785 ..... 8 Second outbreak of small-pox — Exploration of Namaqua- land— Villages in the colony — Tour of Governor Van Plet- tenberg — First Kaffir war — Arrival of French troops — Complaints of the colonists — Agitation in the colony. V Foundation of the Cape Colony . . 32-44 Introduction of slaves — Introduction of Asiatics — The first Flottentot war — The first church — Purchase of territory. Contents The Administration of Lord Charles Somer- set ...... 148-16 Slachters Nek rebellion — Fifth Kaffir war Arrival of British settlers — Success of the British settlers — Signs of progress — Resignation of the governor. X he First British Occupation . . 1 12- Character of the colonists -First days of British rule- Surrender of a Dutch fleet — Insurrection of Graaff-Reinet — The third Kaffir war. The First British Occupation . 1 12-128 Character of the colonists -First days of British rule- Surrender of a Dutch fleet — Insurrection of Graaff-Reinet — The third Kaffir war. VONT XI XII Early Years of English Rule in South Africa ...... 138-147 Powers of the governor — Condition of the Hottentots — Fourth Kaffir war — Establishment of a circuit court — Cession of the colony. PAGE The Colony under the Batavian Republic . 129-137 Dealings with Xosa chiefs — Attack by the English — Capitu- lation of Capetown— Departure of General Janssens. XI XI PAGE The Colony under the Batavian Republic . 129-137 Dealings with Xosa chiefs — Attack by the English — Capitu- lation of Capetown— Departure of General Janssens. XV Events in the Cape Colony from 1826 to 1835 I74-I93 Events in the Cape Colony from 1826 to 1835 I74-I93 Injudicious measures — The Kat river settlement — Condition of the slaves — Emancipation of the slaves — Effects of the emancipation — Treaty with Waterboer — Sixth Kaffir war — The province of Queen Adelaide — Action of LorfLGlenelg. Injudicious measures — The Kat river settlement — Condition of the slaves — Emancipation of the slaves — Effects of the emancipation — Treaty with Waterboer — Sixth Kaffir war — The province of Queen Adelaide — Action of LorfLGlenelg. CONTENTS XI l XIX Events to 'the Close of the Seventh Kaffir War ...... 230-24 Events to 'the Close of the Seventh Kaffir War ...... 230-241 Expedition to aid Adam Kok — Arrangement with Adam Kok — Marks of progress — Seventh Kaffir war — Course of the war— Results of the war. XVI Great Emigration from the Cape Colony. Expulsion of Moselekatse from the Terri- tory South of the Limpopo . . 194-202 Fate of the first party — Attack by the Matabele -The first constitution — Defeat of the Matabele. Fate of the first party — Attack by the Matabele -The first constitution — Defeat of the Matabele. Destruction of the Zulu Power and Founda- tion of the Republic of Natal . . 203-21 Natal and Zululand in 1837- — Arrangement with Dingana — Massacre of emigrants — Desperate fighting — Invasion of Zululand — Death of Peter I'ys Destruction of the Natal army — Arrival of Andries Pretorius — Defeat of a Zulu army — Revolt of Panda — Destruction of the Zulu power — Final defeat of Dingana. Rapid Progress of the Cape Colony under Home Rule ..... 288-301 Railway extension — Harbour works — Ostrich farming — War on the northern border — Kakamas labour colony — Rebellion of Morosi — Annexation of Walvis Bay — Discovery of dia- monds— The Keate award — Annexation of Griqualand West and of Bechuanaland south of the Limpopo. XXIII The Province of British Kaffraria . . 275-2S Policy of Sir George Grey — Self-destruction of the Xosa tribe — Introduction of European settlers — Annexation of the province to the Cape Colony. XX Harry Smith . . . . . 242-2 End of the treaty states— Battle of Boomplaats — Anti-convict agitation — Eighth Kaffir war — -Settlement of British Kafiraria. XI 11 CONT XXVII The Colony of Natal from 1842 to 1873 . 330-3 Influx of Bantu — Small number of European settlers — Account of the Hlubi tribe — Rebellion of Langalibalele — Effects of the rebellion — Introduction of Indians — Pro- duction of sugar — Growth of Durban — Form of legislature. XXV Further Enlargements of the Cape Colony 302-316 Description of Kaffraria — History of its people — Its gradual annexation to the Cape Colony — The ninth Kaffir war — Rebellion of 1880 in Kaffraria — Systems of law, Further Enlargements of the Cape Colony 302-316 Description of Kaffraria — History of its people — Its gradual annexation to the Cape Colony — The ninth Kaffir war — Rebellion of 1880 in Kaffraria — Systems of law, X CONTENTS XXVI PAGE Condition of the Cape Colony in 1910 . 317-329 Area — Industries — Exportation of diamonds — Production of copper — Effects of mission labour — Educational system — Administration of justice — Institutions of various kinds. XXIX Natal from 1873 to 1910 .... 355-361 Construction of an excellent harbour— Railways — Production of coal — Additions of territory — Area— Form of government — Franchise— The Bam bat a rebellion — Industries of Natal. Natal from 1873 to 1910 .... 355-36 Construction of an excellent harbour— Railways — Production of coal — Additions of territory — Area— Form of government — Franchise— The Bam bat a rebellion — Industries of Natal. XXVIII .ululand to its Annexation to Natal . 543-354 Civil war — Supremacy of Ketshwayo — Attitude of Sir Bartle Frere — Invasion of Zululand — Terrible disaster — Progress of the war— Captivity of Ketshwayo — Career of Dinizulu — Annexation of Zululand to Natal. .ululand to its Annexation to Natal .ululand to its Annexation to Natal . 543-354 Civil war — Supremacy of Ketshwayo — Attitude of Sir Bartle Frere — Invasion of Zululand — Terrible disaster — Progress of the war— Captivity of Ketshwayo — Career of Dinizulu — Annexation of Zululand to Natal. The Orange Free State from 1854 to 1899 . 362-374 The Orange Free State from 1854 to 1899 . 362-374 Form of government — Franchise — War of 1858 with the Basuto — Acquisition of Adam Kok:s territory — War of 1865-6 with the Basuto — War of 1867-8— Annexation of Basutoland by Great Britain- Loss of the diamond fields — Industries. Form of government — Franchise — War of 1858 with the Basuto — Acquisition of Adam Kok:s territory — War of 1865-6 with the Basuto — War of 1867-8— Annexation of Basutoland by Great Britain- Loss of the diamond fields — Industries. CONTE XV XXXI PAGE The South African Republic from 1852 to 1884 375-394 Early disorder — Increase of Bantu inhabitants — Adoption of a constitution — War with the Baramapulana — Difficulties with the Barolorg — Administration of President Burgers — War with the Bapedi — Annexation of the republic by Great Britain — Recovery of independence. XXXI PAGE The South African Republic from 1852 to 1884 375-394 XXXIV The Union of South Africa, 1910 to 1916 . 422-431 Amalgamation of the four colonies under one government — Form of the constitution — Passing of the defence force act — Creation of three universities — Population — Area — Public debt — Exports — Imports — Revenue. The Transvaal and Orange Free State from 1899 to 1910 ..... 411- The Transvaal and Orange Free State from 1899 to 1910 ..... 411-421 War between Great Britain and the republics — Terrible losses on both sides — Conclusion of peace — End of the re- publics—Liberal form of government of the new possessions. XXXII The South African Republic from 1884 to 1899 395-410 Discovery of gold — Rise of Johannesburg — Differences between old and new immigrants — Conspiracy to overturn the government — Dr. Jameson's raid — Construction of rail- ways— Form of government — Differences with Great Britain. Portuguese South Afric Occupation of Sofala — Foundation of Sena and Tete — Ex- pedition under Barreto— Mission work — Terribly destructive wars — Importance of Lourenco Marques — Foundation of Beira — Difficulties with the English Company — Creation of the Mozambique Company — Form of government — Conquest of the Matshangana. Basutoland, the Betshuanaland Protectorate, and Swaziland ..... 455-46 Basutoland, the Betshuanaland Protectorate, and Swaziland ..... 455-46 Account of the Basuto — Their rebellion — Area — Population — Tribes in Betshuanaland — Effects of mission work — Area — Population — Account of Swaziland — Area — Population — Total population of British South Africa. (Late) German South-West Africa (Late) German South-West Africa . . 480-5 Description of the territory — Account of the Herero- -Hottentot wars — Occupation of the territory by Germany Display of great energy — Wars with the Hottentots and the Hereros — Discovery of diamonds— Population — Con- quest of the territory by Union forces under Generals Botha and Smuts. Chronological Table of Events . . 507-510 XXXV Rhodesia south of the Zambesi . . . 432-454 Condition of the country before 1890 — Concessions from Lobengula — Creation of the British South Africa Company Rhodesia south of the Zambesi . . . 432-454 Condition of the country before 1890 — Concessions from Lobengula — Creation of the British South Africa Company X COX TEXTS PAGE PA — Occupation of Mashonaland — War with the Matabele — Rapid progress— Rebellion of the Bantu— Exports — Area Population. LIST OF ILLUSTRATIONS PAGE MAP of SOUTH Africa, 1916 .... Facing I GATEWAY OF THE CASTLE OF GOOD HOPE . . FlontlSplCCC SOUTH AFRICA AS OCCUPIED BY BUSHMEN, HOTTENTOTS, AND BANTU IN I65O ...... 7 CROSS ERECTED BY DIAS ON PEDESTAL POINT . . -9 THE CAPE OF GOOD HOPE . . . . .II PORTRAIT OF A BUSHMAN . . . . l6 TABLE MOUNTAIN AS SEEN FROM ROBBEN ISLAND . . 24 PLAN OF THE CASTLE OF GOOD HOPE . . . -41 EXTENT OF THE SETTLEMENT IN l/OO . . . -5$ SOUTH AFRICAN TENT WAGGON . . . . .62 HOUSE ON W. A. VAN DER STEL's ESTATE . . .66 EXTENT OF THE CAPE COLONY IN ] 750 . . .78 THE OLD BURGHER WATCH HOUSE, CAPETOWN . 84 CHURCH OF LAST CENTURY IN CAPETOWN . . . 102 SI.MONSTOWN IN 1795 ...... 106 SOUTH AFRICAN FARMHOUSE OF THE BETTER CLASS IN 1705 I 14 EXTENT OF TERRITORY UNDER EUROPEAN RULE IN l800 . 127 VIEW IN THE KOWIE VALLEY, BELOW GRAHAMSTOWN J 44 FORT WILLSHIRE. BUILT l820 ; ABANDONED 1837 . . 154 A ZULU WARRIOR IN UNIFORM . . . 163 LIST OF ILLUSTRATIONS XVtll XVtll EXPLANATION OF TERMS Explanation of words in common use in South Africa, but that may not be understood elsewhere, at least in the same sense : — Assagai, a javelin or dart used by the Hottentots and Bantu in war and the chase. The word is a corruption of the Portuguese i;azagaya,'' which was derived from the Latin " hasta." Boer, Dutch for a tiller of the ground. The word is applied in this country to cattle breeders as well as to agriculturists, and is fre- quently used in the plural form to signify the whole rural population of European blood speaking the Dutch language. Burgher, a European male, no matter where resident, who is in possession of the franchise and liable to all public duties. It corre- sponds to the civis Romanus of old. Calaha.sk, the hard rind of a gourd, used by the Bantu for various purposes, such as waler-pots, jars, dishes, basins, snufi-boxes, etc. Commando, a body of burghers called out for military purposes. Heemraden, burghers appointed by the government to act as assessors in the district courts of justice. A Dutch word. Induna, an officer of high rank under a Bantu chief. The word is Zulu. Kraal, a cattle-fold. The word is a corruption of the Portuguese " curral."' It is also used to signify a collection of either Hottentot or Bantu huts, as these are usually built in a circle, within which the cattle are kept at night. Lager, a Dutch word meaning an enclosure for protective purposes, such as a circular wall of stone, or a number together. EXPLANATION OF TERMS X I.anddvost, a stipendiary magistrate, who administers justice and receives the revenue of a district. The word is Dutch. receives the Tsetse, a fly whose sting destroys domestic cattle, but has no effect upon wild animals. The word comes from one of the Bantu dialects. The tsetse disappears from a district when the game is exterminated or driven away. Volksraad,* Dutch woid meaning the people's council, an elected legislative body. xcorcs Geographical £s Lab Us hxn ANCIENT INHABITANTS OF SOUTH AFRICA. THE aborigines of South Africa were savages of a very low type. They were pigmies in size, yellow- ish-brown in colour, hollow-backed, and with skins so loose that in times of famine their bodies were covered with wrinkles and flaps. On their heads were rows of little tufts of wiry hair hardly larger than peppercorns, and leaving the greater portion of the surface bald. Their faces were broad in a line with the eyes, their cheeks were hollow, and they had flat noses, thick lips, and receding chins. They anointed their bodies with grease when any was obtainable, and then painted themselves with soot or coloured clay. The clothing of the males was the skin of an animal hung loosely over the shoulders, and often cast aside ; that of the females was little more than a small leathern apron. To the eye of a European no people in any part of the world were more unattractive. - - .••■■< * -Z -Z 16 BAMAHCWA T/0 Ma^hml^Z^tx L A NV D\ B E C H U A N A LAN D •tv/pL&L* fR P R O T E,.C T d» R A T E ^alaxWg4l%^v4 Xropyc of Capricorn ffMMokotloIco Hhohong aJisLrS D es ert Molu jiiMi =J2 f o feexseba °]SFagoais • JV^ Eh p0nv » -«. JBracJiPdfhs -Si B EC .OTia&isl J^lo^ern/bsnteuhKop J Uisip UpvufftOJl-^f, Orange R t PortNollotiiJ ':r"S^J 'SNSJf' CR'"KA Kenhart ^T^Griquatown ' '"'i'1 Telia, fa ' >.UB«7^'» Eorv&Wf Mblopolole B . , °Zo~keng fLxkotLongfj Bl< Taure ■>¥io°siR. 'UmjimAufaJL. ^ (fee'o^'^' 'oMUvrc ^^^Tf^;^?^^ ^f ^ r ■t><Wc\<? ny-f.y?1 4. _Scg3e of E^JA Mies. Heights in,Engli3?i. -feet, Rv€wq\S- Unict of South AB^ca Red. OtJia^Br-itush Territory' Purple, London : T. Fisher Unwin, 1, Adelphi Terrace\V~n~ 16 BAMAHCWA T/0 Ma^hml^Z^tx L A NV D\ B E C H U A N A LAN D •tv/pL&L fR P R O T E,.C T d» R A T E ^alaxWg4l%^v4 Xropyc of Capricorn ffMMokotloIco Hhohong aJisLrS D es ert Molu jiiMi =J2 f o feexseba °]SFagoais • JV^ Eh p0nv » -«. JBracJiPdfhs -Si B EC .OTia&isl J^lo^ern/bsnteuhKop J Uisip UpvufftOJl-^f, Orange R t PortNollotiiJ ':r"S^J 'SNSJf' CR'"KA Kenhart ^T^Griquatown ' '"'i'1 Telia, fa ' >.UB«7^'» Eorv&Wf Mblopolole B . , °Zo~keng zz 26 fLxkotLongfj Bl< Taure ■>¥io°siR. 'UmjimAufaJL. ^ (fee'o^'^' 'oMUvrc ^^^Tf^;^?^^ ^f ^ r ■t><Wc\<? ny-f.y?1 4. _Scg3e of E^JA Mies. Heights in,Engli3?i. -feet, Rv€wq\S- Unict of South AB^ca Red. ANCIENT INHABITANTS OF SOUTH AFRICA. OtJia^Br-itush Territory' Purple, 30 London : T. Fisher Unwin, 1, Adelphi Terrace\V~n~ ANCIENT INHABITANTS OF SOUTH AFRICA. 2 These savages were thinly scattered over every part of the country from a very remote period, for implements — such as stone arrow-heads and knives, similar to those which they had in use when white men first met them — have been found in positions where the overlying materials must have been undis- turbed for an incalculable time. The Bushmen — as the pigmies are termed by Europeans — had no domestic animal but the dog, and they made no effort to cultivate the soil. They lived by the chase and upon wild plants, honey, locusts, the larvae of ants, and carrion. ants, and carrion. They were without other government than parental, and even that was not respected after they were able to provide for themselves. So weak in frame as to be incapable of toil, they possessed great keenness of vision for detecting objects at a distance, and marvel- lous fleetness of foot and power of endurance in the chase. Their weapon of offence was a feeble bow, but the arrow-head was coated with poison so deadly that the slightest wound was mortal. In addition to the Bushmen there lived on South African soil, for a century or two before its discovery by Europeans, a body of people much more advanced towards civilisation, the people now known as Hottentots. They had migrated from the region of the great lakes, and with herds of horned cattle and flocks of sheep had moved slowly to the shore of the Atlantic and then down the coast, keeping always close to the sea, because the pasture there was better and water more plentiful than farther inland; Most probably these people had their origin in a mixture HOTTEN 3 of men of light-coloured hamitic blood with women of Bushman stock. They must have been many centuries on the journey to the southern coast, as they would remain in favourable positions while several generations grew up and passed away. several generations grew up and passed away. The Hottentots frequently captured and detained Bushman girls, so that there were many points of resemblance between the two peoples, as well as great differences. Their languages varied widely in con- struction, though both abounded in clicks. Most of the Bushmen were monogamists, the Hottentot customs admitted of polygamy. ANCIENT INHABITANTS OF SOUTH AFRICA. Then their skull measurements do not correspond. The head of the Hottentot is longer and narrower than that of the Bushman, and his face is more prognathous. The lower jaw of the Bushman is only surpassed in feeble- ness by that of the Australian black, while that of the Hottentot, though far from massive, is much better developed. The Bushman ear is without a lobe, which the Hottentot ear possesses, and the cranial capacity of the Hottentot is higher. capacity of the Hottentot is higher. On the other hand, against these differences several points of resemblance can be placed. The colour of the skin is the same, and the little balls of wiry hair with open spaces between them are in general common to both, though sometimes the head of a Hottentot is more thickly covered. The one has small hands and feet, and so has the other. Their power of imagination is similar, and differs greatly from that of other Africans. The Hottentots were never very numerous, and they occupied only the "strip of" land along the coast ANCIENT INHABITANTS OF SOUTH AFRICA. 4 There was a constant and deadly feud between them and the Bushmen. They took possession of pastures and fountains wherever they pleased, and killed all the game that they could. The Bushmen resented this, and plundered their oxen and sheep, besides murdering every one who strayed from the kraals, so that the feeling of animosity on both sides was intense. They lived in communities under the govern- ment of chiefs, who, however, possessed very limited authority, for public opinion was freely expressed, and was the supreme law. They depended mainly upon the milk of cows and ewes for their subsistence, and did not practise agriculture in any form. Their horned cattle were gaunt and bony, and their sheep were covered with hair — not wool — and had fatty tails of great weight. Their only other domestic animal was the dog. The men lived in almost perfect indolence, moving with their herds and flocks from one place to another as pasture failed ; and when the supply of milk was insufficient it fell to the lot of the women to gather bulbs and roots with which to eke out an existence. ANCIENT INHABITANTS OF SOUTH AFRICA. The huts in which they slept were slender frames of wood covered with mats, and could be taken down and set up again almost as quickly as tents. almost as quickly as tents. These nearly naked people, living in idleness and filthiness indescribable, were yet capable of improve- ment. During the Mast century a vast amount of missionary labour has been concentrated upon the natives of South Africa, and though to the present day there is not a single instance of a -Bushman of BAN 5 pure blood having permanently adopted European habits, the Hottentots have done so to a considerable extent. They have not indeed shown a capacity to rise to the highest level of civilised life, but they have reached a stage much above that of barbarism. reached a stage much above that of barbarism. Before the arrival of Europeans yet another branch of the human family was beginning to press into South Africa. Tribes of stalwart people practising agriculture and metallurgy, under strict government and with an elaborate system of law, were moving down from the north, and by the middle of the seventeenth century had reached the Limpopo river and the mouth of the Umtata. These people formed part of the great Bantu family, which occupies the whole of Central Africa from the Atlantic to the Indian ocean. They were certainly of mixed blood, and one branch of their ancestors must have been of a very much higher type than the other. This is shown in various ways. Among them at the present day are individuals with perfect Asiatic features, born of parents with the negro cast of countenance. In almost any little community may be found men only moderately brown in colour, while their nearest relatives are deep black. Here and there one may be seen with a thick full beard, though the great majority have almost hairless cheeks and chins. And a still stronger proof of a mixed ancestry of very unequal capability is afforded by the fact that most of these people seem unable to rise to the European level of civilisation, though not a few individuals have shown themselves possessed of mental power equal to that of white men. 6 Ancient inhabitants of south Africa. These Bantu were of a healthy and vigorous stock, and were probably the most prolific people on the face of the earth. ANCIENT INHABITANTS OF SOUTH AFRICA. The tribes were seldom at peace with each other, and great numbers of individuals perished yearly through charges of dealing in sorcery, but the losses thus sustained were made good by a custom which provided that every adult female should be married. There was no limit to the number of wives a man could have, and thus in a state of society where the females outnumbered the males, all were provided for. males, all were provided for. The three classes of people referred to in this chapter enjoyed the lives they were leading quite as much as Europeans do, though their pleasures were of a lower kind. Given freedom from disease and a slain antelope, and there could be no merrier creature than a Bushman. He was absolutely devoid of harassing cares. A Hottentot kraal in the clear moonlight of Africa, with men, women, and children dancing to the music of reeds, was a scene of the highest hilarity. The Bantu woman, tending her garden by day, and preparing food in the evening which she may not partake of herself until her husband and his friends have eaten, is regarded as an unhappy drudge by her European sister. In her own opinion her lot is far more enviable than that of the white woman, whom she regards as being always in a state of anxiety. The chief element of disturbance in their lives was war. The hand of the Bushman was always against every man, and every man's hand was against him. The Hottentot tribes were continually robbing each CONSTANT WARFARE. Other of cattle and women, and on their eastern border were struggling in vain against the advancing Bantu. Every Bantu clan was usually at feud with its nearest neighbours, whoever these might be. But life without excitement is insipid to the savage as Wa/fish Bay ^RTuffdcc SOUTH AFRICA AS OCCUPIED BY BUSHMEN, HOTTENTOTS, AND BANTU IN 1650. SOUTH AFRICA AS OCCUPIED BY BUSHMEN, HOTTENTOTS, AND BANTU IN 1650. [Bushmen were the only inhabitants of the parts unmarked, and they lived also in all the rugged and mountainous sections of the parts occupied by Hottentots and Bantu. ANCIENT INHABITANTS OF SOUTH AFRICA. The territory in which the Hottentots roamed with their cattle is marked /fo, and the territory occupied by the Bantu ,$&,.] well as to the civilised man, and these wars and animosities, though sometimes causing great suffering and loss of life, in general provided just that excite- ment which was needed to prevent the minds of the people from sinking into complete stagnation. DISCOVERY OF THE SOUTH AFRICAN COAST BY THE PORTUGUESE. In i486 two little vessels, under command of an officer named Bartholomeu Dias, sailed from Portugal in search of an ocean road to India. Pushing his way down the western coast, Dias passed the farthest point previously known, and sailing onward with the land always in sight came to an inlet of no great depth with a group of islets at its entrance. There he cast anchor, and for the first time Christian men trod the soil of Africa south of the tropic. The inlet is now known as Angra Pequena and Luderitzbucht, but Dias named it Angra dos Ilheos. The surrounding country was a desolate waste of sand, and no signs of human life were seen, nor was other refreshment than seabirds' eggs obtainable. Having set up a cross as a mark of possession for his king, the Portuguese commander proceeded on his voyage. He tried to keep the land in sight, but when he was somewhere near the mouth of the Orange river a gale from the north sprang up, and for thirteen days he was driven helplessly before it. DISCOVERIES OF THE PORTUGUES 9 As soon as it abated the prows of the vessels were turned to the east, with a view of getting near the shore again, but after sailing a long time without reaching land Dias began to think that he must either have passed the end of the continent or" have entered a great gulf like that of Guinea. He therefore changed the course to north, and after a while came in sight of the coast, which he found trending away to the east. The exact spot where he made the land cannot be stated, but it was CROSS ERECTED BY DIAS ON PEDESTAL POINT. {From a Sketch by H. M. Piers.) CROSS ERECTED BY DIAS ON PEDESTAL POINT. {From a Sketch by H. M. Piers.) one of the curves in the seaboard between Cape Agulhas and the Knysna. Large herds of cattle were seen, which the natives drove inland with haste, as they seemed in terror of the ships. It was not found possible to open intercourse with the wild people. people. Sailing again eastward, Dias reached an islet where he landed and set up another cross. It was the islet in Algoa Bay which is still called DISCOVERIES OE THE PORTUGUESE. DISCOVERY OF THE SOUTH AFRICAN COAST BY THE PORTUGUESE. IO on that account Santa Cruz, or, as it is usually written in the French form, St. Croix. Here the sailors objected to proceed farther, and it was with difficulty that they were induced to press on a couple of days longer. At the mouth of a river — either the Kowie or the Fish — the expedition turned homeward, and on its way back discovered a bold headland which Dias named the Cape of Storms, but which was renamed by King John the Second the Cape of Good Hope. As seen from the sea this Cape is much more conspicuous than Agulhas, the true southern extremity of the continent. Ten years passed away after the return of Dias to Portugal before an expedition was fitted out to follow up the discovery he had made. Four small vessels were then made ready, and were placed under command of Vasco da Gama, a man of proved ability. It was not quite five years after Columbus sailed from Palos to discover a new continent in the west, when Da Gama's little fleet put to sea from the Tagus. Five months and a half later he reached a curve in the African coast about one hundred and twenty miles north of the Cape of Good Hope, to which he gave the name St. Helena Bay. Here he landed and by a little strategy managed to obtain an interview with a party of natives, whose friendship he tried to secure by making them presents of trinkets. All went well for a time, but at length a misunder- standing arose, which resulted in the Portuguese attacking the natives, and in a skirmish Da Gama himself and three others were wounded with assagais. DISCOVERIES OF THE PORTUGUESE. \2 Such was the first intercourse between white men and Hottentots. On the 17th of November 1497 Da Gama set sail from St. Helena Bay, and three days later doubled the Cape of Good Hope in fine weather. Turning eastward, he anchored next within a bend of the coast, which he named Agoada de S. Braz, the present Mossel Bay. There he found a number of natives similar in appearance to those he had first seen, but who showed so little symptom of alarm that they crowded on the beach and scrambled for anything that was thrown to them. DISCOVERY OF THE SOUTH AFRICAN COAST BY THE PORTUGUESE. From these people some sheep were obtained in barter, but they would not sell any horned cattle. Keeping within sight of the shore, on the 25th of December Da Gama passed by a beautiful land, to which he gave the name Natal, in memory of the day when Christian men first saw it. On the 6th of January 1498 the fleet reached the mouth of a river which enters the sea on the northern side of Delagoa Bay. Here the Portuguese landed, and found a friendly people, black in colour, who brought copper, ivory, and provisions for sale. During the five days that the fleet remained at this place nothing occurred to disturb the friendly inter- course between the Portuguese and the Bantu residents. Sailing again, Da Gama next touched at Quilimane, where he found people who had dealings with Arabs, and thence he continued his voyage until he reached India. The highway to the East being now open, every year fleets sailed to and from Portugal. In a short FIRST SHIPS IN TABLE BAY 1 3 time the Indian seas fell entirely under Portuguese dominion, and an immense trade was opened up. dominion, and an immense trade was opened up. In 1503 a ship, under command of an officer named Antonio de Saldanha, put into a bay on the African coast that had never been entered before. On one side rose a great mass of rock, over three thousand feet in height, with its top making a level line more than a mile in length on the sky. This grand mountain was flanked at either end with peaks less lofty, supported by buttresses projecting towards the shore. The recess was a capacious valley, down the centre of which flowed a stream of clear sweet water. The valley seemed to be without people, but after a while some Hottentots made their appearance, from whom a cow and two sheep were purchased. whom a cow and two sheep were purchased. Saldanha himself climbed to the top of the great flat rock, to which he gave the name Table Mountain. The bay in which he anchored was thereafter called after him the watering place of Saldanha, until nearly a century later it received from the Dutch sea- captain Joris van Spilbergen its present name of Table Bay. DISCOVERY OF THE SOUTH AFRICAN COAST BY THE PORTUGUESE. No effort was made to explore the interior of the country, and, indeed, setting aside danger from the natives, it would have been beyond the power of any man to have gone far from the coast at this period. The land rises in a series of steps from the seashore to a great interior plain, and until that plain was reached the traveller would have had everywhere a rugged and seemingly impassable range of mountains before him. If by great exertion he had made his way to the sqmrnit of one, he would- have- found him- DISCOVERIES OF THE PORTUGUESE. 14 self on the edge of a broken plateau, with another range — the front of another terrace — shutting in his view. It follows from this conformation of the country that there are no navigable rivers. The streams — even the very largest — are all of the nature of mountain torrents, obstructed with rapids and falls, and varying in volume with rain and drought. There is an utter absence of secure natural harbours on the coast, except in positions where they could be of little service in the early days of exploration. And, in addition to all this, a very large portion of the land along the western seaboard, as well as of the interior plains, is so arid that it could only be traversed by degrees, as its slender resources became known. degrees, as its slender resources became known. In returning with the fleet which left India at the close of 1509, Francisco d'Almeida, first Portuguese viceroy of the eastern seas, put into Table Bay for the purpose of obtaining water and refreshing his people. Some Hottentots appeared on the beach, and a party of ships' people went ashore to barter cattle from them. Traffic was carried on for a time in a friendly manner, but at length a quarrel arose, and two white men were badly beaten. This caused an outcry for vengeance, to which D'Almeida unfortunately lent a willing ear. Next morning, 1st of March 15 10, the viceroy landed with one hundred and fifty men armed with swords and lances. They marched to a kraal and seized some cattle, which they were driving away when the Hottentots, supposed to be about one hundred' and' seventy in '* number, attacked them5. DISCOVERY OF THE SOUTH AFRICAN COAST BY THE PORTUGUESE. The weapons "of the Portuguese were useless against Portuguese map 15 the fleet-footed natives, who poured upon the in- vaders a shower of missiles. A panic followed. Most fled towards the boats as the only means of safety ; a few, who were too proud to retreat before savages, attempted in vain to defend themselves. The viceroy was struck down with knobbed sticks and stabbed in the throat with an assagai. Sixty- five of the best men in the fleet perished on that disastrous day, and hardly any of those who reached the boats escaped without wounds. the boats escaped without wounds. After this event the Portuguese avoided the southern coast as much as possible. With them the country had the reputation of being inhabited by the most ferocious of savages, and of furnishing nothing valu- able for trade. Their fleets doubled the continent year after year, but seldom touched at any port south of Sofala. They made a practice of calling for re- freshment at the island of St. Helena, which had been discovered in 1502, and then pressing on to Mozam- bique without a break, whenever it was possible to do so. They never attempted to form a station below Delagoa Bay. Now and again, however, their ships were driven by stress of weather to seek a port, and occasionally a wreck took place. Curiosity also prompted some of them, and orders from the king required others, to inspect the coast and make tracings of it. The prin- cipal bights and headlands thus acquired names. Nearly all of these have been replaced by others, Dutch or English, but a few remain to our day. Dutch or English, but a few remain to our day. With the belt of land bordering on the sea north pf Zululand the Portuguese were well acquainted, DISCOVERIES OF THE PORTUGUESE. i6 and they had an intimate knowledge of the territory along the lower course of the Zambesi. But of the interior of the country south of the tropic they knew absolutely nothing, and what they imagined and laid down on their maps was so very incorrect that after the territory was explored the whole of their delinea- tions of Africa were regarded as valueless. PORTRAIT OF A BUSHMAN. PORTRAIT OF A BUSHMAN. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. AFTER a long interval English, Dutch, and French ships followed the Portuguese to India. Drake and Cavendish passed in sight of the Cape of Good Hope when homeward bound on their celebrated voyages round the world, but did not land on the African coast. In July 1591 the English flag was seen there for the first time. Three ships — the pioneers of the vast fleets that have since followed the same course — then put into Table Bay on their way to India. Their crews, who were suffering from scurvy, obtained good refreshment, as in addition to wild fowl, shellfish, and plants of various kinds, they bartered some oxen and sheep from Hottentots. One of the ships, commanded by Captain James Lancaster, reached India in safety, another returned to England shorthanded from Table Bay, and the third went down in a gale at sea some- where off the southern coast. In 1601 the first fleet fitted out by the English East India Company, under command of the same Captain James Lancaster mentioned above, put into J7 3 3 RISE OF THE DUTCH REPUBLIC. 1 8 Table Bay on its outward passage. Thereafter for several years the fleets of this Company made Table Bay a port of call and refreshment, and their crews usually procured in barter from the natives as many cattle as they needed. During the closing years of the sixteenth century the people who were destined to form the first European settlement in South Africa were engaged in a gallant struggle for freedom against the powerful Spanish monarchy. The northern Netherland pro- vinces had entered the sisterhood of nations as a free republic which was rapidly becoming the foremost commercial power of the age. While the struggle was being carried on, Portugal came under the dominion of the king of Spain, and the Dutch were then excluded from Lisbon, where they had pre- viously obtained such eastern products as they needed. Some of their adventurous merchants then thought of direct trade with India, but it was not until 1595 that a fleet under the republican flag passed the Cape of Good Hope. It consisted of four vessels, and was under an officer named Cornelis Houtman. This fleet touched at Mossel Bay, where refreshment was procured, the intercourse between the strangers and the natives being friendly. and the natives being friendly. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. After Houtman's return to Europe, several com- panies were formed in different towns of the Nether- lands for the purpose of trading with the Indies. No fresh discoveries on the African coast were made by any of the fleets which they sent out, but to some of the bays new names were given. Thus Paul van Caerden, an officer in the service of the New Brabant THE DUTCH EAST INDIA COMPANY 1 9 Company, when returning to Europe in 1601, gave their present names to Mossel, Flesh, and Fish bays, all of which he entered. The fleets sent out by the different small companies gained surprising successes over the Portuguese in India, but as they did not act in concert no perma- nent conquests could be made. For this reason, as well as to prevent rivalry and to conduct the trade in a manner the most advantageous to the people of the whole republic, the states-general resolved to unite all the weak associations in one great company with many privileges and large powers. The charter was issued at the Hague on the 20th of March 1602, and gave the Company power to make treaties with Indian governments, to build fortresses, appoint civil and military officers, and enlist troops. The Com- pany was subject to have its transactions reviewed by the states-general, otherwise it had almost sovereign power. The subscribed capital was rather over half a million pounds sterling. Offices for the transac- tion of business, or chambers as they were termed, were established at Amsterdam, Middelburg, Delft, Rotterdam, Hoorn, and Enkhuizen. The general control was confided to an assembly of seventeen directors, whose sessions were held at Amsterdam for six successive years, then at Middel- burg for two years, then at Amsterdam again for six years, and so on. The profits made by the Company during the early years of its existence were enormous. The Portuguese ships, factories, and possessions of all kinds in India were fair prize of war, and the most valuable were THE EASTERN TRADE ROUTE. 2 shortly in the hands of the Dutch. Every year fleets of richly laden ships under the flag of the Nether- lands passed the Cape of Good Hope outward and homeward bound. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. In 1619 the directors of the English East India Company proposed to the assembly of seventeen that they should jointly build a fort and establish a place of refreshment somewhere on the South African coast. This proposal did not find favour in Holland, and each company then resolved to form a station of its own. Instructions were issued to the commanders of the next outward-bound fleets of both nations to examine the seaboard and report upon the most suit- able places for the purpose. Thus it happened that in 1620 two English captains, by name Fitzherbert and Shillinge, inspected Table Bay, and believing that no better place could be found, they proclaimed the adjoining country under the sovereignty of King James. They did not leave any force to keep posses- sion, however, and the directors in London having changed their views with regard to a station in South Africa, the proclamation of Fitzherbert and Shillinge was never ratified. English ships still continued oc- casionally to call for the purpose of taking in fresh water, but from this time onward the island of St. Helena became their usual place of refreshment. The assembly of seventeen also allowed its resolu- tion concerning the establishment of a station in South Africa to fall through at this time. Some of the advantages of such a station were already in its possession, and the expense of building a fort and maintaining a garrison might be too high a price to WRECK OF THE HAARL 21 pay for anything additional that could be had. Its fleets usually put into Table Bay for the purpose of taking in fresh water, giving the crews a run on land, catching fish, and getting the latest intelligence from the places they were bound to. Letters were buried on shore, and notices of the places where they were deposited were marked on conspicuous stones. deposited were marked on conspicuous stones. Table Valley was also sometimes occupied for months together by parties of Dutch seal hunters and whale fishers. Among others, in 1611 Isaac le Maire, after whom the straits of Le Maire are named, left his son with some seamen here for this purpose. purpose. Early in 1647 the Haarlem, a ship belonging to the East India Company, put into Table Bay for refresh- ment, and in a gale was driven on the Blueberg beach. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. The crew got safely to land, and succeeded in saving their own effects and the cargo. Whe everything was secured against stormy weather they removed to Table Valley as a better place for an encampment, leaving only a guard with the stores. Beside a stream of fresh water, somewhere near the centre of the present city of Capetown, they made themselves huts, and threw up an earthen bank for shelter around them. The rainy season was setting in, and as they happened to have various seeds with them they made a garden and soon had abundance of vegetables. They were fortunate also in being able to procure in barter from the natives more meat than they needed, so that their experience of South Africa led them to believe that it was a fruitful and pleasant land. After they had been here twelve months a ADVANTAGES OF TABLE VALLEY. 2 2 fleet returning home put in, and took them on to Europe. Upon their arrival in the Netherlands, Leendert Janssen and Nicholas Proot, two of the Haarlem's officers, drew up and presented to the chamber of Amsterdam a document in which they set forth the advantages that they believed the Company might derive from a station in Table Valley. This docu- ment was referred by the chamber of Amsterdam to the directors, who, after calling for the opinions of the other chambers and finding them favourable, in August 1650 resolved to form such a station as was proposed. A committee was instructed to draw up a plan, and when this was discussed and approved of three vessels were made ready to bring the men and the materials to South Africa. The post of commander of the station about to be formed was offered to Nicholas Proot, and upon his declining it, a ship's surgeon named Jan van Riebeek, who had been for some time in the Company's service and had visited many countries, was selected for the office. A better selection could hardly have been made. Mr. Van Riebeek was not a man of high education or of refined manners, but he was indus- trious and possessed of good natural ability. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. He had been in Table Bay with the fleet in which the Haar- lem's crew returned home, and upon the document drawn up by Janssen and Proot being submitted to him for an opinion, he endorsed all that was in it concerning the capabilities of the country. concerning the capabilities of the country. Things were not done in such haste in those days as they are now, and the year 1651 had nearly come LOSS OF LIFE BY SCUR 2$ to an end before the three vessels set sail from Texel. It must not be supposed that they were bringing people to South Africa with the intention of founding a colony, for nothing was then further from the thoughts of the directors of the East India Company. Their object was merely to form a refreshment station for the fleets passing to and from the eastern seas. for the fleets passing to and from the eastern seas. Six months was considered a quick passage between the ports of the Netherlands and the roads of Batavia, where their ultimate destination was made known to the skippers by the governor-general and council of India ; and it was no uncommon circumstance for one-third of the crews to have perished and another third to be helpless with scurvy when the ships arrived there. The loss of life was appalling, as the Indiamen were fighting as well as trading vessels, and usually left Europe with two or three hundred men. The crews were very largely composed of recruits from Germany and the maritime states of Europe, or the population of the Netherlands could not have borne such a tremendous drain of men for any length of time. Table Bay was regarded as two-thirds of the dis- tance from Amsterdam to Batavia, and the directors thought that by establishing a refreshment station on its shores many lives could be saved and much suffering be avoided. The design was first to make a large garden and raise in it vegetables for the supply of the fleets, secondly to barter oxen and sheep from the Hottentots for the same purpose, and thirdly to build a great hospital in which sick men could be left to recover their health. MR. VAN RIEBEEK. 24 Every man at the station was to be a servant of the East India Company. Mr. EVENTS THAT LED TO THE OCCUPATION OF TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. Van Riebeek, who had the title of commander, was to hold a position similar to that of a sergeant in charge of a small military outpost at some distance from the head- quarters of a regiment. Every admiral of a fleet that called was to supersede him for the time being, and he had hardly any discretionary power even when no superior officer was present. TABLE MOUNTAIN AS SEEN FROM ROBBEN ISLAND. TABLE MOUNTAIN AS SEEN FROM ROBBEN ISLAND. FORMATION OF A REFRESHMENT STATION IN TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. In April 1652 Mr. Van Riebeek and his people arrived, and at once set about the construction of a fort by raising banks of earth round a hollow square, within which they erected some wooden sheds brought from Holland. The rainy season at and near the Cape of Good Hope usually commences in the month of April, but this year the summer drought lasted until towards the close of May, and consequently there was a good deal of suffering among the people. Scurvy was showing itself and no sorrel or wild plants of any kind could be found, nor could a garden be made. The only permanent inhabitants of the Cape pen- insula when the Dutch landed were some sixty Hottentots, who were without cattle, and who lived chiefly upon shellfish. Where as many thousands now exist in comfort, these wretched beachrangers — as Mr. Van Riebeek styled them — were barely able to exist at all. The chief man among them was named Harry by the white people. He had once REFRESHMENT STATION IN TABLE VALLEY. 26 been taken to Bantam in an English ship, and he spoke a little broken English, so Mr. Van Riebeek employed him as an interpreter. The others made themselves useful by carrying water and gathering firewood, in return for which they were provided with ships' provisions. with ships' provisions. Two large clans of Hottentots, consisting together of about five thousand souls, roamed over the country within a radius of fifty miles from Table Bay, but they were then far from the peninsula, and very little could be ascertained concerning them. very little could be ascertained concerning them. When the winter rains at last set in, much dis- comfort was the immediate result. The tents and wooden buildings were all found to be leaky. With the change of weather came dysentery, which the people were too weak to resist, and now almost every day there was a death from that disease or from scurvy. By the beginning of June the party was reduced to one hundred and sixteen men and five women, of whom only sixty men were able to per- form any labour. Fresh meat and vegetables and proper shelter would have saved them, but these things were not to be had. They were almost as solitary as if they had been frozen up in the Arctic sea. FORMATION OF A REFRESHMENT STATION IN TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. The two largest of the vessels had gone on to Batavia, leaving the other — a mere decked boat — at Mr. Van Riebeek's disposal, and for many weeks no natives were seen except Harry's miserable followers, from whom no assistance of any kind was to be obtained. But the rain, which had brought on the dysentery, in a very short time brought also relief. Grass sprang TRADE WITH HOTTENTO 2J into existence, and with it appeared various edible plants. They were all correctives of scurvy, and that was mainly what was needed. The strong and the feeble went about gathering wild herbs and roots and declaring there was nothing in the world half so palatable. As soon as the first showers fell, a plot of ground was dug over, in which seeds were planted ; and soon the sick were enjoying such delicacies as radishes, lettuce, and cress. Then they found good reeds for thatch, and when the build- ings were covered with these instead of boards and torn sails, they could almost bid defiance to the rains. The pleasant weather which in South Africa is termed the winter passed away, and in October a large Hottentot clan — called by the Dutch the Kaap- ma"hs — made its appearance in the Cape peninsula with great herds of horned cattle and flocks of sheep, which were brought there for change of pasture. These people and the Europeans met openly on the most friendly terms, though each party was so suspicious of the other that a constant watch was kept. A supply of copper bars, brass wire, and tobacco had been brought from the Nether- lands, and a trade in cattle was now opened. On the European side the commander conducted it in person, assisted only by a clerk and the interpreter Harry. All intercourse between other white men and these Hottentots was forbidden under very severe penalties, with the twofold object of pre- venting any interference with the trade and any act that might lightly provoke a quarrel. REFRESHMENT STATION IN TABLE VALLEY, 28 Parties of the Kaapmans remained in the Cape peninsula nearly three months, during which time Mr. Van Riebeek procured in barter over two hundred head of horned cattle and nearly six hundred sheep. FORMATION OF A REFRESHMENT STATION IN TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. Before they left, they proposed that the commander should help them against a tribe with whom they were at war, and offered him the whole of the spoil, whatever it might be. Mr. Van Riebeek replied that he had come to trade in friendship with all, and he declined to take any part in their quarrels. take any part in their quarrels. By the end of the first year's residence of the Europeans in Table Valley the station had made very satisfactory progress. A large garden had been planted, and the stream that ran down from the mountain had been dammed up in several places, so that the whole of the cultivated ground — several acres in extent — could be irrigated. A plain hospital had been built of earthen walls and a thatch roof, large enough to accommodate two or three hundred men. And another clan had visited the peninsula, with whom a cattle trade had been opened, so that there was plenty of fresh meat for the crews of all the ships that put into the bay. the ships that put into the bay. The second winter was uneventful. Some building was carried on, oxen were trained to draw timber from the forests behind the Devil's peak, and much new ground was broken up. Wild animals gave more trouble than anything else. The lions were so bold that they invaded the cattle pens by night, though armed men were always watching them, and leopards came down from the mountain in broad daylight and THE FIRST CATTLE RAI 2Q carried away sheep under the very eyes of the herds- men. One morning before daybreak there was a great noise in the poultry pens, and when the guards went to see what was the matter, they found that all the ducks and geese had been killed by wild cats. The country appeared to be swarming with ravenous beasts of different kinds. A good look-out was kept over the sea, for the Netherlands and the Commonwealth of England were at war, and it was necessary to guard against being surprised by an English ship. being surprised by an English ship. The Europeans had been living in Table Valley about eighteen months when the first difficulty with the natives occurred. FORMATION OF A REFRESHMENT STATION IN TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. One Sunday while they were listening to a sermon Harry and his people murdered a white boy who was tending the cattle, and ran away with nearly the whole herd. As soon as the event became known pursuing parties were sent out, but though the robbers were followed to the head of False Bay, only one cow that lagged behind was recovered. This occurrence naturally produced an ill-feeling towards the Hottentots on the part of the European soldiers and workmen. One of their companions had been murdered, and his blood was unavenged. The loss of their working oxen imposed heavy toil upon them. The fort was being strengthened with palisades cut in the forests behind the Devil's peak, and these had now to be carried on the shoulders of men. Then for some time after the robbery the pastoral clans kept at a distance, so that no cattle were to be had in barter ; and the want of fresh meat caused much grumbling. 30 REFRESHMENT STATION IN TABLE VALLEY. The directors had given the most emphatic orders that the natives were to be treated with all possible kindness, and so, after a few months, when the run- aways began to return to Table Valley, each one pro- testing his own innocence, no punishment was inflicted upon them for their bad conduct, but they were allowed to resume their former manner of living by collecting firewood and doing any little service of that nature. firewood and doing any little service of that nature. As everything was now in good working order at the station, Mr. Van Riebeek began to send out small exploring parties, not so much to learn the physical condition of the country, however, as to make the acquaintance of Hottentot clans who could be induced to bring cattle to the fort for barter. This object was attained without crossing the nearest range of mountains, and therefore no one tried to go beyond that formidable barrier. During the next few years names were given to the various hills scattered over the western coast belt as far north as the mouth of the Elephant river, the Berg river was inspected from its source to its mouth, and acquaint- ance was made with several Hottentot tribes ; but there was little or no advance in the knowledge of South African geography. FORMATION OF A REFRESHMENT STATION IN TABLE VALLEY BY THE DUTCH EAST INDIA COMPANY. By this time nearly every garden plant of Europe and India was cultivated at the Cape, though potatoes and maize were not yet introduced. Oaks and firs, fruit trees of many kinds, several varieties of vines from Southern Germany and from France, and strawberries and blackberries were thriving. The foreign animals that had been introduced were horses from Java, and pigs, sheep, dogs, rabbits, and poultry from Europe. EXTENSION TO RONDEBOS 3 1 Every season wheat and barley had been sown, but the crop had always failed. Just as it was getting ripe the south-east wind came sweeping through Table Valley, and destroyed it. But it was noticed that even when it was blowing a perfect gale at the fort, nothing more than a pleasant breeze was felt back of the Devil's peak. The commander therefore tried if grain could not be raised in that locality. At a place where a round grove of thorn trees was standing, from which it received the name Rondebosch, a plot of ground was laid under the plough, and some wheat, oats, and barley were sown. The experiment was successful, for the grain throve wonderfully well, and yielded a large return. wonderfully well, and yielded a large return. The Cape establishment was thus answering its purpose admirably, but the expense attending it was found to be greater than the directors of the East India Company had anticipated, so they cast about for some means of reducing its cost. After much discussion they resolved to locate a few burgher families on plots of ground in the neighbourhood of the fort, and instructed Mr. Van Riebeek to carry this design into execution. They were of opinion that the men, though not in their service, would assist in the defence of the station, so that the garrison could be reduced, and that from such persons veget- ables, grain, fruit, pigs, poultry, etc., could be purchased as cheaply as the Company could produce them with hired servants. The plan was to select a few respectable married men from the workpeople, to send their wives and children out to them, and to give them a start as market gardeners or small farmers. FOUNDATION OF THE CAPE COLONY. In February 1657 nine of the Company's servants took their discharge, and had small plots of ground allotted to them along the Liesbeek river at Ronde- bosch. They were the first South African colonists in the true sense of the word. They were provided on credit with everything that was necessary to give them a fair start as agriculturists, and in return they bound themselves to deliver the produce of their ground at the fort at reasonable prices until the debt was cleared off. Within a few months thirty-eight others took their discharge on the same conditions. But it soon appeared that many of these men were not adapted for the life of independent gardeners, and it became necessary to take them back into the Company's service, when the ground that they had occupied was given to others on trial. In this manner a selection was constantly being made, in which only the steady and industrious remained as permanent colonists. There was a rule that only married men of Dutch or German birth should have land assigned to them, INTRODUCTION OF SLAVE 33 but it was not strictly observed, and single men who were mechanics or who would take service with gardeners were frequently discharged by the Com- pany. As soon as a man proved himself able to make a competent living, he had only to apply for his wife and children to be sent out from Europe, and with the next fleet they were forwarded to him. The descendants of many of those who at this time became colonists are now scattered over South Africa from the Cape of Good Hope to the Zambesi and Benguela. Benguela. In this manner the colonisation of South Africa was commenced, but as yet there was no intention of forming a European settlement of any great extent. A few gardeners, fruit growers, and poultry breeders within four or five miles of the fort comprised the whole scheme which the East India Company had in view. These people were to pay tithes of any grain they might produce after twelve years' occupa- tion, but were otherwise to be left untaxed. It was still supposed that as many cattle as were needed could be obtained from the Hottentots. FOUNDATION OF THE CAPE COLONY. In 1658 the great mistake of introducing negro slaves was made, a mistake from which the country has suffered much in the past and must suffer for all time to come. There was no necessity for the intro- duction of these people. The climate for nine months in the year is to Europeans the pleasantest in the world, and even during the other three — excepting from twenty to thirty excessively hot days — white men can labour in the open air without discomfort. The settlement could have been purely European. 4 4 FOUNDATION OF THE CAPE COLONY. 34 But in the seventeenth century it was the custom of all colonising nations to make of the negro a hewer of wood and drawer of water, and the Dutch merely acted in the spirit of the age. merely acted in the spirit of the age. From a Portuguese slave ship captured at sea the first negroes brought to the Cape settlement were taken, and shortly afterwards a number arrived from the coast of Guinea in one of the Company's vessels. Some of them were sold on credit to the burghers, but the greater number were kept by the government to do whatever rough labour was needed. This fixed the character of the colony, for the system once entered upon could only with great difficulty be altered. The white man and the black cannot work side by side on equal terms, hence manual unskilled labour came to be regarded by the Europeans as degrading, a sentiment that has not died out at the present day. The Dutch laws at that time made manumission extremely easy, and freed negroes had all the rights of colonists, though they were without the hereditary training necessary to enable them to make proper use of their privileges. As soon, however, as it was ascertained by experience that they were too indolent and thriftless to put by anything for sickness or old age, when they became a burden upon the commu- nity, a law was made that any one emancipating a slave under ordinary circumstances must not only give security that he or she would not become a dependent upon the poor funds within a certain number of years, but also pay a sum of money into the poor funds as a premium on the risk of his or her requiring aid after that period. FOUNDATION OF THE CAPE COLONY. This checked manu- merely acted in the spirit of the age. From a Portuguese slave ship captured at sea the first negroes brought to the Cape settlement were taken, and shortly afterwards a number arrived from the coast of Guinea in one of the Company's vessels. Some of them were sold on credit to the burghers, but the greater number were kept by the government to do whatever rough labour was needed. This fixed the character of the colony, for the system once entered upon could only with great difficulty be altered. The white man and the black cannot work side by side on equal terms, hence manual unskilled labour came to be regarded by the Europeans as degrading, a sentiment that has not died out at the present day. a sentiment that has not died out at the present day. The Dutch laws at that time made manumission extremely easy, and freed negroes had all the rights of colonists, though they were without the hereditary training necessary to enable them to make proper use of their privileges. As soon, however, as it was ascertained by experience that they were too indolent and thriftless to put by anything for sickness or old age, when they became a burden upon the commu- nity, a law was made that any one emancipating a slave under ordinary circumstances must not only give security that he or she would not become a dependent upon the poor funds within a certain number of years, but also pay a sum of money into the poor funds as a premium on the risk of his or her requiring aid after that period. This checked manu- INTRODUCTION OF ASIAT 35 mission considerably, still, as many slaves were emancipated by the testaments of their owners, and others obtained their liberty under special circum- stances, the free negroes would have become a large body in course of time if they had not been swept away by imported diseases, to which their habits of living made them an easy prey. living made them an easy prey. Besides negroes, the East India Company at this time began to introduce Asiatics — chiefly natives of Malacca, Java, and the Spice islands — into the settle- ment. FOUNDATION OF THE CAPE COLONY. These persons were criminals sentenced by the high court of justice at Batavia to slavery either for life or for a term of years, and then sent to South Africa to undergo their punishment. In in- telligence they were far above the natives of Guinea or Mozambique, many of them being able to work as masons, harness makers, coopers, and tailors. They all professed the Moslem religion. Being without women of their own nationality, they formed con- nections with African slave girls, and thus arose one of the many classes of mixed breeds in the country. of the many classes of mixed breeds in the country. At a little later date the Company made of South Africa a place of banishment for Indian political prisoners of high rank, who were often accompanied by their families and numerous attendants, male and female. These people had fixed allowances from the government for their support. Sometimes families of their dependents became attached to the country, and preferred to remain here when the time came for their return to Java if they had chosen to go back. A race of pure Asiatics thus arose, never very numerous, though for more than a century political FOUNDATION OF THE CAPE COLONY, 36 offenders continued to be sent from Batavia to Cape- town. The last were some natives of high rank in the islands of Tidor and Ternate, who made their escape from this country in 1781. escape from this country in 1781. One of these exiles was the Sheikh Joseph, who took a leading part in the Bantamese civil war of 1682, and who was a determined opponent of the Dutch. He was a man of great repute for sanctity, and was believed by his Moslem followers to have performed some extraordinary miracles. He died near the head of False Bay, and his tomb there is still a place of pilgrimage for Mohamedans in South Africa. Thus early in the history of the Cape Colony three varieties of human beings were introduced : Europeans, negroes, and Javanese or Malays. The Hottentots formed a fourth variety. As years passed away mixed breeds of every colour between these four extremes were to be seen, side by side with the pure races, so that nowhere else could such a diversity of hue and of features be found as in the Cape peninsula. FOUNDATION OF THE CAPE COLONY. The crosses between Europeans and the lower races did not increase in number as rapidly as pure breeds, however, owing to a lack of high fertility among themselves, and unless connected again with one of their ancestral stocks, they often died out altogether in the third generation. The same may be said of the half-breed Asiatics and negroes, but the cross between the others was more fertile. The pastoral Hottentot clans looked upon the European settlement along the Liesbeek with a THE FIRST HOTTENTOT WAR %J good deal of jealousy. It was not alone the ground under cultivation that was lost to them, for the government had given the burghers a free right of pasture, and thus the former owners were excluded from the best patches of grass along the base of the mountain. Early in 1659 the two clans that had always been accustomed to visit the Cape peninsula in the summer season with their flocks and herds appeared there as usual, and were informed that they must keep away from the grass that the Company and the burghers needed. This announcement was not at all to their liking. They had ample pasture left in the peninsula for ten times the number of cattle in their possession, and all the country stretching away beyond the isthmus as far as could be seen was theirs to roam over ; but they were like other people, they did not relish being deprived by force of any- thing that they regarded as their own. So they commenced to drive off the burghers' cows, and murdered a white herdsman. The beachrangers in Table Valley, though they were certainly gainers by the presence of the Europeans, and though they had a long-standing feud with the pastoral clans, now joined their countrymen. In this manner what the colonists termed the first Hottentot war began. the colonists termed the first Hottentot war began. In fact, however, it could hardly be termed a war, for the natives were careful to avoid a pitched battle, and the Europeans were unable to surprise any large body of them. On two occasions only were small parties met, when six or seven were killed and a few more were wounded. The pastoral clans then abandoned the peninsula, and the beachrangers, FOUNDATION OF THE CAPE COLONY. 3* upon begging for peace, were allowed to return to Table Valley. FOUNDATION OF THE CAPE COLONY. A strong fence, through which cattle could not be driven, was now made along the outer boundary of the settlement, three watchhouses were built to defend it, and in these were stationed companies of horse- men, whose duty it was to patrol the border. Some powerful dogs were imported from Java, and the Europeans then considered themselves secure. The Hottentots were the first to make overtures for a restoration of friendship. About a twelvemonth after the first breach of the peace they sent messengers to the fort to propose a reconciliation, and as these were well received the chiefs followed, when terms were agreed to. These were that neither party was thereafter to molest the other, that the lately hostile clans were to endeavour to induce those living farther inland to bring cattle to the fort for sale, that the Europeans were to retain possession of the land occupied by them, that roads were to be pointed out along which the Hottentots could come to the fort, and that any European who molested a Hottentot should be severely punished. After the conclusion of peace the cattle trade went on briskly. Bartering parties were sometimes sent out, but Hottentots often came from a distance of eighty or a hundred miles with troops of oxen and flocks of sheep for sale. They were very eager to obtain bright coloured beads and other trifles. The quantity of beads given for an ox cost only from eight to ten pence, but there were other and larger expenses connected with the trade. Presents, consisting of flat THE FIRST CHUR 39 pieces of copper, wire, iron rods, axes, tobacco, pipes, and other articles, were frequently made to the chiefs to secure their friendship, and all who came to the fort were liberally entertained. The burghers were strictly prohibited from holding intercourse of any kind with other Hottentots than the beachrangers, but in defiance of the law some of them found means to carry on a petty cattle trade. to carry on a petty cattle trade. After ten years' service in South Africa Mr. Van Riebeek was sent on to India, where he received promotion, and Mr. Zacharias Wagenaar took his place at the Cape. During this commander's term of office — which extended over four years — a few events occurred that are worthy of notice. FOUNDATION OF THE CAPE COLONY. In earlier years a catechist, who was also a school- master, held services on Sundays, and the chaplains of ships that called administered the sacraments. But now the settlement was provided with a resident clergyman. From this time onward there was a fully organised church, subject to the spiritual control of the classis or presbytery of Amsterdam. the classis or presbytery of Amsterdam. In 1664 tne island of Mauritius was taken into possession by the Dutch East India Company to keep it from falling into other hands, and it was made a dependency of the Cape station. A few men were sent there to cut ebony logs, and once a year a packet took supplies to them from Table Bay and brought back a cargo of timber. Owing to the threatening attitude of England, the directors resolved to build a strong fortress in Table Valley, as the walls of earth, which were considered ample protection against Hottentots, FOUNDATION OF THE CAPE COLONY. 40 would be a poor defence if a British force should land. The castle of Good Hope, which is still standing, was commenced in 1666, and was com- pleted in 1674. It is now useless for military purposes, but for some time after its construction it was considered almost impregnable. The directors, who were beginning to realise that the French and the English might prove formidable rivals in the eastern seas, had come to regard their station in the Cape peninsula as a strategic point of great importance. " The castle of Good Hope," they wrote, " is the frontier fortress of India," and as such they provided it with a strong garrison. they provided it with a strong garrison. Commander Wagenaar's successors for some time were men of very little note, and nothing of much con- sequence occurred to disturb the quiet course of life in the settlement. All was bustle and activity when the outward or homeward bound Indian fleets were in the bay, but after their anchors were raised there was nothing to create excitement. The workmen engaged in building the castle and the garrison when it was completed increased the demand for food, so that more servants of the Company took their discharge and set up for themselves as market gardeners. FOUNDATION OF THE CAPE COLONY. Soon the best plots of land within the boundary fence were all taken up, and then the fence was disregarded and the settlement spread out to the present village of Wynberg. The proportion of men who took their discharge and succeeded in making a living on their own account was, however, always very small. It was probably not ten per cent of the whole. The others after a trial had to be taken back into the FOUNDATION OF THE CAPE COLONY. 42 Company's service as soldiers or sailors, most of them with debts that they could never wholly clear off. The system was thus a very unsatisfactory one. The directors thought of improving upon it by sending out families accustomed to agriculture in the Netherlands, who would serve as models for the others ; but though they offered free passages, grants of land without payment, exemption from the tithe for twelve years, and supplies of necessaries on easy credit, very few people of the class required could be induced to migrate to South Africa. The Cape was too far away and too little was known of it to tempt men and women to leave a country where there was no difficulty in making a comfortable living. In 167 1, however, five or six families arrived, and thereafter during several years one or two came out occasionally. occasionally. Twenty years after Mr. Van Riebeek landed and took possession of as much ground as he needed, without thinking of asking the consent of any one, a member of the high court of justice at Batavia on his way back to Europe called at the Cape, and being superior in rank to any one here, took command during his stay. This officer — Arnout van Overbeke by name — considered it advisable to make a formal purchase of territory from the nearest Hottentot chiefs, and these petty potentates, on being applied to, very readily gave their consent. FOUNDATION OF THE CAPE COLONY. If they reasoned at all about the matter, they probably thought that the price offered was clear gain, for the white people would certainly take as much ground as they needed, whether sold to them or not, PURCHASE OF TERRITO 43 At any rate the principal chief of one tribe and the regents of another affixed their marks to documents that are still in existence, in which they ceded to the East India Company the whole territory from Saldanha Bay to False Bay, reserving to themselves and their people, however, the right to move freely about and make use of any part of it not occupied by Europeans. They received nominally in return goods to the value of £\,6oo, actually — according to the accounts furnished to the directors — the articles transferred cost £g 12s. gd. transferred cost £g 12s. gd. A few months after this transaction an outpost was formed at Hottentots-Holland, near the head of False Bay, on the eastern side of the isthmus. One of the objects in view was to raise a large quantity of wheat, for which purpose the ground at that place appeared specially suitable, but the chief design was to pro- vide a retreat for the garrison in case it should be needed. The Free Netherlands were then engaged in the most unequal struggle that modern Europe has witnessed, for Louis XIV of France, Charles II of England, and the ecclesiastical princes of Cologne and Munster were united against them. On two occasions shortly before the war broke out the admirals of French fleets had taken possession of Saldanha Bay, though without leaving any men there to guard it, and it was believed that the French king might make an effort to seize the Cape penin- sula. An attack by the English was equally probable. As events turned out, the Dutch got the better of their opponents in Europe, and the Cape settlement was not molested, so that the outpost at Hottentots- FOUNDATION OF THE CAPE COLONY. 44 Holland became simply a farming establishment where the wheat and other grain needed by the Company was grown. Company was grown. By this time the belt of land along the sea coast as far eastward as Mossel Bay had been thoroughly explored by parties sent out to obtain cattle. FOUNDATION OF THE CAPE COLONY. In 1658 some members of a trading expedition climbed to the top of the mountain barrier near the ravine through which the Little Berg river flows ; but the land as far as they could see appeared to be un- inhabited, so there was nothing to induce further search in that direction. Nine years later another way over the barrier was found at the place now known as Sir Lowry's pass, sixty miles south of the ravine of the Little Berg river. The Hottentot tribes termed the Hessequa, Gauriqua, Attaqua, and Oute- niqua were then successively reached and traded with. Those previously known along the western coast were the Chainouqua, the Goringhaiqua (or Kaapmans), the Cochoqua, the Grigriqua, and the Namaqua. the Cochoqua, the Grigriqua, and the Namaqua. Bushmen had been met on several occasions, and their manner of living was pretty well known. Some of these wild people had once attempted to seize the merchandise belonging to a European trading party, when a number of them were shot down, greatly to the satisfaction of the Hessequa and other Hottentots who lived in the neighbourhood of the place where the event occurred. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. ONE of the most powerful of the Hottentot tribes near the Cape peninsula was called the Cochoqua, or by some name which the Europeans wrote in that form, for probably it had clicks in it. This tribe was composed of two great clans, the larger of which was under a chief named Gonnema. Gonnema had an evil reputation among all the other Hottentots with whom the Dutch were acquainted, for he was in the habit of swooping down upon them unawares and helping himself to their daughters and their cattle, and they were too weak to resist him. He had sold a good many oxen to the white people, but they did not like him either, for his bearing was never very friendly. They usually termed him the black captain, on account of his habit of using soot instead of clay to paint himself with. In 1673 a war broke out between Gonnema and the Europeans, the only war that has ever taken place between white people and natives in South Africa of which we have not the versions of both parties to 45 45 THE SECOND HOTTENTOT WAR. 46 form a judgment from. The Cochoqua clan has left no story, nor is there a plea on its behalf on record. But the Dutch accounts are full of details, and it is easy from them to ascertain how Gonnema came to feel himself aggrieved. The country was teeming with game, antelopes of many kinds, elephants, rhinoceroses, and hippopotami. Hunters were sent out from the fort, and brought back waggons laden with dried meat, which was supplied to the garrison instead of beef. The hippo- potamus, or sea-cow as it was called, was specially sought after, for its flesh was regarded as equal to pork, and whips made of its hide commanded a very high price everywhere. Parties of burghers were in the habit of getting leave from the government and going out hunting elands and sea-cows, sometimes being away from home three or four weeks together. It never seems to have occurred to the white people that the Hottentots might object to the destruction of so much game, but very likely that was the cause of Gonnema's hostility. of Gonnema's hostility. In 1672 he came upon some hunters at Riebeek's Kasteel, and took their waggons and other property, but allowed them to escape with their lives. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. In the following year he made prisoners of eight burghers and a slave who were hunting near the same place, and after detaining them some days murdered them all. At the same time one of his sub-captains surprised a little trading outpost of the Company at Saldanha Bay, plundered it, and murdered four Europeans. Bay, plundered it, and murdered four Europeans. A mixed force of soldiers and burghers was then sent against the Cochoqua clan, and as it was partly THE SECOND HOTTENTOT WAR 4? composed of horsemen it had the good fortune to cut off Gonnema's people from a strong position to which they tried to retreat and to seize eight hundred of their horned cattle and nine hundred sheep. The Hottentots followed the expedition when returning to the fort, but did not succeed in recovering their stock. Ten or twelve of them were shot, and on the other side one burgher was wounded. Various clans now offered their aid against Gonne- ma, and were accepted as allies by the Europeans. For several months the Cochoqua kept out of the way, but at length they were so nearly surrounded that they barely managed to escape, leaving all their cattle behind. The spoil was much greater than on the first occasion, and was divided between the Europeans and the Hottentot allies. Gonnema after this loss kept to the mountains for nearly two years, avoiding his enemies, but prevent- ing all intercourse between them and the tribes beyond. Then he pounced suddenly upon some of the Hottentot allies of the Europeans, and with the loss of only fifteen of his own men killed a good many of them and swept off the greater part of their herds. He was pursued by all the soldiers and burghers that could be mustered, but he got safely away to his mountain fastnesses. No expedition sent against him after that time managed to surprise him, for his scouts were always on the alert. The Euro- peans found that they were wearying themselves to no purpose in trying to find him, so they desisted from the fruitless task. 48 THE SECOND HOTTENTOT WAR. For four years the settlement was kept practically in a condition of blockade on the land side, when Gonnema sent to ask for peace, as he was tired of living like a Bushman in the mountains. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. His messengers were well received, and were followed by three of his chief men, who agreed in his name to the terms proposed. They were that there should be peace and friendship between all the parties engaged in the war, and that Gonnema should pay to the Company a yearly tribute of thirty head of cattle. Presents were then exchanged, and the land was once more at rest. Perhaps it was never intended that tribute should really be paid, at any rate it was considered prudent not to refer to the subject again, and the Cochoqua clan was left untroubled about it. This was the last war with Hottentots during the rule of the Dutch East India Company in South Africa. It was a trifling affair if considered by the number of combatants or the quantity of spoil, and not a single hand-to-hand engagement had taken place ; but it had very important consequences. During four years Gonnema had cut off the cattle trade, so there was no fresh beef or mutton for the crews of the fleets that called, and even the hospital could not be supplied, as the oxen and sheep that were captured and that the clans in alliance with the Europeans were able to furnish were soon exhausted. The Company was not disposed to run the risk of a second experience of this kind. The expense of the station had grown very far beyond original ex- pectations, there was now a huge fortress to be kept This was the last war with Hottentots during the rule of the Dutch East India Company in South Africa. It was a trifling affair if considered by the number of combatants or the quantity of spoil, and not a single hand-to-hand engagement had taken place ; but it had very important consequences. ORIGIN OF THE CATTLE FARMERS 49 up and a large garrison to be maintained in addition to victualling charges properly so called ; and such an outlay could only be justified by the perfect efficiency of the establishment. If a supply of fresh meat could not be depended upon, one of the main objects of its existence was a failure. And as trade with the Hottentots beyond the nearest mountains might at any time be cut off again, European cattle breeders must be introduced. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. The great difficulty in the way was the scarcity of Europeans with the habits needed. They would be obliged to live far apart, and would be exposed to plunder by the natives and losses from the ravages of wild animals. The country was swarming with lions, leopards, hyenas, and jackals ; and with the clumsy firelocks of those days it was a risky matter to go out alone into the wilds. As a commencement the Company established several cattle posts on its own account on the eastern side of the isthmus, to which cows and ewes were sent as they could be procured in barter, and at each a corporal was stationed with a few soldiers to guard the stock. Then offers were made to the gardeners at Ronde- bosch and Wynberg to improve their prospects by turning cattle breeders. They could select land near the Company's posts, so that they would not be alto- gether without companionship, no taxes of any kind would be demanded from them till they were in a good position, and breeding cattle would be lent to them to take care of, half the increase of which would be their own. The view of the government was that if gardeners and small farmers could not b^ 5 5 EXTENSION OF THE SETTLEMENT. 50 procured as emigrants from Europe, they must be trained in the old way of selection to take the place of those who should become stock breeders. The prospect, however, did not appear very attrac- tive, for before the close of 1679 only eight burghers accepted the Company's offer and took up their residence beyond the isthmus. residence beyond the isthmus. At this time a very energetic man, named Simon van der Stel, arrived from Amsterdam as commander. No one could have been better qualified to carry out the new project, and he threw himself heart and soul into it. He had, however, a particular desire that only Netherlanders should settle in the country, for he believed that whatever was Dutch was good, and whatever was not Dutch was not worth bothering about. But the directors in Holland were not of this opinion. They were very glad to obtain the services of competent men of all nationalities, and provided the majority of the settlers were Dutch they were quite willing to give equal privileges to others. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. Except in this respect Simon van der Stel was allowed to carry out the plan in his own way. He began by inducing a party of eight families to re- move from Rondebosch to a fertile and beautiful valley beyond the isthmus, where he gave them large plots of ground in freehold, with extensive grazing rights beyond. This settlement, which was named Stellenbosch, he intended to be the centre of a dis- trict in which all kinds of farming pursuits should be carried, on, where vineyards .should be planted and . wine be made,. - where wheat, should be grown ARRIVAL OF HUGUENO 5 I and cattle be reared. When the fleets from India put into Table Bay on their way home, the com- mander's agents ingratiated themselves with the people on board, and whenever a man likely to make a good colonist was discovered, inducements were held out to him to remain in the country. In this way the vacancies were filled up in the Cape peninsula as fast as they arose, and many new- comers could be located beside experienced men at Stellenbosch. A few years later a settlement was formed in a similar manner at Drakenstein, in the valley of the Berg river, one of the most charming situations in South Africa. The directors were doing all that was in their power to get suitable people to migrate from the Netherlands. Among others they sent out a few- young women from the orphan asylums in Amster- dam and Rotterdam, who were carefully protected and provided for until they found husbands in the colony. And now an event took place in Europe which enabled them to secure over a hundred families of the very best stamp. This was the revocation by Louis XIV of the edict of Nantes, which drove many thousands of Protestant refugees from France into Holland. Their presence in some of the pro- vinces so greatly reduced the demand for labour that industrious Dutch families were more willing to remove than they had previously been, and the Company was able to send to South Africa nearly two hundred Huguenots and about the same number EXTENSION OF THE SETTLEMENT. 52 of Dutch people of both sexes and all ages. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. Upon their arrival they were scattered over the country between the Groenberg, the Koeberg, and Hotten- tots-Holland, the larger number of the French being located in the valley of the Berg river. Care was taken, however, to mix them together, so that the nationalities would speedily become blended. nationalities would speedily become blended. Having now a base to fall back upon if necessary, a few stragglers began to push their way in one direction down the Berg river, and in another beyond the Koeberg. Still, at the close of the seventeenth century there was no white man living more tha'n forty-five miles from the castle, and the whole terri- tory occupied by Europeans was within the range of mountains visible from ships in Table Bay. The colonists, with their wives and children, were then some fourteen hundred in number. The French Huguenots were about one-sixth of the whole, a rather larger proportion consisted of Germans from the borderland between the high and low Teutons, and nearly two-thirds were Dutch from the different Netherland provinces. The Germans were, almost without exception, men who were married to Dutch women. Intermarriages between the Huguenots and other colonists were common, and in another generation distinctions of nationality were entirely lost. The language used in common conversation was Dutch made as simple and expressive as possible, so as to be understood by slaves with only the mental capacities of children. Grammatical rules were dis- regarded. In the pulpit and in family devotions, FORM OF GOVERNME 53 however, correct Dutch was used, as it is very generally to the present day. generally to the present day. There were three churches provided with clergy- men : in Capetown, at Stellenbosch, and at Draken- stein. At each of these places there was also a public school, in which children were taught to read the bible, to cast up simple accounts, to repeat the Heidelberg catechism, and to sing the psalms. The Dutch reformed was the state church, and no other public services were allowed, but in their own houses people might hold any kind of worship that they pleased. pleased. The head of the settlement was now termed the governor. Public matters of all kinds were regulated by a council of eight individuals, who were the highest officials in rank in the country. In this council the governor sat as president. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. In Capetown there was a court for the trial of petty cases, and a high court of justice, from whose decisions there was an appeal to the supreme court at Batavia. Three burghers had seats in the high court of justice whenever cases affecting colonists were tried. They were the spokes- men between the colonists and the government, and were consulted upon all matters affecting the settle- ment, but they had no votes outside the court of justice. They were called burgher councillors. At Stellenbosch there was a court which had cog- nisance of all petty cases beyond the Cape peninsula. It was presided over by an official termed a landdrost, who also collected the revenue and looked after the Company's interests generally. Eight burghers — termed heemraden — had seats and votes. This court FORM OF GOVERNMENT. 5 acted further as a district council, in which capacity it saw to the repair of roads, the distribution of water, the destruction of noxious animals, and various other matters. It raised a revenue by erecting a mill to grind corn and leasing it to the highest bidder, by collecting a yearly tax of one shilling and fourpence for every hundred sheep or twenty head of horned cattle owned by the farmers, and by sundry other small imposts. Further, it had power to compel the inhabitants to supply waggons, cattle, slaves, and their own labour for public purposes. their own labour for public purposes. In Capetown there was an orphan chamber, which acted as trustee of property belonging to children when a parent died. There was also a matrimonial court, before which every person in the settlement — male and female — who wished to be married had to appear and show that there were no legal impedi- ments to the union. All these bodies — burgher councillors, petty court of justice, heemraden, orphan chamber, and matri- monial court — as also the consistories of the churches, were in a manner self-perpetuating corporations. Every year some of their members retired, but before doing so double lists of names were sent by the boards to the government, and from these lists their successors were appointed. Such a thing as popular election to any office was unknown. The system worked well on the whole, and the people were satisfied with it. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. The burghers were required to meet at stated periods for drill and practice in the use of arms, and all were held liable for service in case of the FORM OF GOVERNME 55 appearance of an enemy. They were formed into companies of cavalry and infantry, each with its standard of a particular colour. Most of them were excellent marksmen, owing to their habits of fre- quently hunting game ; but their firearms were very clumsy weapons compared with those of our day. To reduce the cost of the garrison, numbers of soldiers were permitted to engage as servants to burghers, on condition that they could be called back to their colours at any time. Capetown at the close of the century contained about eighty private houses. The great garden of the Company, which Simon van der Stel had partly converted into a nursery for European, Indian, and native plants, was regarded as something marvellous by visitors of all nations, so great a variety of vege- tation was to be seen and admired there. This governor, upon his retirement in 1699, went to live upon a beautiful estate which the Company gave him a little beyond Wynberg, where he planted the vine- yards that in later years produced the celebrated Constantia wines. He was an enthusiast in the matter of cultivating oak-trees, and during the twenty years of his government many scores of thousands were planted by his orders all over the settlement. The system of taxation was as bad as could be devised, so far as effect upon the character of the people was concerned. The exclusive right to sell various articles — among others spirituous liquors — was sold by auction as a monopoly to the highest bidder. Upon anything that the government re- quired— such as beef and mutton — a price per pound SYSTEM OF TAXATION. 5 was fixed to the residents in Capetown, and a much higher price to foreigners ; the monopoly was then put up for sale, and whoever offered to supply the Company at the lowest rate became the purchaser. Thus beef might be a penny a pound to a mechanic in Capetown, twopence a pound to the captain of an English ship, and a halfpenny a pound to the Com- pany's hospital. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. The tithe of grain and the district tax upon cattle were both paid upon returns made by the farmers themselves, so that a premium was offered for falsehood. This system was soon dis- covered to be vicious, but it was continued without modification until towards the close of the next century. The government exercised the right of fixing the price of anything that it needed, and prohibiting the sale of the article to any one else until its own wants were supplied. Thus, if half-a-dozen Dutch ships and two or three English Indiamen were lying at anchor in Table Bay at a time when the meal in the Com- pany's stores was exhausted, the Englishmen would certainly get no bread until the Hollanders' tables were covered. But, upon the whole, strangers were much better treated here than the Dutch were treated in foreign ports, and it was only in times of scarcity that they had cause to complain of anything except high prices. high prices. The Company was supposed to be the only whole- sale merchant in the country. From its stores in Capetown shopkeepers were supplied with imported goods, and everything that was exported in bulk passed through its hands. But in point of fact a SYSTEM OF TRA S7 large proportion of the trade of the country was carried on with ships' people, Dutch and foreign. From the captain of an Indiaman of any nationality down to the youngest midshipman, every one had some little venture of his own that he was always ready to trade with. It might be a bale of calico, or it might be a slave, for many bondsmen were brought to South Africa in this way. Even among the fore- mast hands the spirit of commerce was strong. A sailor when homeward bound was commonly accom- panied by monkeys and parrots and cockatoos and various descriptions of birds and beasts till the fore- castle was often like a menagerie ; in his chest he had fancy articles from Japan, silk handkerchiefs from China, or perhaps some curiosities picked up at an Indian isle. This kind of commerce was very petty, but in the aggregate it must have amounted to some- thing considerable, for the inhabitants of Capetown during more than a hundred years lived and throve upon it. THE SECOND HOTTENTOT WAR AND ITS CONSEQUENCES. Nowhere in the world, we read again and again in accounts of travellers of many nationalities, could a greater variety of goods be purchased and sold. The traffic was carried on openly, for though there was a law that goods should not be imported by foreigners, it was not applied to ventures by ships' people — English, French, or Danish — on their own account. By this time the knowledge of South African geography had very greatly increased. Simon van der Stel himself with a large party of attendants had visited the copper mountain of Little Namaqualand, and had received information from the natives there EXPLORA 59 of the great river now known as the Orange. Some Dutch and English sailors, shipwrecked on the eastern coast, before they were rescued had travelled in one direction to Delagoa Bay, and in the other to the Buffalo river. From them as accurate an account of the southern Bantu tribes was obtained as any we have at the present day. Parties of cattle traders had brought back information of every Hottentot tribe in existence except the Korana. But as yet no white man had set foot upon the plain of the Karoo, and consequently nothing was known of the far interior. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. In the first year of the eighteenth century the second terrace upward from the sea began to be occupied by Europeans. Wilhem Adrian van der Stel, eldest son of Simon van der Stel, was then governor. He made a tour of inspection through the settlement, and afterwards crossed the mountain range close to the ravine of the Little Berg river, for the purpose of examining the country beyond. It was the beautiful tract of land now called the Tulbagh basin which the governor and his party entered, and it seemed to them to invite human occupation. The settlement on the coast belt had been a success, but it had not been able to supply many cattle. The land was adapted for cultivation, there was a market within easy reach, and the ideas of the people favoured the plough. To this day the inhabi- tants of that part of the country depend upon their crops of wheat, their fruit, and their wine, and keep no more oxen and sheep than are required for their own use. LIFE OF THE EARLY SETTLER 6l On the other side of the mountain range agriculture was not then possible. The Tulbagh basin is fertile ; its extensive corn lands, its vineyards, and the gardens and orchards round its homesteads form now a pleasant sight in the early summer ; but in those days, when there was no other way of access to it than over a mountain that could only be crossed with the greatest difficulty, no man would think of making a living by the plough. Cattle breeding alone could be depended upon there. A small military outpost was formed in the basin to protect the settlers from the Bushmen who had their haunts in the surrounding mountains, and then several families were induced to try their fortune in the lonely vale. A few individuals were still being sent out from Europe every year, and the old system of discharging servants of the Company continued in practice, so that settlers were obtainable. But as a rule new-comers were located near the Cape peninsula, and young people born in the country commenced life for themselves beyond the mountain range. life for themselves beyond the mountain range. Gradually they spread beyond the Tulbagh basin, down the valley of the Breede river, and over the Witsenberg to the high plateau called the Warm Bokkeveld. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. Others crossed the range at Hottentots- Holland, and pushed their way along the banks of the Zonderend and down towards the sea at Cape Agulhas. Others again kept up the western coast belt, passed the Piketberg, and in course of time reached the mouth of the Elephant river. The life led by these- pioneers of civilisation was rough and wild, but it had its own peculiar charm. 63 LIFE OF THE EARLY SETTLER Cattle breeding was found to pay fairly well. Some- times Bushmen would drive off oxen or sheep, and a little excitement was caused by the pursuit of the marauders, but otherwise the time passed away quietly. The best of health was commonly enjoyed, and there was the most perfect freedom. The only direct tax towards the general revenue was £$ a year, which each grazier was required to pay, and for which he had a legal right to the use of six thousand acres of ground, with the privilege of moving into the Karoo for a couple of months every year to give his stock a change of pasture. During these migra- tions he and his family slept in a great tent waggon, and passed the day in the open air, usually selecting a patch of trees on the bank of a stream for a camp- ing place. A distaste for town life, with its restraints and all the nameless annoyances to which simple people are exposed when in contact with men of sharper intellect, soon became part of the nature of a cattle breeder, and grew stronger with each succeed- ing generation. The children and grandchildren of Dutch gardeners, German mechanics, and Huguenot tradesmen by force of circumstances reverted in habits and in thought to the condition of semi- nomads. In the language of South Africa these people were called trekboeren, that is wandering farmers. Many of them became expert elephant hunters, and travelled great distances in search of ivory. Behind them, as they moved onward, a more settled class of people occupied the country, though very thinly. -These built better- houses- than the others; as CAPE COLONY FROM I7OO TO 1750. 64 soon as rough roads were made they combined agri- culture with cattle breeding, and generally they led more stable lives. In this manner hardly a year passed by without an expansion of the European settlement. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. Wilhem Adrian van der Stel, who was governor when the colonists crossed the first mountain range, was a man of culture and ability, but he had an inordinate passion for making money, a fault that was common to many of the Company's servants. To get together a fortune with which to return to the Netherlands was an object ever before the eyes of these men, and the system under which they served favoured the accomplishment of their plans. The Company paid its officers the smallest of salaries, but allowed them perquisites of various kinds. In the preceding chapter mention has been made of the trade carried on under its sanction by the people of its fleets. Its officials on shore had even greater privileges. Some of them received commissions on sales and purchases of goods, others held monopolies of lucrative duties, others again were allowed to trade in specified articles on their own account. Spices only were guarded with the most extreme jealousy, and if any one had ven- tured to buy or sell a pound of pepper or a dozen nutmegs except at the Company's stores, dismissal from the service would have been the lightest part of his punishment. his punishment. It followed from this system that in the early days of the settlement, the officials in South Africa were in general exceedingly anxious to get on to iYidia, WILHEM ADRIAN VAN DER ST EL 65 because there was little or nothing to be made here. Some of them had been allowed to carry on trade on their own account, the landdrost of Stellenbosch had a monopoly as an auctioneer, the secretary of the council had a monopoly of making out certain legal documents,, and several others had exclusive privi- leges ; but what were these petty gains, they thought, to the wealth that was gathered by others in the Indian isles? Wilhem Adrian van der Stel looked about for some means to fill his purse, but could devise nothing else than a farm. There was a law that neither the governor nor any other high official should carry on farming or cattle dealing on his own account, and on several occasions the directors reminded him of it, but by fraudulent means and in direct opposition to the policy of the Company he obtained possession of a large tract of land at Hottentots-Holland. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. There he erected extensive buildings, planted nearly half a million vines, and laid out groves, orchards, and corn lands to a corresponding extent. In the open country beyond the mountain he kept from six to eight hundred breeding cattle and eight or ten thousand sheep. Of this extensive establishment the directors were kept in entire ignorance, and there is no mention whatever of it in any official docu- ment until complaints against the governor reached Holland. The burghers looked upon the big farm with very lively indignation. Their principal gains were derived from the sale of produce to foreigners, and they saw that market being practically closed to; 6 6 6 j •€ < c C 5 c j • < c C 5 c WILHEM ADRIAN VAN DER STEL 6j them. The governor, they believed, would manage to secure the larger part of any profitable business for himself, and whatever escaped him would fall either to his father, who was farming at Constantia, or to his younger brother, who was farming below Stellenbosch. There have never been people less willing to submit silently to grievances, real or imaginary, than the colonists of South Africa. In 1705 some of them sent a complaint of what was going on to the governor-general and council of India, but at Batavia nothing was done in the matter. Probably they did not expect redress from that quarter, for before there was time to receive a reply, a memorial to the directors was drawn up and signed by sixty- three of the best men in the settlement. In this document Wilhem Adrian van der Stel was accused of misconduct and corrupt practices tending to the serious loss and oppression of the colonists. Similar charges, but in a lower degree, were made against the officer next in rank to the governor and against the clergyman of Capetown. These persons also had been neglecting their public duties, and devoting their attention to farming. With the arrival of the homeward-bound fleet at the beginning of 1706 the governor learnt of the complaint sent to Batavia, and immediately suspected that a similar charge would be forwarded to Holland. The danger of his position now drove him to acts of extreme folly as well as of tyranny. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. He caused a certificate to be drawn up, in which he was credited with the highest virtues and the utmost satisfaction CAPE COLONY FROM I7OO TO 1750. 68 was expressed with his manner of ruling the colony. The residents in the Cape peninsula were invited to the castle, and were then requested to sign this cer- tificate. The landdrost of Stellenbosch was directed to proceed with an armed party from house to house in the country, and get the residents there to sign it also. By these means two hundred and forty names in all were obtained, including those of a few Asiatics and free blacks. Many, however, refused to affix their signatures, even under the landdrost's threat that they would be marked men if they did not. that they would be marked men if they did not. The governor suspected that a farmer at Stellen- bosch named Adam Tas was the secretary of the disaffected party, and the landdrost was directed to have him arrested. Early one morning his house was surrounded by an armed party, he was seized and sent to the castle, his premises were searched, and his writing-desk was carried away. There could be no truce after this between the. governor and his opponents, for if a burgher could be treated in this manner, upon mere suspicion of having drawn up a memorial to the high authorities, no man's liberty would be safe. Bail was at once offered for the appearance of Tas before a court of justice, but was refused. He was committed to prison, where he was kept nearly fourteen months. kept nearly fourteen months. In his desk was found the draft from which the memorial to the directors had been copied. It was unsigned, but papers attached to it indicated several of those who had taken part in the matter. Within the. next few days seven of these were arrested, two of whom were committed to-, prison, one was sent to WILHEM ADRIAN VAN DER STEL 6g Batavia, and four were put on board a ship bound to Amsterdam. The governor hoped to terrify them into signing the certificate in his favour and denying the truth of the charges against him, but not one of them faltered for a moment. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. Their wives petitioned that the prisoners might be brought to trial at once before a proper court of justice, and when it was hinted that if they would induce their husbands to do what was desired, release would follow, these true- hearted women indignantly refused. In the mean- time the memorial had been committed to the care of a physician in the return fleet, and after the ships sailed he gave it to one of the burghers who were banished. The governor continued to act as if his will was above the law of the land. Further arrests of burghers were made by his direction, the properly constituted courts were abolished, and in their stead his creatures were appointed to office. The people of Stellenbosch, men and women, announced their determination to maintain their rights, upon which a body of soldiers was sent to support the Landdrost. body of soldiers was sent to support the Landdrost. Meantime three of the burghers sent to Europe arrived at Amsterdam, the other having died on the passage, and they lost not a day before presenting the memorial to the directors and making their own complaint. In a matter of this kind it was necessary to act with promptitude as well as with justice. The Company had numerous and powerful enemies always watching for a chance to attack it before the states- general, and a charge of oppression of free Nether- landers in one of its colonies would be a weapon of CAPE COLONY FROM I7OO TO 1 7 50. 70 which they would not fail to make good use. A commission was therefore appointed to investigate the matter, and a report was sent in by it that the charges were fully substantiated. In consequence the governor, the officer next in rank, the clergyman of Capetown, and the landdrost of Stellenbosch were ordered to return to Europe by the earliest opportunity, and as their guilt was fully proved they were dismissed from the service. Wilhem Adrian van der Stel's farm, as having been obtained by fraud and in direct opposition to their order, was confiscated. From the documents connected with this case the views of the directors and of the colonists concerning the government of the country and the rights of its people can be gathered with great precision. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. The directors desired to have a large body of freemen living in comfort, loyal to the fatherland, ready and willing to assist in the defence of the colony if attacked, enjoying the same rights as their equals in Europe, and not differing much from each other in rank or position. They issued orders that no official, from the highest to the lowest, was to own or lease land in the colony or to trade in any way in corn, wine, or cattle. The burghers were to be governed in accordance with law and justice. governed in accordance with law and justice. On their part, the colonists claimed exactly the same rights as if they were still living in the Nether- lands. They expressed no wish for a change in the form of government, what they desired being merely that the control of affairs should be placed in honest hands. In their opinion they forfeited nothing by FIRST OUTBREAK OF SMALL- POX J\ removal to South Africa, and the violence displayed by the governor towards Adam Tas and his associates was as outrageous as if it had taken place in the city of Amsterdam. They asserted their undoubted right to personal liberty, to exemption from arrest unless under reasonable suspicion of crime, to admission to bail, to speedy trial before a proper court of justice, to freedom to sell to any one, burgher or foreigner, except under special circumstances when restriction was needed for the good of the community, whatever their land produced, after the tithes had been paid and the Company's needs had been supplied. And these claims, made in as explicit terms as they could be to-day by an Englishman living in a crown colony, were not challenged by the directors or even the partisans of the late governor, but were accepted by every one as unquestioned. every one as unquestioned. In 1 710 the island of Mauritius was abandoned by the Dutch East India Company, the directors having come to the conclusion that it was not worth the cost of maintaining a large garrison, and that with a small garrison it was not secure. A few colonists who were there had the choice of removal to South Africa or to Java, and nine families elected to come to this country. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. A few months later the French took possession of the island, and under them it soon became a place of importance. In 17 1 3 a terrible evil came upon the country. In March of that year the small-pox made its first appearance in South Africa. It was introduced by means of some clothing belonging to ships' people who had been ill on the passage from India, but CAPE COLONY FROM t?00 TO I/^O. 72 who had recovered before they reached Table Bay. This clothing was sent to be washed at the Com- pany's slave lodge, and the women who handled it were the first to be smitten. The Company had at the time about five hundred and seventy slaves of both sexes and all ages, nearly two hundred of whom were carried off within the next six months. From the slaves the disease spread to the Euro- peans and the natives. In May and June there was hardly a family in the town that had not some one sick or dead. Traffic in the streets was suspended, and even the children ceased to play their usual games in the squares and open places. At last it was impossible to obtain nurses, though slave women were being paid at the rate of four and five shillings a day. All the planks in the stores were used, and in July it became necessary to bury the dead without coffins. During that dreadful winter nearly one- fourth of the European inhabitants of the town perished, and only when the hot weather set in did the plague cease. the plague cease. The disease spread into the country, but there the proportion of white people that perished was not so large as in the town. It was easier to keep from contact with sick persons. Some families living in secluded places were quite shut off from the rest of the colony, and the farmers in general avoided moving about. The death-rate among the free blacks was very high, but it was among the Hottentots that it reached its maximum. Whole kraals in the neigh- bourhood of the Cape peninsula disappeared without FIRST OUTBREAK OF SMALL-PO J$ leaving a single representative. The unfortunate clans when attacked, believing that they were be- witched, gave way to despair, and made no effort to save themselves. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. The beachrangers in Table Valley did not even remove the dead from their huts, but sat down and awaited their own turn without a gleam of hope. without a gleam of hope. When the disease ceased there were only dejected remnants left of the old tribes of the Hessequa, Chainouqua, Goringhaiqua, Cochoqua, and Grigri- qua. Beyond them it had not spread. After this date these tribal titles are not found in official records or accounts by travellers, and the Hottentot clans that remained within a hundred miles of the castle ceased to be regarded as of any importance. They continued as before to be governed by their own chiefs without interference from the European autho- rities except when they committed crimes against white people or slaves, there were reserves specially set apart for their use, and they were at liberty to roam over any land not occupied by colonists ; but they were without influence or power, and their friend- ship was no longer courted nor their enmity feared. ship was no longer courted nor their enmity feared. It does not appear that the Bushmen suffered from the small-pox, for by some chance they did not come into contact with other people while it was prevalent. In general there was war between them and the colonists. As the game was destroyed or retreated, they turned to the oxen and sheep of the graziers for sustenance, and it was then a matter of necessity to expel them from their haunts. They would not, or could not, accommodate themselves to the new CAPE COLONY FROM 17OO TO I75O. 74 order of things that was growing up around them, and therefore they were doomed to perish. But the struggle was a severe one, and there were times when it almost seemed as if the wild people would be able to turn back the wave of colonisation that was spreading over the country. They managed to inflict heavy losses upon the Europeans by burning houses and driving off cattle, and occasionally a man or a woman died from the poison of their arrows ; but in the long run the combined action of the farmers and the superiority of the flintlock over the bow decided the question against them. bow decided the question against them. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. In 1 72 1 the Company established a station at Delagoa Bay, with the object of opening up a trade along the eastern coast. This station was a depen- dency of the Cape government, just as Mauritius had been. But the place proved exceedingly unhealthy, and the trade in gold, ivory, copper, and slaves was very much smaller than had been anticipated. On one occasion, also, the factory was surprised and plundered by pirates. Attempts were repeatedly made to explore the country and find the place from which a little alluvial gold was brought by occasional black visitors, but the parties sent out never suc- ceeded in getting beyond the Lebombo mountains, as either fever attacked them or hostile Bantu barred the way. In every respect the station was a failure, and therefore, after maintaining it for nearly ten years at a great sacrifice of life and money, the Company abandoned it. After the punishment inflicted upon Wilhem Adrian van der Stel and his associates, the govern- SYSTEM OF ADMINISTRATI ?$ ment of the Cape Colony was conducted for nearly three-quarters of a century in a fairly honest manner, and no complaint of tyranny or oppression was made by the people. The system of administration, in- deed, opened a door to abuses that in the same form would not be tolerated now, and they were certainly of a grave nature. Thus a perquisite of the store- keeper was to buy at one rate of exchange of silver money and to sell at another rate, by which he gained a commission of nearly eight pence in the pound. The victualler was allowed to require a few pounds overweight in every bag of grain that the Company purchased from a farmer, and to place the surplus to his own credit. The police magistrate, as his perquisite, kept half the fines which he inflicted for contravention of simple regulations as well as for crimes. The governor himself and the officer second in rank had as perquisites a fixed sum deducted from the purchase amount of every cask of wine brought to the Company's stores. But as this was the established order of things, the colonists submitted to it without complaint. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. Some- times they grumbled about bad seasons, or the destruction of their crops by locusts, or the low prices given for farm produce ; and cattle diseases of one kind or other often caused them much loss. When foreign vessels were in Table Bay, too, there was always much dissatisfaction if the Company required anything that was saleable at a large profit. There was never any distress, however, through want of the necessaries of life, nor was there any inter- CAPE COLONY FROM I7OO TO I75O. y6 ference by the government with the recognised rights of the people. Experiments in the cultivation of various plants were frequently made by order of the directors, in the hope of finding something beside wheat and wine that would pay the farmers to grow and the Com- pany to export. The olive was tried again and again, but always without success. Tobacco, indigo, and flax were also fruitlessly experimented with. Great expectations were once raised by the production of eight pounds of raw silk, but that also proved a failure, as the returns were so trifling that people would have nothing to do with it. would have nothing to do with it. In the winter season Table Bay was unsafe, being exposed to the fury of north-west gales, and the Company had often sustained heavy losses by ship- wreck there. Thus, in a terrible gale during the night of the 16th of June 1722 seven Dutch and three English vessels were driven ashore, and six hundred and sixty men were drowned. On this occasion property valued at nearly ;£ 2 50,000 was destroyed. And on the 21st of May 1737 nine vessels belonging to the Company were wrecked, and two hundred and eight lives were lost. The cost price of the cargo alone which was strewn on the beach was ;£ 160,000. These and many other disasters caused the directors to issue orders that a mole should be constructed in Table Bay, so as to form a safe harbour, and in the meantime their ships were to refresh at Simon's Bay from the 15th of May to the 15th of August, the season when gales from the EFFORT TO IMPROVE TABLE BAY 7 J north-west are common. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. Simon's Bay offered secure shelter during the winter season, but there was a drawback to its use in difficulty of access by land, which made the supplying a ship with fresh provi- sions very expensive. In 1742 it was first used as a port of call. A village then sprang up on its southern shore, which received the name Simons- town. The mole in Table Bay was commenced in Feb- ruary 1743. As it was held to be a work of importance to the colony as well as to the Company, a tax was levied upon all the white people in the settlement. Those in the Cape peninsula were assessed at the labour of one hundred and fifty-three stout slaves for two months in the year, and those in the country at ^293 in money or provisions. All the Company's slaves and all the waggons and cattle that could be spared from other work were employed upon the mole. A strong gang of convicts was sent from Batavia to assist in its construction. By the close of 1746 it was three hundred and fifty-one feet in length from the shore, but the work was then stopped. The convicts from Java had nearly all died from change of climate and excessive fatigue, and the burghers declared that they could not pay their quota any longer. The expense was found to be beyond the means of the Company, though it was believed that if the work could be completed Table Bay would be a perfectly safe harbour. The base of the mole is still to be seen like a reef running out from the shore, and its site is called Mouille Point on that account to- the present day. GROWTH OF THE SETTLEMEN 79 By this time the settlement extended so far that it was considered necessary to provide two more churches in the parts most thickly populated and a court of justice for the colonists on the frontier. In 1743 a clergyman was stationed at Roodezand, now the village of Tulbagh, and a church was established there. Two years later a clergyman and a school- master were appointed to Zwartland, and went to reside at the warm bath, the site of the present village of Malmesbury. PROGRESS OF THE CAPE COLONY FROM 1700 TO I750. In 1745 a landdrost was stationed at a place thereafter known as the village of Swellendam, and a board of heemraden was ap- pointed, just as at Stellenbosch. A dividing line was laid down between the districts of Stellenbosch and Swellendam, and also between the Cape and Stellen- bosch; but on the north and the east the boundary was declared to be " where the power of the honour- able Company ends." COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. FROM 175 i to 1771 the colony was governed by Ryk Tulbagh, a man of very high moral character and considerable ability, who had risen by merit alone from the humblest position in the Company's service. Though two great troubles fell upon the country within this period, it was always regarded by the old colonists as the brightest time during Dutch rule in South Africa. The governor was firm, but just and benevolent, and was so beloved that he was commonly called Father Tulbagh. One of the troubles referred to was an outbreak of small-pox: — the second that occurred in South Africa — that terrible scourge having been introduced at the beginning of the winter of 1755 by a home- ward-bound fleet from Ceylon. At first it was supposed to be a kind of fever, but after a few days there were cases that admitted of no doubt. It assumed, however, various forms, and among some of the distant Hottentot tribes differed in appearance so much from what was held to be true small-pox that the Europeans termed it gall sickness. 80 gall 80 SECOND OUTBREAK OF SMALL-PO 8 1 In Capetown hardly a single adult who was attacked recovered. In July the weather was colder than usual, and during that month over eleven hundred persons perished. If that death-rate had continued, before the close of the year there would have been no one left, but as soon as the warm weather set in the disease became milder. Two great hospitals were opened : one for poor Europeans, supported by church funds, the other for blacks. To the latter all slaves who were attacked were sent, the expense being borne by their owners. Those who recovered were employed as nurses. In Capetown from the beginning of May to the end of October nine hundred and sixty-three Europeans and eleven hundred and nine blacks died. In the country the white people did not suffer very severely, as they kept so secluded on their farms that for several months hardly a waggon load of produce was taken to town for sale. The government excused the muster of the burghers for drill, and even the services in the churches were not attended by people from a distance. With the Hottentot tribes that escaped on the former occasion the disease created fearful havoc. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. Not one was left unscathed, except the Korana along the Orange and its branches. On the coast north- ward to Walvis Bay and eastward until Bantu were reached, the tribes as such were utterly destroyed. The individuals that remained were thereafter blended together under the general name of Hot- tentots, and their old distinguishing titles became lost even among themselves. 7 7 CAPE COLONY FROM I75O TO 1 785. 82 How far the disease extended among the Bantu cannot be stated with certainty. These people had pushed their outposts as far westward as the Keis- kama, and at least one Hottentot tribe — the Damaqua — had been incorporated by them, while another — the Gonaqua — was beginning to be assimilated. According to Tembu traditions the territory between the Kei and the Bashee was almost depopulated by the small-pox, and the clans between the Kei and the Keiskama must have suffered severely, but to what extent those beyond the Bashee were affected is not known. The other trouble was a depression in the price of agricultural produce, especially of wine, that brought the colonists to the verge of bankruptcy. The Company had been in the habit of purchasing a considerable quantity of wine for sale and consump- tion in India, where it was served out instead of spirits to the soldiers and sailors, but so many com- plaints were made about its quality that in 1755 lts use there was nearly abandoned. By some chance just at this time very few foreign ships put into Table Bay, so that the market was glutted with produce for which there was no demand. Prices fell lower than had ever been known before, and the farmers saw nothing but ruin before them. nothing but ruin before them. But South Africa has always been a country of sudden reversions from adversity. In this instance matters were at their worst when in December 1758 a large fleet of French men-of-war and transports with troops arrived from Mauritius, purposely to refresh and lay in a supply of provisions. At once EXPLORATION OF N AM AQU ALAND 83 the price of produce doubled or trebled, and all the surplus stock was disposed of. There was then war between Great Britain and France, and the repre- sentatives of both nations in India soon came to look upon the Cape Colony as a convenient source of supply. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. The officers of French packets from Mauritius and of English packets from St. Helena bid against each other in Capetown for cattle and meal and wine, so that until March 1763, when tidings of the conclusion of peace were received, the farmers enjoyed unusual prosperity. farmers enjoyed unusual prosperity. During Mr. Tulbagh's administration some pre- viously unexplored parts of the country were care- fully examined. One expedition travelled along the coast eastward to the Kei, and in returning kept as close as possible to the foot of the Amatola mountains and the Winterberg. West of the Tyumie river this party found no inhabitants except Bushmen. A botanist who was with the expedition brought back many specimens of plants then strange to Europeans, which he afterwards cultivated in the garden in Cape- town. Another exploring party made its way far into Great Namaqualand. Among its spoils was the skin of a giraffe, an animal that never roamed south of the Orange river, and consequently was unknown to the colonists. The skin was sent by Governor Tulbagh to the museum of Leiden, together with many specimens of the animals and plants of South Africa. Capetown at this time contained six or seven thousand inhabitants, rather more than half of whom were slaves. It had but one church building, but VILLAGES IN THE COLO 85 there were three clergymen. The houses were com- modious, though commonly but one story in height. In front of each was an elevated terrace, called a stoep, on which the inhabitants lounged in the evenings. In the principal streets were miniature canals, that served for drainage purposes, and along which were rows of trees as in the Netherlands. The town was patrolled at night by a burgher watch. The watch-house, built while Mr. Tulbagh was governor, is still standing, and until recently served as municipal offices. It is now used as an art gallery, and in it is kept a magnificent collection of pictures presented to South Africa by a gentleman named Michaelis. The village of Stellenbosch had grown to be a very pretty place, and was quite embowered with oak- trees and rose hedges. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. It was a custom for old people to live there, so as to be near the church and to provide homes for their grandchildren attending school, while men and women in the prime of life were occupied in their vineyards and orchards or on their farms. Simonstown contained several large buildings be- longing to the Company, but had not many private residences of much importance. Most of the families, indeed, moved to Capetown for the summer, and the place was then almost deserted. Swellendam and Paarl were mere hamlets, con- sisting of large gardens, orchards, and vineyards along a single street. Both were in charming situations as far as scenery was concerned, and their beauty was increased by a row of oak-trees on each CAPE COLONY FROM I75O TO 1 785. 86 side of the road. Svvellendam had no clergyman, but was provided with a school, and the teacher conducted religious services. Its most prominent building was the drostdy, as the courthouse and residence of the landdrost was termed. The clergy- man of the Drakenstein congregation resided at Paarl, and the church building was there. The hamlet had a school, but no courthouse, as it was within the magistracy of Stellenbosch. within the magistracy of Stellenbosch. While Mr. Tulbagh was governor the colonists spread out eastward to the Little Fish river and northward to the headwaters of many of the streams that flow into the Orange. They did not indeed occupy one-tenth of all the land embraced within these limits, but they took possession of the choicest spots for grazing cattle, and went beyond extensive tracts that were less suited to their needs. Governor Tulbagh died in 1771. His successor, Mr. Joachim van Plettenberg, was a man of very different character. He was not devoted to money- making like Wilhem Adrian van der Stel, but he allowed his subordinates to do what they pleased, and many of them were not ashamed to resort to nefarious practices to increase their possessions. In conse- quence the residents in Capetown and the burghers who lived by agriculture were soon in a state of unrest. The graziers, who seldom came in contact with an official, were much less subject to oppressive treatment, and in general did not concern themselves much with what went on at the seat of government. In 1778 the governor made a tour through the colony. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. On the Zeekoe river, near the present TOUR OF GOVERNOR VAN PLETTENBERG. 87 village of Colesberg, he set up a beacon to mark the extent of his journey and the north-eastern limit of .the colony. When returning to Capetown by another route, he inspected the bay which still bears his name, and caused another beacon to be erected there. At Willem Prinsloo's farm on the Little Fish river, the site of the present village of Somerset East, the governor stayed several days. There the frontier graziers and hunters assembled to meet him and make him acquainted with their condition and wants. Their principal requests were very commendable, for what they desired most earnestly was that a magis- trate and a clergyman might be stationed with them. The governor forwarded a report of this interview to the directors, with a recommendation that the desires of the frontiersmen should be complied with. The result was that in 1786 a landdrost — as head of a new district — was stationed at a place which received the name Graaff-Reinet, and soon afterwards a clergyman went to reside there. From Prinsloo's farm Governor Van Plettenberg sent messengers to invite the nearest Bantu chiefs to visit him. The Bantu tribe farthest in advance was the Xosa, and some of its clans were then living on the Tyumie and Kat rivers, while nearer the sea the remnant of the Gonaqua tribe of Hottentots, whose territory was between the Fish river and the Keis- kama, had by mixture of blood become practically incorporated with it. Several of the chiefs accepted the governor's invitation, and a friendly conference took place, at which it was arranged that the lower course of the Fish river should be a dividing line between CAPE COLONY FROM 1750 TO 1 785. 88 the Bantu and the Europeans. In November 1780 this agreement was formally sanctioned by the council, and thereafter for many years the lower Fish river was regarded as the eastern boundary of the colony. was regarded as the eastern boundary of the colony. On the northern border the struggle between the colonists and the Bushmen was incessant. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. The wild people had been obliged to retire before the ad- vancing wave of colonisation, and they seemed now to be massed in the mountains bordering the great plain south of the Orange river, while the graziers were scattered over the choicest pastures along the same range. Horned cattle, sheep, and goats were driven off in hundreds together, the herdsmen were murdered, and from several places the Europeans were obliged to retire. In May 1774 a commandant was appointed for the northern border, and a plan was made to eject the Bushmen from their strong- holds and restore the farmers to the places from which they had been driven. At the beginning of summer a combined force of burghers, half-breeds, and Hottentots, in three divi- sions acting in concert, took the field. The country for more than three hundred miles along the great mountain range was scoured, and all the Bushmen found who would not surrender were shot. Accord- ing to the reports furnished to the government, five hundred and three were killed and two hundred and thirty-nine taken prisoners. Some of these were afterwards released, and others were bound to the farmers for a term of years. It was hoped that this punishment would deter the Bushmen from thieving, but it had no such effect, FIRST KAFFIR WA 89 They became even more troublesome than before, and it was with difficulty that the graziers held their own. The wild people were really fighting for existence, as the Europeans destroyed the game wherever they settled and left the aborigines nothing to exist upon. settled and left the aborigines nothing to exist upon. x-\nother enemy also had now to be reckoned with, for the extension of the settlement had brought the Europeans face to face with the Xosas, a people who might be called civilised when compared with Bush- men, but who were almost as expert stock-lifters. The arrangement made by Governor Van Pletten- berg and the chiefs who met him at Prinsloo's farm was not observed for a single year. In 1779 several Xosa clans crossed the Fish river and spread them- selves over the present districts of Albany and Bathurst. They said they did not want to quarrel with the Europeans, and to prove the truth of their assertions they murdered a number of Hottentots and took their cattle, without molesting the colonists. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. But shortly they began to drive off the herds of the white people also, and in September 1779 the farmers of the invaded districts, together with those along the right bank of the Bushman's river, were obliged to withdraw to a place of safety. withdraw to a place of safety. Two commandos took the field against the in- truders. The Xosas were attacked and defeated on several occasions, but they were not entirely driven to their own side of the Fish river. In the winter those who were supposed to have been subdued crossed again into the colony, together with many others, and it became evident that a grand effort must be made to expel them. CAPE COLONY FROM 1/50 TO 1785. 90 The government then appointed a farmer named Adrian van Jaarsveld commandant of the eastern frontier. He gave the Xosas notice that they must retire at once, or he would shoot them. One of the clans thought it prudent to remove, and was there- fore not molested, but the others remained where they were. they were. The commandant thereupon collected all the European and Hottentot families of the frontier in a couple of lagers formed by drawing up waggons in a circle and filling the spaces between the wheels with thorn-trees. Leaving a few men to defend these camps, with ninety-two burghers and forty Hottentots, all mounted and well armed, he fell upon the Xosas and smote them hip and thigh. He was in the field less than two months, and when he disbanded his force there was not a Xosa west of the Fish river, and the first Kaffir war was over. While the colony was in a state of disaffection and confusion, owing to misgovernment and strife with barbarians, tidings were received — March 1781 — that Great Britain had declared war against the Nether- lands, and that the republic was in alliance with France. The East India Company at this time was declining in prosperity, and was unable to maintain a large garrison in this country, where its yearly outlay was greater by about £25,000 than its income. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. Practically, therefore, Capetown was almost defence- less, and Great Britain had cast a covetous eye upon it as a half-way station to the great empire she was building up in Hindostan, ARRIVAL OF FRENCH TROOP 9 1 As soon as war was proclaimed, an English fleet with a strong body of troops was despatched under Commodore George Johnstone to seize the colony, but the object of the expedition was made known to the French government by a spy, and a squadron was hastily fitted out to thwart it. Pierre Andre de SufTren, in later years vice-admiral of France, was in command of the French ships. Commodore Johnstone put into Porto Praya to take in a supply of fresh water, and anchored without any arrangements for defence, as he believed his destination was unknown to every one except the British government and himself. One of Suffren's ships was also in want of water, so he too steered for Porto Praya, and not expecting to find the English fleet there, made no preparations for action. Upon rounding a point of land he caught sight of his opponent, and pressed on with only half his ships to secure the advantage of surprise. to secure the advantage of surprise. A sharp action followed, which ended by the French being beaten off, but some of the English ships were badly damaged. SufTren now pressed on under all the sail his vessels could carry, and upon his arrival at the Cape landed a strong body of French troops, who speedily made the peninsula secure against attack. As soon as his fleet was refitted Johnstone followed, but learning the condition of things from the crew of a prize, he made no attempt upon the colony. He inflicted great damage upon the East India Company, however, by seizing several richly laden Indiamen that were waiting in CAPE COLONY FROM I750 TO 1 78 5. 92 Saldanha Bay for men-of-war to escort them homeward. To the great losses sustained during this war the bankruptcy of the Company has usually been attri- buted, but it may be doubted whether the corruption of its officials in the Indian islands had not as much to do with its downfall. As far as South African history is concerned, the cause is immaterial, the fact remains that the government of the Cape Colony now found itself unable to meet the calls upon it. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. It tried to borrow money on interest, but did not succeed in getting as much as it needed, and it then issued paper notes without any security excepting a promise to pay when possible, at the same time declaring these notes a legal tender for payment of debts. Some of them were redeemed a few years later, but others were afterwards issued, and then gold and silver disappeared from circulation and unsecured paper took their place. paper took their place. The disaffection of the colonists in the vicinity of Capetown towards the government of Mr. Van Plettenberg was openly and fearlessly shown. In 1779 they sent four delegates to Holland to represent their grievances and endeavour to obtain redress. And now for the first time the burghers asked to be represented in the government, for they had been told by travellers of the events that had taken place on the other side of the Atlantic, and had begun to apply to themselves the political doctrines which the young republic was teaching. They asked also for free trade with the mother country and its eastern dependencies, and liberty to sell their produce to- COMPLAINTS OF THE COLONIST 93 foreigners without a license from the fiscal, as the chief law officer of the colony was termed. The practice of requiring a license had grown from a simple quarantine regulation to a source of great oppression, as the fiscal would do nothing unless he was heavily bribed. Many of the officials were openly keeping shops, and the burghers asked that this should be prohibited. Besides these they had several other grievances, most of which, however, can be summed up that they desired closer connection with Holland and less dependence upon Batavia. Holland and less dependence upon Batavia. But matters in the Netherlands were not as they had been in the time of Wilhem Adrian van der Stel. Then the East India Company was prosperous, and had many enemies always attacking it and bring- ing its transactions to light, so that there was a guarantee for the good government of its possessions. Xow the Company was tottering to its fall, and men of all shades of opinion were doing their utmost to prop it up, as its crash might ruin the state. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. The directors, therefore, did not enter with alacrity into the unpleasant matter brought before them, though they appointed a committee to investigate the com- plaints. This committee sent copies of all the docu- ments received from the delegates to the officials in the colony to report upon, and awaited the replies without doing anything further. The officials natu- rally tried to put their case in as good a light as possible, and the war gave them ample time for consideration. Thus four full years elapsed before the committee sent in a report, and then it was to the effect that CAPE COLONY FROM 1750 TO 1 78 5. 94 the charges had not been proved. Almost the only relief recommended was that the high court of justice should consist of an equal number of officials and of burghers. The directors adopted this report, and thus the efforts of the burghers to obtain redress were so far a failure. At this time the colonists were thriving, and it was supposed by the directors that they would not make much effort to disturb an order of things in which money was easily made. There had never before been such a demand for produce as that created by the large garrison and the French forces in the East. The Company's needs were very small during the war and for some time afterwards, so that little was taken at low prices. Many new trading houses had been opened by burghers. In Capetown there was a display of prosperity which astonished strangers. European and Indian wares in the greatest variety were introduced in large quantities by Danish ships, and though the prices asked were very high, they commanded a ready sale. commanded a ready sale. But the burghers of South Africa, though relishing keenly the pleasure of making money, have at every period of their history shown a firmer attachment to what they hold to be their political rights and liber- ties. If at times a few men have been found to waver between money and freedom from misrule, the women have never hesitated to reject wealth at the price of submission to wrong. On this occasion neither men nor women were disposed to let the question rest. COURSE OF EVENTS IN THE CAPE COLONY FROM I750 TO 1785. The government resorted to various petty acts of tyranny, but the party opposed to it AGITATION IN THE COLON 95 grew in strength, and resolved now to appeal to the states-general. states-general. The delegates were still in Holland, so documents were sent to them from the colony to be laid before the supreme authority of the republic. But as the directors now announced that they intended to replace the principal officials with other men, and to make a few small changes in the system of government and of carrying on trade, the states- general declined to take up the cause of the burghers until the effect could be seen. The colonists sent home other delegates to push their case, but these quarrelled with each other, and could therefore effect nothing. The agitation in South Africa did not cease, however, until the rule of the East India Company came to an end. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. Though the condition of the East India Company was that of hopeless insolvency, as was afterwards seen, the directors managed to obtain large sums of money on loan, and among other expensive projects they resolved to fortify the Cape peninsula so that it should not again tempt an invader. With the consent of the states-general they sent out as governor an engineer officer named Cornelis Jacob van de GraafT, in order that he might direct the work, and they stationed here a large body of mercenary troops, chiefly German and Swiss regiments in their pay. pay. Colonel Van de Graaff took as little trouble to restrain the officials from acting unjustly as his pre- decessor had taken, and the new men were soon as corrupt as the old. They all knew that the end of the Company was at hand. Most of them were trying to make as much money as they could before the final crash, no matter by what means, and the governor, though free of that vice, had no scruple in fiECA'LESS EXPENDIT 97 squandering the property entrusted to his care. No one at the Cape had ever before lived in such style. The horses, carriages, and servants at his town and country houses would have sufficed for the governor- general of India. There was reckless waste in every- thing that he took in hand. The public expenditure was made to exceed the revenue by nearly £92,000 a year, and though much of this was expended on military works, more was utterly thrown away. military works, more was utterly thrown away. In 1790 the money borrowed by the Company was exhausted, and as it was impossible to raise another loan, an immediate and great reduction of expendi- ture was unavoidable. The spendthrift governor was recalled, military works of every kind were stopped, and nearly the whole of the troops were sent to India. The states-general now appointed a commission to examine the Company's affairs and report upon them, with the result that a supreme effort was made to prevent a collapse. Two men of ability — Messrs. Nederburgh and Frykenius — were sent to South Africa and India with power to reform abuses, increase revenue, and reduce expenditure. In June 1792 they arrived at the Cape and assumed control of affairs. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. By increasing some of the old taxes and imposing new ones, the commissioners raised the revenue to rather over £30,000 a year. The white people in South Africa of all ages at this time were about fifteen thousand in number, so that on an average each paid £2 a year to the government. With this increase of revenue, and by reducing expenses in 8 8 8 END OF FAST INDIA COMPANIES RULE. 98 every way that seemed possible, the balance of loss to the Company was brought down to .£27,000 a year. Distress, consequent upon the reduction of the garrison and an almost total cessation of trade, was now general. Professedly to relieve it, and at the same time to increase the revenue, the commissioners established a loan bank in connection with the government. Paper was stamped to represent different sums, amounting in all to ,£135,000, was declared a legal tender, and was issued through the bank to applicants at six per cent interest on good security. By this means relief from pressure of debt was obtained by many landowners ; but the effect of adding such an amount to the cartoon money already in circulation, with no gold to redeem it, was highly disastrous. The commissioners redressed a few of the grievances of which the burghers complained, but they made no change in the form of government. They fixed the price at which the Company could demand as much wheat as it needed at about five shillings the hundred pounds, and gave the colonists permission to export the surplus to India or the Netherlands, provided it was sent in Dutch ships. They also threw open the trade in slaves with Mada- gascar and the east coast of Africa. At the same time they forbade the landing of any goods what- ever from foreign vessels. Trade with strangers was restricted to the sale of provisions for money, un- less special permission was first obtained from the government. SECOND KAFFIR WA 99 Against this order the residents in Capetown pro- tested in the strongest language. " We live from God and the foreigners," they said, " and if the trade is stopped we must perish." The commissioners declined to cancel the regulation, but they were at length induced to suspend it for three years, which, as events turned out, amounted to the same thing. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. as events turned out, amounted to the same thing. The graziers on the eastern frontier were in as great trouble as the residents of Capetown. A power- ful Xosa chief had recently died, leaving as his heir a boy of tender years, named Gaika. The counsellors of the tribe selected Ndlambe, an uncle of the lad, as regent, but some of the clans refused to submit to him, and in March 1789 they suddenly crossed the Fish river into the colony. The farmers fled before them, but were unable to save the whole of their cattle. The landdrost of GraafT-Reinet then called the burghers of the district to arms, and sent an express to Capetown with a request that the government would assist him with a hundred soldiers. The government decided that war with the Xosas must be avoided at any cost. A commission was appointed to induce them to make peace, and was plainly instructed to purchase their good will. In the meantime the burghers had taken the field, when the Xosa clans, without waiting to be attacked, fell back to the Fish river. They were lying on the western bank, and the burghers were approaching, when the instructions of the government were received by the landdrost. The commando was at once discharged. Not a shot had been fired, nor a END OF EAST INDIA COMPANY'S RULE. 100 single head of cattle recovered, so the burghers were indignant and almost mutinous when they were required to disband. required to disband. The commission then sought an interview with the Xosa chiefs, and tried by means of large presents and smooth words to induce them to retire to their own country; but as this did not succeed, an arrangement was made that they might occupy the land between the Fish river and the Kowie during good behaviour. Of course they attributed such a concession to the weakness of the white people, and in a short time they sent out parties to steal cattle far and wide. This condition of things lasted four years, until May 1793, when a reprisal was made upon a kraal by a party of farmers. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. The clans in the colony were then joined by many of their tribe beyond the Fish river, all eager for plunder, and in a very short time they spread over the whole of the coast lands as far as the Zwartkops river, burning the houses, driving off the cattle, and murdering all the farmers that fell in their way. There were fully six thousand warriors west of the Fish river, and over sixty-five thousand head of cattle, taken from colonists, had been driven across that stream. The government therefore had no option, but was obliged to call out the burghers of Swellendam, and attempt to drive the intruders back and recover the booty. The control of operations, however, was entrusted to a man who professed to believe in the guilelessness of children of nature and who had more sympathy with the Xosas than with the Europeans, so that the campaign ended in CHURCHES IN THE COLON IOI utter failure. The commandant then managed to get the chiefs to promise that they would live in peace with the white people, and upon this the government declared the second Kaffir war at an end. The burghers were naturally dissatisfied, but the government took no notice of their request that some one in whom they could have confidence should be placed in command, and the war be prosecuted until the intruders were expelled from the colony. They were obliged to disperse, and they did so in a spirit which needed very little provocation to induce a revolt against the East India Company. revolt against the East India Company. The Dutch reformed still continued to be the state church, but it was not now the only one in the colony. In 1780 the Lutherans were permitted to have a clergyman in Capetown, and in 1792 the Moravians founded the mission station Genadendal for the benefit of the Hottentots. This society had sent an evangelist to South Africa many years before, and he had met with nothing but kindness until he baptized some converts, when the government inter- fered, as in its opinion religious strife would follow the creation of a rival church. Now, however, more liberal views were entertained, and the Moravian clergymen met with hearty encouragement. clergymen met with hearty encouragement. A great change was taking place in the Dutch reformed church itself, by the introduction of the teaching usually termed evangelical. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. The reverend Helperus Ritzema van Lier, a correspondent of the reverend John Newton, of Olney, and who was imbued with the same spirit as that celebrated END OF EAST INDIA COMPANY'S RULE. 102 clergyman, created almost a revolution in Capetown. For the cold formal services of two or three hours' length, which constituted the principal duty of the earlier ministers, he substituted shorter sermons and prayers, more visitation of parishioners, frequent meetings for religious purposes, and incitement to acts of benevolence and charity. At this date CHURCH OF LAST CENTURY IN CAPETOWN. [From a Sketch by G. Thompson.) CHURCH OF LAST CENTURY IN CAPETOWN. [From a Sketch by G. Thompson.) mission work among the heathen was commenced by the colonial church, and it has ever since gone on increasing in volume. At this time also the philan- thropic labours of a band of ladies in Capetown began, which resulted a few years later in the establishment of an orphan asylum, a mission chapel AFFAIRS IN EURO IO3 and school, and a fund from which to the present day aged women in poor circumstances draw weekly allowances. The reverend Mr. Vos, of Tulbagh, belonged to the same school of thought as Mr. Van Lier, and his congregation set an example in mission work, which was shortly followed by others in the country. Western Europe was now in the throes of the mightiest convulsion of modern times. France had become a republic. The people of the Netherlands were divided into two parties, one of which was in sympathy with the French, while the other favoured a stadtholderate with very large powers and the con- tinuance of the alliance with England which had existed since 1788. The first was termed the patriot, the second the Orange party. An appeal to arms was unavoidable, and on the 1st of February 1793 a declaration of war with Great Britain and the stadt- holder's government was issued at Paris. holder's government was issued at Paris. Upon tidings of the outbreak of hostilities reaching South Africa, the commissioners formed all the clerks and junior officers in the civil service into a military company, which they termed the pennist corps, and they raised a company of half-breeds and Hottentots, put them in uniform, and set them to learn to be soldiers. This corps was termed the pandours. No other means could be devised of strengthening the colony. Messrs. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. Nederburgh and Frykenius then appointed an old Indian official, named Abraham Josias Sluysken, head of the Cape government, and as soon as he took over the duty they proceeded to Java. END OF EAST INDIA COMPANY 'S RULE. 104 During 1794 the complaints of the burghers of GraarT-Reinet were unceasing with regard to the paper money, the stagnation of trade, the new taxes, and, above all, the arrangement with the Xosas which the authorities termed peace. The landdrost took no notice of their statements, so they requested the government to recall him, but Mr. Sluysken would not even listen to them. By this treatment their patience was at length exhausted. In February 1795 they expelled the landdrost, and set up a republic of their own. No more absurd form of government than that which they established has ever existed, but it served their purpose. Adrian van Jaarsveld was appointed military commander of the new state. The burghers declared that they were not in rebellion against the Netherlands, but that they would be governed by the East India Company no longer. Mr. Sluysken had no force to send against them, so they had everything their own way. In June the people of Swellendam followed the example of those of GraarT-Reinet. They too ex- pelled their landdrost, declared themselves a free republic, and elected a governing body which they termed a national assembly. In Stellenbosch and in Capetown there were many persons who sympathised with these movements, though they themselves did not proceed to the length of open rebellion. It is highly improbable that the puny states thus called into existence could have held their own for any length of time, as their commerce could easily be cut off; but, on the other hand, the East India Company ARRIVAL OF A BRITISH FORC 105 could not establish its authority over the distant colonists again. The country was really in a state of anarchy. anarchy. The troops in the Cape peninsula consisted of six hundred and twenty-eight infantry, four hundred and thirty engineers, and two hundred and ten pandours. The head of the whole force was Colonel Robert Jacob Gordon. The infantry regiment was termed the national battalion, though it was composed of men of various countries. It was commanded by Lieutenant-Colonel De Lille. Lieutenant-Colonel De Lille. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. While matters in South Africa were in this condition the French were meeting with astonishing success in Europe. The winter of 1794-5 was so severe that towards the end of January the rivers were frozen hard, and their armies crossed into Utrecht and Gelderland, compelling the English forces to retire to Germany. The patriot party in the Netherlands gave them an enthusiastic welcome. The government was changed in form, the stadtholder made his escape to England in a fishing-boat, and the Batavian Republic, as the country was now named, entered into close alliance with France. The British government immediately fitted out an expedition to seize the Cape Colony, and in hope of facilitating the conquest a mandate was obtained from the fugitive stadtholder requiring the authorities in Capetown to admit English troops into the castle and forts. In June 1795 the expedi- tion arrived in Simon's Bay. Admiral Elphinstone and Major-General Craig, who were in command respectively of the sea and land forces, presented the FRUITLESS NEGOTIATIO 107 mandate to Mr. Sluysken and the council, who were in entire ignorance of recent events in Europe. in entire ignorance of recent events in Europe. With hardly an exception the officials in South Africa sympathised with the Orange party, but they could not in decency openly obey an order issued in a foreign country by a fugitive prince. They therefore made many protestations of their duty to their country and of their determination to resist an invading force to the utmost, but their actions did not correspond with their words. There was but one way in which they could oppose the British forces with any hope of success, and that was by repudiating the East India Company and declaring for the patriot faction. The colonists, almost to a man, favoured that faction, as did the engineer corps and the few Dutch soldiers in the national battalion. The foreign soldiers in that battalion were disaffected, owing to being paid in paper money, and would not fight under any circum- stances. But, with the engineers, from five to six hundred trained men were available, and at least two thousand burghers would have responded to an appeal to aid the patriot cause. Rather than this, however, Sluysken, Gordon, and De Lille were willing to let the English get possession of the country. the English get possession of the country. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. A long correspondence with the British officers followed, but it ended in nothing. Eighteen days after the arrival of the fleet in Simon's Bay the Dutch abandoned Simonstown, and concentrated their force at Muizenburg, a very strong natural position on the road to Capetown. A fortnight later eight hundred English soldiers were landed, and were quartered in deserted buildings. END OF EAST INDIA COMPANY'S RULE. TOS On the 7th of August General Craig, at the head of sixteen hundred men, marched from Simonstown to attack the Dutch camp at Muizenburg. That position could easily have been made impregnable, but little or nothing had been done to strengthen it. De Lille, who was in command, did not even attempt to defend it, but fell back towards Capetown as the English approached. He would not resist the friends of the prince of Orange, and, indeed, shortly afterwards entered the English service. Some artillerymen under Lieutenant Marnitz and some burghers made a brief stand, but being abandoned by their commander and the national battalion, they were driven from the post. Besides securing the only obstacle to an advance upon Capetown, General Craig thus got possession of the greater part of the Dutch military stores and of a quantity of provisions, which he much needed. Two days later three hundred and fifty soldiers arrived from St. Helena to strengthen the force under his command. Up to this time the burghers believed that the government was in earnest in opposing the English, and though they had little confidence in the military leaders and none at all in the national battalion, nearly fifteen hundred of them assembled in arms and were eager to defend the country. Even Swellendam sent a contingent, for the people there knew very well that if the English were masters of Capetown their republic would not last long. But now a belief began to spread that they were being betrayed, and every day some of those in arms left their colours and returned home, FEEBLE DEFENCE OF THE COLONY 109 On the 4th of September a fleet of English ships entered Simon's Bay with three thousand soldiers on board, under command of General Sir Alured Clarke. Some of them were destined for India, but as matters stood, they were all landed and sent on to Muizenburg. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. On the 14th two columns were formed, together between four and five thousand strong, and marched towards Capetown, sixteen miles distant by the road to be followed. The Dutch forces, military and burgher, under Captain Van Baalen, were stationed at Wynberg, half way between Muizenburg and Capetown. Some burgher cavalry tried to harass the English troops on the march, and succeeded in killing one man and wounding seventeen, but the force to which they were opposed was too strong to be checked by any efforts that they could make. Van Baalen drew up his troops as if he meant to stand firm, but as soon as the English were within range of his guns he retreated with the greater part of the national battalion. The burghers cried out that they were being betrayed and sold. It was a scene of confusion. One company of infantry and most of the engineers made a stand for a few minutes, and then fled towards Capetown, abandoning the camp with everything in it. The burghers, strongly impressed with the idea that Mr. Sluysken and Colonel Gordon, as well as the officers of the national battalion, were traitors at heart, and considering that if they fell back to Capetown they would be in a trap and must become prisoners of war, dispersed and returned to their homes. END OF FAST INDIA COMPANY'S RULE. HO The council then sent a messenger to the British officers, requesting a suspension of arms in order to arrange conditions of surrender, and at midnight General Clarke consented to an armistice for twenty- four hours. Next morning General Craig met the Dutch commissioners — Messrs. Van Ryneveld and Le Sueur — at Rondebosch, and after some discussion articles of capitulation were agreed to. These pro- vided for the surrender of the Dutch troops, but the officers were to be at liberty either to remain in Capetown or to return to Europe, upon giving their word of honour not to serve against England while the war lasted. The colonists were to retain all their rights, including the existing form of religion. No new taxes were to be levied, but the old imposts were to be reduced as much as possible. Everything belonging to the East India Company was to be handed over to the English officers, but all other property was to be respected. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. The lands and buildings belonging to the Company were to be considered as security for the paper money in circulation. security for the paper money in circulation. At three o'clock in the afternoon of Wednesday, the 1 6th of September 1795, fourteen hundred British soldiers under General Craig arrived at the castle and drew up on the open ground in front. The Dutch troops marched out with colours flying and drums beating, passed by the English, and laid down their arms, surrendering as prisoners of war. In the evening General Clarke arrived with two thousand infantry and a train of artillery. Thus ended the rule of the Dutch East India Company in South Africa, after an occupation of a REVIEW OF THE COMPANY S RULE Ill little over a hundred and forty- three years. The Company itself had ceased to exist before the symbol of its authority disappeared from the castle of Good Hope. Its administration until a quarter of a century before its fall, though by no means admirable, was as just and honest as that of any English foreign possession at the same time, because it had powerful opponents who kept a vigilant eye upon its proceed- ings ; but when that wholesome restraint was removed, its rule became corrupt and ruinous. Yet none of its acts even then were so unjust as prejudice has made them appear. Thus one English writer of eminence — Sir John Barrow — represented a regulation con- cerning the apprenticeship of children of slaves and Hottentot women living on farms as if it applied to the whole Hottentot race, and succeeding compilers copied his statement without question or doubt. Worse still, two English commissioners of inquiry, without taking the trouble to investigate the matter, reported upon a law concerning degraded Hottentot women and vagrants in Capetown as if the Hottentots everywhere had been made subject to its provisions ; and their report has been quoted again and again as proof of the merciless misgovernment of the East India Company. Now that its records are open to inspection, such charges are known to be incorrect. THE END OF THE EAST INDIA COMPANY S RULE IN SOUTH AFRICA. It governed South Africa with a view to its own interests, its method of paying its officials was bad, its system of taxation was worse, in the decline of its prosperity it tolerated many gross abuses ; but it cannot in fairness be accused of overbearing tyranny or cruelty towards either Europeans or Hottentots. THE FIRST BRITISH OCCUPATION. THE surrender of the Cape Colony to the British forces brought together two branches of the same race, for conquerors and conquered were of one stock. Of all the nations of Europe the inhabitants of the northern Netherlands are the closest in blood to the people of England and Scotland. During the cen- turies that they had been separated, however, their training had been different, so that many slight variations had arisen. Though in the most im- portant features their characters were the same, each regarded the variations in the other as blemishes, and often made more of them than was fair or honest. If this can be said of Englishmen and Dutchmen in Europe, it can be asserted more strongly of Englishmen and Dutchmen when they first came in contact in South Africa, for in this country circumstances had tended greatly to develop a few traits. The system of taxation had been pernicious in its effects upon the character of the people. There were exceptions, but in general the farmers had come to CHARACTER OF THE COLONIST II3 regard very lightly the giving in the number of their cattle and the produce of their lands at less than a third of the true quantity. A man, whose word under other circumstances might be depended upon, in this matter would utter deliberate falsehoods without any twinges of conscience, and even thought he was justified in doing so because the returns he was supplying were for taxation purposes. This trait in the character of the burghers was at once detected by the Englishmen with whom they came in contact, and made a very bad impression. in contact, and made a very bad impression. On the other hand, the habit of most Englishmen of that time of distorting accounts of national events made an equally bad impression upon the South African burghers, and thus each regarded the other as untruthful. The system of perquisites by which the East India Company's officials were paid had caused another ugly trait to be unduly developed in the character of many of the colonists. Accustomed to be mulcted of petty amounts in every transaction, they had come to consider it rather a proof of cleverness than an immoral act to get the better of those with whom they were bargaining. THE FIRST BRITISH OCCUPATION. It was regarded as nothing more than fair retaliation to cheat the government and its officers whenever and by whatever means it could be done. The tendency to dishonest and deceitful practices was made much of by unfriendly critics, though it was far from general, and at its worst was not greater than that of traders else- where who sell a bad article at the price of a good one. CHARACTER OF THE COLONIST II $ The burghers were charged with being very igno- rant. Excepting those in Capetown, they had hardly any education from books, and knew nothing more than how to read, write, and compute a little. All had bibles, the psalms in metre, and the Heidelberg catechism ; but few possessed any books on secular subjects. Yet no people on earth were less stupid. They filled the offices of elders and deacons in the churches, of heemraden in the courts of law, of commandants in war, with as much ability as educated people in Europe could have shown. educated people in Europe could have shown. The colonists at a distance from Capetown were described as living in a very rough style. Their houses were small, poorly furnished, and untidy, said English visitors. It was true that the frontier farmers did not build large houses, for they were constantly liable to be plundered and driven away by savages. As soon as a district became tolerably safe, however, comfortable dwellings were put up by all who had means. The untidiness complained of was the result of the employment of coloured servants. The an- cestors of the colonists brought to South Africa the cleanly and orderly habits of the people of the Netherlands ; but in many instances families had been unable to sustain the effort of compelling their servants to be neat and clean, and had fallen into the way of letting things take their course. But this was not peculiar to the Cape Colony : it was the case wherever coloured people were employed as domestics. Mrs. Stowe's picture of Aunt Dinah's kitchen is just as faithful with the scene laid in Louisiana as if it had been laid in South Africa. THE FIRST BRITISH OCCUPATION. Il The other faults attributed to the colonists were those of country people all the world over. THE FIRST BRITISH OCCUPATION. They were inclined to bigotry in religious matters, were very plain in their language, and loved to impose marvellous tales upon credulous listeners. They were accused of indolence by some English visitors, but that was not a charge that could fairly be made. The man who managed either a grain or wine or cattle farm so as to make it pay had sufficient occupation without doing much manual labour. occupation without doing much manual labour. On their side, the colonists found just as great faults in the English character. They pictured Englishmen as arrogant above all other mortals, as insatiable in the pursuit of wealth, as regardless of the rights of others, and as viewing everything with an eye jaundiced by national prejudice. an eye jaundiced by national prejudice. And yet, with all these harsh opinions of each other, there was really so little difference between English people and South Africans that as soon as they came together matrimonial connections began to be formed. The attractions of blood were stronger after all than prejudices born of strife and want of knowledge. In the blemishes of the colonial character that have been described, there was nothing that education of a healthy kind would not rectify, and against them could be set several virtues possessed in a very high degree. The colonists were an eminently self-reliant people, and seldom lost heart under difficulties. In tenacity of purpose they were without equals. Their hospitality was admitted even by those who were determined to see in them nothing else that was FIRST DAYS OF BRITISH RULE 11/ praiseworthy, and their benevolence towards persons in distress was very highly developed. There was no part of the world where a well-behaved and trust- worthy stranger more readily met with assistance and genuine friendship. Though the British troops were in possession of Capetown, the people of the country districts were not disposed to acknowledge the new authorities. The greater number of the farmers retired to their homes, declaring that they did not consider them- selves bound by the acts of the late government. Under these circumstances every possible effort to soothe the colonists was made by the British com- manders. THE FIRST BRITISH OCCUPATION. The people of Capetown were treated in such a manner as to dispel their anxiety, and they were assured that they would presently be in the enjoyment of such liberty and good fortune as they had never known before. Many of the old servants of the East India Company, who were willing to take an oath to be faithful to the king of England as long as he should hold the coiony, were retained in employment, and most of the clerks in the different offices were allowed to keep their situations. offices were allowed to keep their situations. The paper money in circulation amounted to rather more than a quarter of a million pounds sterling, and was a source of much anxiety to its holders. The British commanders announced that it would be received at the public offices at its full nominal value. They also abolished a very obnoxious tax on auction accounts, and substituted for the old burgher coun- cillors a popular board termed the burgher senate. Two days after the capitulation they sent a document THE FIRST BRITISH OCCUPATION. II 8 over the country, in which a promise was made that every one might buy from whom he would, sell to whom he would, employ whom he would, and come and go whenever or wherever he chose, by land or by water. The farmers were invited to send their cattle and produce to Capetown, where they could sell whatever they wished in the manner most profitable for themselves, and the English would pay for any- thing purchased in hard coin. They were also invited to send persons to confer with the British com- manders, if there was any matter upon which they wished for explanation. These measures had the desired effect in the Cape and Stellenbosch districts, and no opposition was made there to the new authorities. In Swellendam also, after a short time, the people decided to abolish the republic, and to submit to the English. An attempt to hold out was, however, made by the burghers of Graaff-Reinet, acting chiefly under guidance of a man named Jan Pieter Woyer. THE FIRST BRITISH OCCUPATION. Sup- plies of ammunition and goods of every kind were therefore cut off from them, with the result that before the close of 1796 they too were obliged to tender a nominal submission, though they were in hope that before long aid from abroad would enable them to recover their independence. recover their independence. Woyer had left the country in a Danish ship bound to Java, that put into Algoa Bay, where vessels were then very rarely seen. Six French frigates happened to be at anchor in Batavia Roads when he arrived there. The admiral sent one with a supply of powder and lead for the Graaff-Reinet farmers, but when she SURRENDER OF A DUTCH FLEET \\§ reached Algoa Bay an English ship of war happened to be there, and after a short action the frigate was obliged to retire. The government of Java also sent a vessel laden with munitions of war, clothing, sugar, and coffee, for the use of the farmers. It was intended that her cargo should be landed at Algoa Bay, but in a storm the vessel was so much damaged that she put into Delagoa Bay to be repaired, and in that port was seized by the crew of an English whaler, aided by a few Portuguese. by a few Portuguese. A fleet of nine ships, sent from Holland under command of Admiral Lucas, also failed in the object of aiding the colonists against the English. The admiral put into Saldanha Bay, and was there caught as in a trap between a much stronger British fleet on one side and a large British army on the other. On the 17th of August 1796 he was obliged to surrender his ships and nearly two thousand soldiers and sailors, without even an attempt to resist. sailors, without even an attempt to resist. Admiral Elphinstone and General Clarke only remained in South Africa a few weeks after the capitulation. They then went on to India, leaving General Craig at the head of the Cape government. This officer did his utmost, to place English rule before the colonists in as favourable a light as pos- sible, and though as a conqueror he could not be loved, as a man he was highly respected. loved, as a man he was highly respected. THE FIRST BRITISH OCCUPATION. When tidings of the conquest reached England, the high authorities resolved that the Cape Colony should be ruled by a man of rank, who should have all the power held by the governor and the council under the Dutch East India Company. A very strong garrison THE FIRST BRITISH OCCUPATION. 12 was to be maintained in Capetown, and the officer in command was to act as administrator in case of the governor's death or absence. governor's death or absence. Accordingly the earl of Macartney, an old Irish nobleman who had done good service in India, was sent out as governor, and took over the duty in May 1797. His administration was free of the slightest taint of corruption, but was conducted on very strict lines. Those colonists who professed to be attached to Great Britain were treated with favour, while those who preferred a republic to a monarchy were obliged to conceal their opinions, or they were promptly treated as guilty of sedition. There never was a period in the history of the country when there was less freedom of speech than at this time. All the important offices were given to men who could not speak the Dutch language, and who drew such large salaries from the colonial treasury that there was little left for other purposes. An oath of allegiance to the king of England was demanded from all the burghers. Many objected, and a few did not appear when summoned to take it. The governor was firm, dragoons were quartered upon several of those who were reluctant, and others were banished from the country. The free trade promised in 1795 also came to an end. Commerce with places to the east of the Cape of Good Hope was restricted to the English East India Company, and heavy duties were placed upon goods from the westward brought in any but English ships. British goods brought from British ports in British ships were admitted free of duty. The government INSURRECTION IN GRAAFF-RRINET 121 resumed the power to put its own prices upon farm produce, and to compel delivery at those rates of all that was needed for the garrison and the ships of war frequenting Simon's Bay. THE FIRST BRITISH OCCUPATION. The prices fixed, however, were fair and reasonable, and the burghers did not object to sell at such rates, though among themselves they spoke very bitterly of the arbitrary rule to which they were subjected. they were subjected. In November 1798 the earl of Macartney returned to Europe on account of his health. Major-General Francis Dundas then acted as administrator until December 1799, when Sir George Yonge arrived from England as governor. England as governor. During this interval there was a petty insurrection by a party of farmers in Graaff-Reinet. The arrest of Adrian van Jaarsveld on a charge of forgery and setting a summons of the high court of justice at defiance was the immediate cause of the outbreak. The old commandant was being conveyed to Cape- town for trial when he was rescued by a band of frontiersmen, the same who had been the last to submit to British authority. A strong military force, consisting of a squadron of dragoons, a regiment of infantry, and a Hottentot corps, was at once sent to quell the disturbance. This was an easy matter, as the great majority of the people of the district of Graaff-Reinet declined to aid the insurgents, who thereupon sent in a petition for pardon. The officer in command of the troops replied in writing that they must lay down their arms before he would have any dealings with them, and named a place where they could do so. THE FIRST BRITISH OCCUPATION. 12 One hundred and thirteen men appeared at the place appointed, and gave up their arms to the troops. There was no promise of any kind in the document sent to them, but they were under the impression that pardon was implied in its terms, and therefore pro- tested when they were made prisoners. Ninety-three were released upon payment of fines, and the remain- ing- twenty were sent to Capetown, where they were placed in close confinement. Forty-two others after- wards gave themselves up, and were pardoned ; but seven of the most violent fled into Kaffirland, where they were joined by a band of deserters from the British army, and lived for several years under pro- tection of a powerful chief. THE FIRST BRITISH OCCUPATION. Those who were sent to Capetown were brought to trial before the high court of justice, when two were condemned to death and the others to various kinds of punishment ; but with the exception of one who was flogged and banished and two who died in confinement, they were all released by the high commissioner De Mist in 1803. released by the high commissioner De Mist in 1803. The appearance of the pandours on the frontier gave rise to a disturbance of another kind. Some of the Hottentots of that part of the country, seeing men of their own class in arms against colonists, very naturally felt an inclination to aid them, and began to plunder the farmhouses of guns, powder, and clothing. They shed no blood, however, and when they had secured what appeared to them to be sufficient booty, they repaired to the British camp with their wives and children, in the belief that they would be regarded as having acted in a praiseworthy manner. General Vandeleur, the officer in command, did not know TMM THIRD A'ARF/R WAR 12$ what to do with them. He allowed a hundred of the young men to enlist in the Hottentot regiment, and the others — about six hundred of both sexes and all ages — he sent to Algoa Bay with an escort to wait there until he could receive instructions concerning them from the government in Capetown. them from the government in Capetown. A matter of much greater importance than either of these petty insurrections had unexpectedly arisen, and was claiming all his attention. Gaika, who was a boy at the time of the second Kaffir war, had recently attained manhood, and had then claimed the chieftainship to which he was by birth the heir. His uncle, the regent Ndlambe, was unwilling to resign, and a large party in the tribe declared its readiness to support him. Gaika appealed to arms, and a battle was fought, in which he was not only victorious but had the good fortune to take his uncle prisoner. Ndlambe was carelessly guarded, however, and in February 1799 he managed to escape, when with a great number of followers he crossed the Fish river into the colony. All the clans that had been living between the Fish river and the Kowie since the previous war, except one, joined the powerful refugee. THE FIRST BRITISH OCCUPATION. The white people who were in or near the line of his march took to flight, some losing all they had, others who could collect their cattle in time driving them off and leaving everything else behind. In a few days the invaders were in full possession of the whole country along the coast to the Sunday river. country along the coast to the Sunday river. General Vandeleur had no intention of employing British soldiers against the Xosas, but as he was marching towards Algoa Bay, with a view of return- THE FIRST BRITISH OCCUPATION. 12 ing to Capetown, he was attacked by them in a thicket on the bank of the Sunday river. He beat them off, and then fell back a little and formed a camp to enable a patrol of twenty men to join him. But this patrol had already been surrounded, and after a gallant defence all were killed except four men who managed to escape. The camp was hardly formed when it was attacked by the Xosas, who rushed on in masses with their assagai shafts broken short so that they could be used as stabbing weapons. These charges were met with volleys of musket balls and grape shot, that covered the ground with bodies, until at length the Xosas turned and fled. until at length the Xosas turned and fled. The general then marched to Algoa Bay. After fortifying a camp on the Zwartkops river, he sent some of his soldiers to Capetown by sea, and called out a burgher commando to expel the invaders. The Hottentots who had plundered the frontier farm- houses were still at the bay drawing rations, and he thought it prudent to disarm them ; but upon the attempt being made they fled in a body and joined the Xosas. At the beginning of June a burgher commando assembled at the Bushman's river, but instead of attacking the intruders, General Vandeleur tried to persuade them to retire. Thus the farmers lost heart by being kept waiting, and many dispersed, while the Xosas came to believe that the white men were afraid of them. They and the insurgent Hottentots then overran and pillaged the country far and wide. THE FIRST BRITISH OCCUPATION. By the close of July twenty-nine white people had lost their lives, there was hardly a house left standing east THE THIRD KAFFIR WAR 12$ of the Gamtoos, and nearly all the cattle were in the hands of the marauders. In August a large burgher force was got together, and five hundred soldiers were sent to Algoa Bay, General Dundas, however, was determined to make, another attempt to come to a friendly arrangement, so he proceeded to the disturbed district himself, and sent a confidential agent named Maynier to parley with the hostile chiefs. Six or seven hundred soldiers and three strong divisions of burghers were in the field. There was nothing left within reach to plunder. So when Maynier offered not to molest the Xosas in the coast belt east of the Bushman's river, if they would promise not to trespass beyond that territory, they readily pledged their word, and accepted as a mark of friendship the presents which he offered them. To get a parallel to either this transaction or the dealings of the East India Com- pany with the Xosas in the previous war, we must go back in English history to the time of the heathen Danes. The hearts of the farmers sank within them when peace was proclaimed, but they were obliged to abide by the decision of their rulers, and thus for a short time there was a kind of truce which was observed in an indifferent manner. Things remained in this state for nearly three vears, during which time the farmers of Graaff-Reinet ,vere in a condition of great poverty and distress. The depredations of the Xosas and Hottentots were then carried so far that for very shame's sake it Was necessary to renew hostilities. A burgher force was called out, and placed under a very brave and highly THE FIRST BRITISH OCCUPATION. 12 respected farmer named Tjaart van der Walt. Some success attended the early operations of this force, but in August 1802 the commandant was killed in action, and the burghers then dispersed. Five months later they were brought together again, but as the Xosas now asked for peace and promised to return to their own country as soon as possible, terms were concluded with them. THE FIRST BRITISH OCCUPATION. They and the Hottentots engaged not to roam about the country and plunder the farmers' cattle, and the Europeans engaged to give them time to remove without disturbing them. disturbing them. The government of Sir George Yonge was thoroughly corrupt. It could not indeed be proved that he received bribes for his own benefit, but he could only be approached through his favourites, and they — the most prominent of them being his private secretary who was his near relative — were unscrupulous to the last degree. In a short time so many complaints reached England from people of both the English and Dutch nationalities at the Cape that he was recalled. He left the colony in April 1801, and after his arrival in London was not again employed in the public service. Major- General Dundas for the second time acted as administrator, and held that office until the restora- tion of the colony to Holland. tion of the colony to Holland. In 1799 the first agents of the London missionary society arrived in South Africa. Unfortunately almost from the day of their landing some of them took a more prominent part in politics than in elevating the heathen, and as they advocated socia THE FIRST BRITISH OCCUPATION. 12 equality between barbarians and civilised people, they were speedily at feud with the colonists. they were speedily at feud with the colonists. Terms of peace, which afterwards proved to be nothing more than a short truce giving the com- batants a little breathing time, between Great Britain, France, and the Netherlands — then the Batavian Republic — were signed at Amiens on the 27th of March 1802, one of the conditions being that the Cape Colony should be restored to its former owners. Accordingly, after a delay in carrying out this condition which gave Napoleon an excuse for resuming hostilities a little later, in February 1803 a Dutch garrison of rather over three thousand men replaced the British troops, and General Dundas transferred the government to the Batavian com- missioner De Mist. THE COLONY UNDER THE BATAVIAN REPUBLIC. THE Cape settlement was now a direct dependency of the states- general as the governing body of the Batavian Republic, and liberal measures were adopted regarding it. The executive power was entrusted to a governor, who was also commander-in-chief of the garrison. For this office Lieutenant-General Jan Willem Janssens — an able military officer and a man of high moral v/orth — was selected. A legislative and executive council was provided, consisting of four members and the governor as president. The high court of justice was made independent of the other branches of the government, and consisted of a president and six members, all versed in law. Trade with the possessions of the republic every- where was allowed on payment of a small duty for revenue purposes. An advocate of good standing — Mr. Jacob Abraham de Mist — was sent out as high commissioner, to receive the colony from the English, to instal the new officials, and to draw up such regulations as he might find necessary, which, after approval by the states-general, were to be embodied in a charter, THE Cape settlement was now a direct dependency of the states- general as the governing body of the Batavian Republic, and liberal measures were adopted regarding it. The executive power was entrusted to a governor, who was also commander-in-chief of the garrison. For this office Lieutenant-General Jan Willem Janssens — an able military officer and a man of high moral v/orth — was selected. A legislative and executive council was provided, consisting of four members and the governor as president. The high court of justice was made independent of the other branches of the government, and consisted of a president and six members, all versed in law. Trade with the possessions of the republic every- where was allowed on payment of a small duty for revenue purposes. An advocate of good standing — Mr. Jacob Abraham de Mist — was sent out as high commissioner, to receive the colony from the English, to instal the new officials, and to draw up such regulations as he might find necessary, which, after approval by the states-general, were to be embodied in a charter, I29 1 UXDER THE BAT A VI AN REPUBLIC. 130 The ist of March 1803 was observed as a day of thanksgiving to Almighty God for the restoration of the colony to its ancient owners. THE COLONY UNDER THE BATAVIAN REPUBLIC. In the morning service was held in all the churches, and at noon the commissioner De Mist installed Lieutenant-General Janssens as governor. The other officials also who had arrived from Europe had their duties formally assigned to them. The landdrosts and most of the clerks under the English rule retained their posts. clerks under the English rule retained their posts. In April the governor left Capetown to visit the eastern part of the colony, and ascertain how matters were standing with the white people, the Xosas, and the Hottentots. At Algoa Bay he found a party of Hottentots under the guidance of Dr. Vanderkemp, a missionary of the London society ; and for their use he assigned a tract of land in the neighbourhood, ever since known as Bethelsdorp. Locations of ample size were also assigned to the Hottentot captains who had recently been in arms against the colonists, but with the improvidence of their race most of them with their people soon wandered away to other parts of the country, and the land set apart for their benefit was regarded as waste by succeeding governors. For the time being, however, matters were placed on a satisfactory footing with the people of this race who had lately been hostile. The governor then proceeded to the Sunday river, where he had a conference with Ndlambe and the other Xosa chiefs who were living in the colony. The chiefs, who of course knew nothing of the rela- tive strength of England and Holland, were under the impression that the Dutch government must be DEALINGS WITH XOSA CHIEF 131 much more powerful than the other, because it was apparent to them that it had supplanted its opponent, and they knew that the colonists were supporting it with enthusiasm. The farmers were in high spirits, and had sent them word that they must not think the old times had come back again, for the great person called the Batavian Republic was immeasurably superior to the poor creature John Company, who had been ill a long time and was now dead. They therefore expressed a desire for peace and friendship with the white people, and there was no difficulty in settling minor matters with them. THE COLONY UNDER THE BATAVIAN REPUBLIC. But the all- important question of their return to their own country could not be arranged so easily, for though they admitted the Fish river as the boundary, they declared they could not cross it through fear of Gaika. Shortly after this the intruding clans began to quarrel among themselves. Two of them joined Gaika in an attack upon Ndlambe, but the old chief succeeded in beating them back. The Xosas thus remained in occupation of the belt of land along the coast east of the Bushman's river. The other parts of the district of Graafif-Reinet, however, enjoyed for a season a fair amount of tranquillity, so that the farmers were able to carry on their ordinary occupations. occupations. Mr. De Mist also, like the governor, made a tour through the colony, in order to become acquainted with the condition and wants of the people. The settlement was previously divided for magisterial and fiscal purposes into four districts — the Cape, Stellen- UNDER THE BAT A VI AN REPUBLIC. 13 bosch, Swellendam, and Graafif-Reinet, — he now divided it into six of smaller size, and stationed landdrosts at Tulbagh and Uitenhage. landdrosts at Tulbagh and Uitenhage. Among the many regulations which he made was one giving full political equality to persons of every creed who acknowledged and worshipped a Supreme Being. Another provided for the creation of state schools, but this was an idea in advance of the times in South Africa, for the great majority of the colonists objected to schools that were not in connection with the church. The country did not remain long enough under the Batavian flag to test this question, but the probability is that state schools could not have succeeded, as the antipathy to them was so strong. Yet another regulation permitted marriages to take place before the landdrosts, and required them to be registered in the district courts. be registered in the district courts. In 1805 the European population of the colony consisted of between twenty-five and twenty-six thousand individuals, exclusive of soldiers. They owned nearly thirty thousand slaves, and had in their service about twenty thousand free coloured people. It is impossible to say how many Hottentots were living at kraals, or Bushmen roaming about on the border, for these people paid no taxes, and there- fore no notice was taken of them by the census framers. THE COLONY UNDER THE BATAVIAN REPUBLIC. Capetown had a population of rather over six thousand Europeans and nearly eleven thousand persons of colour. persons of colour. In May 1803, less than three months after the restoration of the colony, war broke out again be- tween Great Britain and the Batavian ■Republic, ATTACK &Y THE ENGLI 1 33 On receiving this intelligence, General Janssens de- voted all his attention to putting the Cape peninsula in a condition for defence. But soon instructions were received from Holland that he must send his best regiment to Batavia, as the mother country was unable to furnish more men, and troops were urgently needed in Java. All that the governor could do to make up for its loss was to increase the Hottentot corps, which had been transferred to him by General Dundas, to six hundred rank and file, and to form the Asiatics in and about Capetown into a volunteer corps, termed the Malay artillery. corps, termed the Malay artillery. No one doubted that the English would attempt to seize the colony again, but a state of suspense continued until the last week of 1805, when tidings were received that a great fleet was approaching. Signals were at once made to the different drostdies, summoning the burghers to arms, and though the heat was so intense that they could only ride at night, hundreds came trooping to Capetown. But there were no means of feeding them long after they arrived, for the two previous seasons had been ex- ceptionally bad, and it had not been possible to lay up a store of grain. At this time, though the government made desperate exertions to obtain corn, there was never more than sufficient flour in Capetown for two days' consumption of the garrison and the inhabitants. Under these circumstances a large force, however devoted to the cause it was striving for, could not be kept together long. In the evening of the 4th of January 1806 the fleet — which consisted of sixty-three ships — came to UNDER THE BAT AVI AN REPUBLIC. 13 anchor east of Robben Island, at the entrance of Table Bay. There were on board nearly seven thousand soldiers, under command of Major-General David Baird, an officer who was well acquainted with the Cape and its fortifications, having served here in 1798. THE COLONY UNDER THE BATAVIAN REPUBLIC. On the 6th and 7th six regiments were landed, with some artillery and provisions, at a little cove about eighteen miles by road from Capetown. about eighteen miles by road from Capetown. As soon as it was known that the English were landing on the Blueberg beach, General Janssens marched to meet them, leaving in Capetown a con- siderable burgher force and a few soldiers under Lieutenant-Colonel Von Prophalow to guard the forts. He had an army rather over two thousand strong, but composed of a strange mixture of men. There were mounted burghers, Dutch soldiers, a German mercenary regiment, the crews of two wrecked French ships, Malays, Hottentots, and even slaves. He had sixteen field-guns. slaves. He had sixteen field-guns. At three o'clock in the morning of the 8th this motley force was under arms, when the scouts brought word that the English were approaching. Two hours later the British troops came in sight on the side of the Blueberg. General Baird had with him about four thousand infantry, besides artillery- men and five or six hundred sailors armed with pikes and drawing eight field-guns. pikes and drawing eight field-guns. As soon as the armies were within range, the artillery on both sides opened fire. A few balls from the English guns fell among the German mercenary troops, who at once began to retreat. The burghers, the French corps, the remainder of CAPITULATION OF CAPETOW I 35 the troops, and the coloured auxiliaries behaved well, receiving and returning a heavy fire of artillery and musketry. But the flight of the main body of regular troops made it impossible for the mixed force left on the field to stand a charge which was made by three Highland regiments, and by order of General Janssens the remnant of the army fell back. the remnant of the army fell back. The loss on the English side in the battle of Blueberg was fifteen killed, one hundred and eighty- nine wounded, and eight missing. The roll-call of the Dutch forces when the fugitives were rallied shows the killed, wounded, and missing together. When it was made that afternoon three hundred and thirty-seven men did not answer to their names. General Janssens after his defeat sent the foreigners in his army to Capetown, and with the burghers and Dutch troops retired to the mountains of Hottentots- Holland. THE COLONY UNDER THE BATAVIAN REPUBLIC. In the morning of the 9th General Baird resumed his march towards Capetown. It was not in Colonel Von Prophalow's power to resist with any prospect of success, so he sent a flag of truce to request a suspension of arms in order to arrange terms of capitulation. General Baird granted thirty-six hours, but required immediate possession of the outer line of defence, including the fort Knokke at its extremity on the shore. His demand could not be refused, and that evening an English regiment was quartered in Fort Knokke. In the afternoon of the 10th articles of capitulation were signed. The regular troops and the Frenchmen of the wrecked ships were to become prisoners of UNDER THE DATA VI A. V REPUBLIC. I3 war. Colonists in arms were to return to their former occupations. Private property of all kinds was to be respected, but everything belonging to the Batavian government was to be given up. The inhabitants were to preserve all their rights and privileges, and public worship as then existing was to be maintained. The paper money was to continue current until the pleasure of the king could be known, and the public lands and buildings were to be regarded as security for its redemption. The inhabitants of Capetown were to be exempt from having troops quartered on them. The force opposed to General Janssens was so great that he could not hope to make a long resist- ance, but his position in the mountains of Hottentots- Holland was more favourable for obtaining terms than if he had fallen back upon Capetown after the defeat at Blueberg. General Baird proposed that he should capitulate on honourable conditions, and on the 1 8th arrangements to that effect were made. They provided that the troops should not be con- sidered prisoners of war, but be sent to Holland at the expense of the British government, and that the inhabitants of the colony were to enjoy the same rights and privileges as had been granted to those of Capetown, except that the right of quartering troops upon them was reserved, as the country had not the same resources as the town. Seven transports were prepared, and the troops — ninety-four officers and five hundred and seventy- three rank and file — were embarked in them. THE COLONY UNDER THE BATAVIAN REPUBLIC. One of the best was placed at the disposal of General DEPARTURE OF GENERAL JANSSENS 1 37 Janssens, who had liberty to select such persons as he wished to accompany him. Thirty-one of the civil servants under the Batavian administration desired to return to Europe, and were allowed passages. Fifty-three women and the same number of children also embarked. All being ready, on the 6th of March 1806 the squadron, bearing the last representative of the dominion of the Netherlands over the Cape Colony, set sail for Holland. over the Cape Colony, set sail for Holland. General Janssens had endeared himself to the South African colonists, whom he held in strong regard. His last act before embarking was to write to General Baird recommending them to his care as a people who could be led to any good. He was a brave and skilful military commander, but he was unfortunate in having always to deal with an enemy of vastly superior strength. It was his destiny to surrender to the English at a later date the valuable island of Java, a possession then regarded as of much greater importance than the Cape Colony. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. CONQUEST is a grievous thing for any people, no matter how lightly the conqueror imposes his rule. Apart from all other considerations, it wounds their pride and reduces their energy, for it is everywhere seen that a man of a leading race will do without second thought what one of subject nationality will never do at all. It was thus only natural that the colonists should feel dejected when the English flag was again the symbol of authority in South Africa. They had been ardently attached to the Batavian Republic, and had enjoyed three years of good government combined with ample liberty : now all they had cherished was gone. General Baird, indeed, used the most consoling language ; but they remembered that General Craig had done the same, and a hard unsympathetic rule had followed. They saw all authority again centred in one man, for the council was abolished, and the independence of the high court of justice was destroyed. The members of that court — the presi- dent only excepted — were now ordinary civil servants no POWERS OF THE GOVERNO 1 39 who were appointed by the governor and held office during his pleasure. Even religious freedom came to an end, for a Roman catholic clergyman who had been chaplain to some of the foreign troops in the Dutch service was not permitted to remain in the colony. His expulsion, however, was not felt as a grievance, for, in truth, the great majority of the burghers desired his presence less even than General Baird. There was one hope left, and that rested on the chance of war. If Napoleon should succeed in the struggle with England, which seemed very probable in 1806, they would once more be connected with their fatherland. And so in a spirit of despondency, but not of absolute despair, they submitted to the power that they could not resist. For some time there was fear of actual famine in Capetown. The inhabitants were restricted to a small daily allowance of bread, but with all haste wheat and rice were imported from India, and as the crops of the following season were remarkably good, the danger passed away. As soon as possible the colony was again placed under the same form of government as during the first British occupation, and under the same com- mercial regulations. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. The earl of Caledon, an Irish nobleman only twenty-nine years of age, was sent out as governor with very great authority, though in matters of primary importance he was to act under instructions from the secretary of state in London. He could fix prices for any produce required by the army, and assess the quantity each farmer was com- ENGLISH RULE IN SOUTH AFRICA. 1 40 pelled to deliver. He directed and controlled the different departments. His proclamations and notices had the force of law. With the lieutenant-governor he formed a court of appeal in civil cases of over £200 value, and with two assessors he decided appeals in criminal cases. He had also power to mitigate or suspend sentences passed by the inferior courts. Some of the orders of the earl of Caledon, such as forbidding the farmers of certain districts to keep African sheep, read strangely to-day ; but though he was very strict, he was an amiable and upright man, and was guided in all his doings by a desire to improve the country. His benevolence was almost unbounded, and, indeed, his last act when leaving South Africa was to present a thousand pounds in currency to the orphan asylum. currency to the orphan asylum. The most important measure of his administration had reference to the Hottentots. These people had always in theory been regarded as independent of the European government, and subject to chiefs of their own race. Only in cases where white people or slaves were concerned were they liable to be tried before courts of justice, and they were neither taxed nor called upon to perform public services except when of their own accord they enlisted as pandours. In reality they lived in a state of anarchy. Whoever believed that men of all colours and conditions were equal, the Hottentots certainly did not. They re- spected the poorest and weakest white man far more than they did their own nominal chiefs, for whose authority they cared nothing at all. Many of their women formed connections with slaves, and the farmers CONDITION OF THE HOTTENTOT I4I were obliged to maintain them, or the slaves would run away. Children born of such connections could be apprenticed to the farmers for a certain number of years, when through their mothers' rights they became free to go where they chose. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. With this exception, all but those who lived in Capetown or one of the villages or mission stations could assault or plunder one another without fear of punishment. As far as land was concerned, there were reserves set apart for their benefit in the long-settled parts of the country, and they could use ground not occupied by farmers anywhere. But many of them preferred to live as dependents of a white man, though they seldom remained long in the service of the same person. To obtain brandy and tobacco they were willing to perform light labour occasionally, but nothing could induce them to adopt a life of regular industry. In short, they had become rovers and vagrants. vagrants. The earl of Caledon issued a proclamation which removed all vestiges of chieftainship from the Hottentots in the colony, made them subject to European law, and restrained them from wandering over the country at will. Any one found without a pass from a landdrost or an employer was to be treated as a vagabond. Certain missionaries of the London society raised a great outcry in England against this proclamation and another giving the landdrosts power to apprentice children of destitute Hottentots, which was issued by Sir John Cradock ; but no measures could be devised of greater benefit to the people affected. It is true ENGLISH RULE IN SOUTH AFRICA. 14 that some small bands, rather than submit to control, moved over the Orange river into Great Namaqua- land ; but the vast majority of the Hottentots were rescued by these apparently harsh proceedings from utter ruin, if not from extinction. In 1811 Sir John Cradock succeeded the earl of Caledon as governor. He too was a man of very high principle, so that autocratic rule at this period was presented to the colonists in its best form. Ever since the return of the English the Xosa clans within the colony had been restless, probably because they saw that the burghers were not attached to the new rulers, and in consequence were less capable of resistance. They not only sent out plundering parties to drive off cattle, but they were constantly taking more territory, and only laughed at the remonstrances of the white people. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. When Sir John Cradock reached South Africa, he found reports awaiting him from the landdrost of Uitenhage, in which he was informed that there was only one farm still occupied east of the drostdy, and that no other choice was left than the expulsion of the Xosas by force or the abandonment of the remainder of the district. A strong body of burghers was therefore called out, and some European soldiers with the Hotten- tot regiment were sent to the front. Lieutenant- Colonel John Graham was placed in command of the whole force. He was instructed to try to persuade the Xosas to retire peacefully ; but if they would not do so he was to take the most effectual measures to compel them to return to their own country. Major FOURTH KAFFIR WA 1 43 Cuyler with an escort of twenty-five farmers and an interpreter was therefore sent to the most advanced kraal to hold a parley with the chiefs. Close to the kraal some men were observed, and the major tried to speak to them, but the old chief Ndlambe advanced a few paces from the others, and, stamping his foot on the ground, shouted : " This country is mine ; I won it in war, and intend to keep it." Then shaking an assagai with one hand, with the other he raised a horn to his mouth. Upon blowing it, two or three hundred men rushed from a thicket towards Major Cuyler's party, who owed their escape solely to the fleetness of their horses. There was thus no alternative to the employment of force. Everything was arranged for an attack upon the Xosas, but before it was made the land- drost of Graaff-Reinet and eight farmers were treacherously murdered during a conference with a party of warriors. In January 1812 an advance was made by the burghers and Hottentots in six divisions, that swept the country before them, while the European soldiers occupied strong positions in the rear. This plan succeeded admirably, for the Xosas, about twenty thousand in number, after a brief resistance fled to their own country. Some women who were made prisoners were then sent to inform them that on their own side of the boundary they would not be molested, but if they returned to the colony they would be shot. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. By the beginning of March the fourth Kaffir war was over, and it had ended — as neither the second nor the third had— favourably for the Europeans, ESTABLISHMENT OF A CIRCUIT COURT. 145 A line of military posts, garrisoned partly by Euro- pean soldiers, partly by the Hottentot regiment — which was shortly afterwards raised to eight hundred men, — and partly by burghers, was now formed from the sea to the second chain of mountains, to prevent the return of the people expelled. The principal post in the line, where the headquarters of the troops on the frontier were stationed, was named Grahams- town, in honour of the officer in command. During recent years several governors had thought of establishing a circuit court, but the various changes which had taken place prevented the completion of the design until 181 1. Three members of the high court of justice then left Capetown on the first circuit, with instructions to try important cases, to ascertain whether the landdrosts performed their duties correctly and impartially, to inspect the dis- trict chests and buildings, and to report upon the condition of the people and all matters affecting public interests. Their proceedings were conducted with open doors, and no distinction was made between persons of different races or colour, either as accusers, accused, or witnesses. Throughout South Africa satisfaction was expressed with the establish- ment of a circuit court of this kind, and everywhere the judges were received with respect. Unfortunately, however, the reverend Messrs. Vanderkemp and Read, missionaries of the London society, had given credence to a number of stories of murder of Hottentots and other outrages said to have been committed by colonists, and their reports — in which these tales appeared as facts — were published 11 ed a 11 ed a 11 ENGLISH RULE IN SOUTH AFRICA. I4 in England. By order of the British government, the charges thus made were brought before the second circuit court, which held its sessions in the last months of 18 12. In this, the black circuit as it has since been called, no fewer than fifty-eight white men and women were put upon their trial for crimes alleged to have been committed against Hottentots or slaves, and over a thousand witnesses — European, black, and Hottentot — were summoned to give evidence. The whole country was in a state of commotion. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. The serious charges were nearly all proved to be without founda- tion ; but several individuals were found guilty of assault, and were punished. The irritation of the relatives and friends of those who were accused with- out sufficient cause was excessive ; and this event, more than anything that preceded it, caused a last- ing unfriendly feeling between the colonists and the missionaries of the London society. missionaries of the London society. In 1813 the French met with great reverses in Europe, and one of the first results was that the prince of Orange, who had been in exile in England since 1795, returned to the Netherlands and was received by the people as their ruler. To this time the British government regarded the Cape Colony not as a national possession, but as a conquest that might be restored to its original owner on the conclu- sion of peace. But now an agreement was made with the sovereign prince of the Netherlands that for a sum of six million pounds sterling he should cede to Great Britain the Cape Colony and some Dutch provinces in South America. This agreement was CESSION OF THE COLON Itf embodied in a convention signed at London in August 1814, when the claim of the Netherlands to South Africa was extinguished for ever. to South Africa was extinguished for ever. And so the hopes that the colonists entertained of coming again under the flag of Holland were dissi- pated, but time had done much to soften their regret. To say that they were reconciled to English rule would be incorrect. They were, however, becoming accustomed to it, and as yet, excepting the statements of the London missionaries, nothing had occurred to cause any friction. Their language was still used in the courts of law and the public offices. Their churches had been increased to nine, and their clergy- men were paid by the state. Six new magistracies — George, Clanwilliam, Caledon, Grahamstown, Cradock, and Simonstown — had been established. The financial condition of the government, bad as it subsequently proved to be, was not yet causing much alarm. In the Cape peninsula, where alone Englishmen were met in considerable numbers, intermarriages were already so common that race antipathies were rapidly dying out. EARLY YEARS OF ENGLISH RULE IN SOUTH AFRICA. After the absorption of Holland by France, also, the colonists lost the enthusiastic at- tachment which they had felt for the Batavian Republic, so that altogether the prospect was fair that in course of time the Europeans in South Africa would forget their old aversion to British rule, unless something untoward happened to revive it. THE ADMINISTRATION OF LORD CHARLES SOMERSET. THOUGH the colony had now become a permanent British possession, no change in the form of its govern- ment was made, nor was there any reduction of the excessively high salaries paid to the officials sent from England. Lord Charles Somerset, who succeeded Sir John Cradock in 1814, drew a salary of ;£io,ooo a year, and was provided at the public expense with a residence in town, a country house at Newlands, a marine villa at Camp's Bay, and a shooting lodge at Groenekloof. He and the heads of departments among them absorbed more than one-fourth of the entire revenue of the country. Buildings needed for landdrosts' offices in the country districts and for various purposes in Capetown had been provided during recent years, but the cost had been de- frayed by the creation of paper money, not from surplus funds in the treasury. Such a system could only end in disaster, but no one saw trouble ahead, and the secretary of state took no steps to correct it. SLACHTER S NEK REBELLI 1 49 Lord Charles Somerset had been in the colony a little longer than a year when an event took place which stirred the smouldering fire of disaffection to British rule. There was a farmer named Frederik Bezuiden- hout living on the eastern frontier, in a secluded dell in the valley now called Glen Lynden. This man was summoned to appear before a court of justice on a charge of ill-treatment of a servant, but did not attend, so a company of pandours was sent to arrest him. When they were seen approaching he fired upon them, and then took shelter in a cavern close by, where, as he refused to surrender, he was shot dead. On the following day his relatives and friends assembled for the funeral, when one of his brothers declared that he would never rest until the Hottentot regiment was driven from the frontier. The others present expressed themselves of the same mind, and a plan of insurrection was made. An attempt to induce others to join them failed, however, and they were never able to muster more than fifty men. Within a very short time the government became acquainted with what was taking place, and as a strong force of burghers who had no sympathy with lawlessness assisted the troops sent to restore order, the revolt was suppressed without difficulty. THE ADMINISTRATION OF LORD CHARLES SOMERSET. .Most of those who had taken part in it surrendered, but a few tried to escape to Kafifirland. These were followed by a party of pandours, and all were captured except Jan Bezuidenhout, who would not surrender, and, i5o with his wife and little son helping him, stood at bay till he was shot dead. The prisoners — thirty-nine in number — were tried by a special commission of the high court of justice, and six were sentenced to death, the others to various kinds of punishment. Lord Charles Somerset would only mitigate one of the death sentences, and five of the insurgents were hanged in presence of their com- panions. The burghers who had assisted the govern- ment were greatly shocked by this severe punishment, for they had not thought they were helping to bring their misguided countrymen to death. By them, as well as by the families of those who took part in the disturbance, the event was long remembered with very bitter feelings towards the British authorities. very bitter feelings towards the British authorities. It is now necessary to cast a glance at the clans east of the Fish river, for movements were taking place among them that brought on another war with the white people, apparently a most unjustifiable war on the part of the European government, but really one for which a good reason was not wanting. one for which a good reason was not wanting. After Ndlambe's expulsion from the colony, bands of his followers found means to get through the line of military posts and plunder the farmers beyond. His young athletes, good-natured when not in a state of excitement, fleet of foot, daring, and capable of long abstinence from food, made their way from thicket to thicket through the country they had lived in nearly thirteen years, and the first notice of their presence in any locality was an empty fold from which the cattle had been driven at night. The more expert the robber, the greater hero was he FIFTH KAFFIR WA I 5 I among his companions, and the prouder were his relatives of him. It was their way of earning glory and gain at the same time. and gain at the same time. THE ADMINISTRATION OF LORD CHARLES SOMERSET. Occasionally a band of soldiers would appear at one of their kraals and take compensation for the losses of the farmers, and then another account would be run up in the same way. Thus there was a feeling of hostility on both sides, with no prospect of a change for the better. change for the better. Ndlambe and Gaika were all the time quarrelling with each other, and in 1818 the elder chief suddenly became the stronger of the two. A large and im- portant clan, previously neutral, went over to his side, and a famous seer, named Makana, declared in his favour. This Makana was a man of conspicuous ability among his countrymen. If he had been of chieftain's blood, there is little doubt that he would have made a great position for himself, but his parents were commoners, and therefore in Kaffirland he could never be the head of a tribe. He took the only way to power open to him, and became a religious teacher. The people believed that he was in communication with the spirits of the mighty dead, and that his visions and dreams were inspired. His precepts were of a highly moral nature, for he had learned a good deal of Christianity from mission- aries, and adapted it to his own ideas. In time Makana acquired enormous influence, which he used in an attempt to solidify the western section of the Xosa tribe, by bringing the half- independent clans of which it was composed into complete subjection to one head. Gaika, sunk in LORD CHARLES SOMERSET S ADMINISTRATION. 152 drunkenness and sensuality, was incapable even of comprehending such a purpose ; so he declared for the manly and clear-headed Ndlambe, though that chief must then have been nearly eighty years of age. The nominal head of the tribe, who was named Hintsa, resided far away beyond the Kei, and usually troubled himself very little about the western clans, over whom he had hardly any authority. But on this occasion he too pronounced in favour of the old chief, and sent a band of warriors to aid him. By a stratagem of Makana, the greater number of Gaika's adherents were drawn into an ambush on the Debe flats, where after a desperate battle they were driven from the field with frightful slaughter. THE ADMINISTRATION OF LORD CHARLES SOMERSET. The defeated chief fled to the Winterberg, and sent to the colony to beg for aid. colony to beg for aid. Now comes the question : Was Lord Charles Somerset justified in assisting him ? The quarrel was between two rivals in a tribe over which he had no right of control, what business had he to interfere in it? The answer is that the governor could not permit a formidable hostile power to grow up on the border of the colony. To those who do not consider that reason sufficient, his action must appear unjustifiable. Regarding Ndlambe as an implacable and danger- ous enemy, he issued instructions to Lieutenant- Colonel Brereton to proceed to Gaika's assistance with a combined force of burghers and soldiers. In December 1818 Colonel Brereton crossed the Fish river, and being joined by Gaika's adherents, attacked Ndlambe, who was believed to be at the head of eighteen thousand men, colony to beg for aid. Now comes the question : Was Lord Charles Somerset justified in assisting him ? The quarrel was between two rivals in a tribe over which he had no right of control, what business had he to interfere in it? The answer is that the governor could not permit a formidable hostile power to grow up on the border of the colony. To those who do not consider that reason sufficient, his action must appear unjustifiable. FIFTH KAFFIR WA 153 Ndlambe and his followers, however, did not ven- ture to make a stand on open ground, but retired to dense thickets, which afforded them shelter. Their kraals were destroyed, and twenty-three thousand head of cattle were seized. The British commander found it impossible to restrain the savage passions of Gaika's followers, who were mad with excitement and joy at being able to take revenge, and were unwilling to show mercy when any of their enemies fell into their hands. He withdrew/therefore, before Ndlambe was thoroughly humbled, and on reaching Grahams- town the burghers were disbanded and permitted to return to their homes. Ndlambe at once took advantage of the oppor- tunity. Falling upon Gaika, he put that chief to flight, and then he poured his warriors into the colony. The inhabitants of the district between the Fish and Sunday rivers, unless close to military posts, were compelled hastily to retire to lagers, and lost nearly all their property. THE ADMINISTRATION OF LORD CHARLES SOMERSET. Seventeen white people and thirteen Hottentots were murdered. A burgher force was called out, but before the farmers could take the field Grahamstown was attacked. In the early morning of the 22nd of April 1 8 19 between nine and ten thousand warriors, led by Makana, made a sudden rush upon that post, which had then a garrison of only three hundred and thirty-three men. They were met with a deadly fire of musketry and artillery, and after a short struggle were driven back with heavy loss. Three months later a strong army of colonists and soldiers crossed the Fish river, drove Ndlambe's LORD CHARLES SOMERSETS ADMINISTRATION. 154 adherents eastward to the bank of the Kei, killed many of them, seized all their cattle, and burned their kraals. The old chiefs power was completely broken. The fifth Kaffir war ended by the surrender of Makana, who gave himself up in the hope that his friends would then be spared. He was sent a prisoner to Robben Island, and three years afterwards was drowned when trying to escape. Though there was no more fighting, the forces were kept in the field for several months. The FORT WILLSHIRE. BUILT l820 ; ABANDONED 1837. (From a Sketch by A. Steedman.) FORT WILLSHIRE. BUILT l820 ; ABANDONED 1837. (From a Sketch by A. Steedman.) governor then resolved to try to prevent the Xosas from entering the colony again by keeping a belt of land beyond the border unoccupied except by soldiers, who were to patrol constantly up and down it. The military officers recommended that the Keiskama and Tyumie, as being a better line of defence than the Fish river, should be made the limit of Kaffirland, and the governor accepted their advice. In October he met Gaika, who was so dependent upon his good will that when he ARRIVAL OF BRITISH SETTLER I 55 proposed his scheme the chief at once agreed to it. On the right bank of the Keiskama a defensible barrack was then built, which was named Fort Willshire, and there a body of European troops was stationed. A little later another barrack was built on the Kat river, and was named Fort Beaufort. The territory between the Fish river and the new line was kept without inhabitants, but it was easily tra- versed by the Xosas, who knew every thicket and jungle in it. THE ADMINISTRATION OF LORD CHARLES SOMERSET. In 1820 it was ceded by Gaika to the colony. We have now arrived at an important period in the history of South Africa. To this time the colonists outside of Capetown were almost entirely Dutch- speaking, henceforward the English language is to be heard in many farmhouses as well as in the villages and towns throughout the country, and English customs and ideas are to come into rivalry with the customs and ideas of the earlier settlers. For several years after the general peace which followed the fall of Napoleon much distress was felt by the labouring classes in Great Britain, and emigra- tion was commonly spoken of as the only effectual remedy. In the Cape Colony in 18 19, according to the census, there were only forty-two thousand white people ; so it seemed to the imperial government that the country invited settlers, and parliament without demur granted ,£50,000 to defray the cost of sending out a large party. Heads of families representing nearly ninety thou- sand persons applied for passages, and from these I56 LORD CHARLES SOMERSETS ADMINISTRATION. a selection was made of the number required. The ships in which they left England and Ireland, with one exception, reached South Africa safely, and in April 1820 the immigrants commenced to land on the sandy beach of Algoa Bay. A few hundred who arrived a little later were located first at Clanwilliam, but in a short time most of them abandoned that part of the colony and followed the others to the eastern frontier. Several were people of some means, who brought out a number of servants and appren- tices, the others were of various callings, a large proportion being artisans, men who had worked in factories in England, clerks, and storemen. There were nearly twice as many male as female adults. The imperial government defrayed the cost of ocean transit, and each head of a family was promised a plot of ground one hundred acres in extent, on condition of occupying it for three years. Those who brought out servants were to have an additional hundred acres for each. Nothing more than this was promised, but means of transport to the land on which they were located were provided by the government, and for more than eighteen months rations of food were supplied to all who needed them. THE ADMINISTRATION OF LORD CHARLES SOMERSET. With few exceptions, the immigrants were located between the Bushman's and Fish rivers, the Zuurberg and the sea, a pleasant land to look upon, with its waving grass and many streamlets and patches of dark evergreen forest in the recesses of the mountains. It was part of the territory that Ndlambe had occupied for thirteen years, and that he had vainly tried to hold in 181 2. SUCCESS OF THE BRITISH SETTLERS t$? At the same time that these people were being sent from Great Britain at the expense of the government, a few came to South Africa without any aid, on the assurance of the secretary of state that they would receive larger grants of land if they paid for their passages. Altogether, nearly five thousand indivi- duals of British birth settled in the colony between March 1820 and May 1821. For several years the immigrants were subject to much distress. Most of them knew nothing about tilling ground, but they tried to live upon their little farms until they could get title-deeds, in order to be able to sell. Season after season their wheat crops were destroyed by rust. Then there was a great flood, which washed away many cottages and gardens. In addition to other troubles, roving Xosas made their way into the district, and robbed the poor people of many of the cattle that they had purchased. At the end of 1821 the artisans began to disperse. In different villages throughout the colony they obtained plenty of work, at prices that soon placed them in a good position. They were followed from the locations by many others, who were not qualified to make farmers, but who easily found openings in other pursuits. The government then enlarged the farms of those who knew how to make use of them, and better times for all set in. It was about five years after their arrival before each one found himself in the sphere for which he was best adapted, and in another five years it began to be questioned whether a similar party had ever succeeded so well in any other countrv. I58 LORD CHARLES SOMERSET'S ADMINISTRATION. Grahamstovvn and Port Elizabeth owe their im- portance to these British immigrants. In 1820 neither of these places was more than a hamlet attached to a military post, but a few years later both were flourishing towns. both were flourishing towns. THE ADMINISTRATION OF LORD CHARLES SOMERSET. About one-eighth of the European inhabitants of the colony were now English-speaking, and theirs was a language which quickly spreads. When a man from the British Islands and one from any other country live together, their intercourse is conducted in the language of the Briton, for he refuses to learn a speech that was strange to him in youth. In South Africa this matter might with great advantage have been left to settle itself. But the deepest feelings of the old colonists were stirred by an order of the imperial government that after the 1st of January 1825 all official documents, and after the 1st of January 1828 all proceedings in courts of law should be in English. In Simonstown, Grahamstown, and Port Elizabeth, the exclusive use of the English language in the courts of law was not objected to ; but in other places, where Dutch was spoken by nearly the whole people, the order was regarded as a very serious grievance. Many requests were made to the government to annul it, but to no purpose, and upon the dates named English became the official language of the country. It would have been difficult to devise a measure more calculated to irritate the Dutch inhabitants. Just at this time also great distress was caused to many people by an order concerning the paper money. There were in circulation notes to the SIGNS OF PROGRE I 59 nominal value of a little over ^"700,000, of which about one-third had been created by the English government, one-seventh had been forged so cleverly that they could not be separated from those that were genuine, and the remainder were of Dutch origin. The existence of this paper was certainly a very great drawback to commerce, and it was necessary for the advancement of the country that it should be got rid of. But when an order came from England reducing it to three-eighths of its nominal value, and making British silver money a legal tender at that rate of exchange, it was felt as a crushing blow by many people. Not a few were entirely ruined. But commerce was placed on a safe footing, for the old rixdollar notes were replaced by others at the reduced rate, on which the value was marked in pounds sterling, and the imperial treasury was responsible for their redemption in gold. THE ADMINISTRATION OF LORD CHARLES SOMERSET. Notwithstanding the widespread discontent and the drawbacks to prosperity which have been men- tioned, the colony showed many signs of progress during the administration of Lord Charles Somerset. The villages of Beaufort West, Bathurst, Worcester, Somerset East, and Somerset West were founded, the first lighthouse on the coast was built, a good waggon road was opened through a cleft in the mountain range behind French Hoek, and the South African public library was established. The breed of cattle, and especially of horses, was greatly im- proved, mainly through the importation of thorough- bred stock by the governor himself. Wine was the principal article of export, but mules were now LORD CHARLES SOMERSET'S ADMINISTRATION. l6o sent to Mauritius and horses to India in considerable numbers. The clergymen of the Dutch church were increased to sixteen, and of the English church to five. A Wesleyan clergyman who was sent out from Eng- land in 1 8 14 was not permitted by the governor to conduct services publicly, so his society appealed to the secretary of state, with the result that religious liberty was secured for the colony. A Roman catholic clergyman was now resident in Capetown, and Protestant clergymen of various denominations were scattered over the country and carrying on mission work beyond the borders. At each drostdy a high-class government school was established, to which parents were invited to send their children free of charge. In the eastern part of the colony these schools were of the utmost service, but as instruction was given through the medium of the English language only, they were regarded with much anti- pathy in the western districts, and were not there as useful as they might otherwise have been. useful as they might otherwise have been. In 1825 a council was established to advise the governor in such affairs of importance as he might choose to submit to it for discussion. It consisted of six officials appointed by the secretary of state, and was intended to modify the despotic power of the governor ; but practically it was a very slight check upon the authority of a man of strong will like Lord Charles Somerset, who treated in a most arbitrary manner all who professed democratic principles or who ventured to oppose him in any way. THE ADMINISTRATION OF LORD CHARLES SOMERSET. Among other acts which caused much clamour was the sup- RESIGXATIOX OF THE GOVERNOR IOI pression by his order of a liberal newspaper called the Commercial Advertiser, and the virtual confisca- tion of the press with which it was printed. tion of the press with which it was printed. The later years oi his administration were marked by distress among the farmers — owing to bad seasons, — by a decreasing revenue, by much grumbling about the burden of taxation and the excessive cost of government, and by numerous complaints of his tyranny made to the secretary of state and to the imperial parliament. But he had influential friends, for he was a brother of the duke of Beaufort and of that Lord Fitzroy Somerset who afterwards became Lord Raglan, and his party was then in power. In 1826, however, he was obliged to return to England to defend his conduct against charges by the liberal leaders in the house of commons, who were making capital of him in their attacks upon the treasury benches, and as there was a change of ministry shortly afterwards, he considered it prudent to resign the government of the Cape Colony, a course of action that prevented his case coming on for hearing. 12 THE WARS AND DEVASTATIONS OF TSIIAKA. At this period nearly the whole of South Africa beyond the borders of the Cape Colony was in a state of violent disturbance, owing to wars among different Bantu tribes. At this period nearly the whole of South Africa beyond the borders of the Cape Colony was in a state of violent disturbance, owing to wars among different Bantu tribes. About the year 1783, or perhaps a little later, one of the wives of the chief of a small tribe living on the banks of the river Umvolosi gave birth to a son, who was named Tshaka. Before he was fully grown the boy excited the jealousy of his father, and was obliged to flee for his life. He took refuge with Dingiswayo, head of a powerful tribe, who in his early years had gone through many strange adven- tures, and had by some means come to hear of the European military system. When Tshaka fled to him, Dingiswayo was carrying on war with his neighbours, and had his followers regularly drilled and formed into regiments. The young refugee became a soldier in one of these regiments, and by his bravery and address rapidly rose to a high position. Time passed on, Dingiswayo died, and the army raised Tshaka, then its favourite general, 162 162 GENIUS OF TSHAKA, to supreme command. This was the origin of the terrible Zulu power. Tshaka was a man of great bodily strength and of unusual vigour of mind, but he was utterly merciless. ^f*ft ■J- -.:__ _..?_.&■ v.y 7 y^.-'A-'- ■"' A ZULU WARRIOR IN UNIFORM. (Sketch by Captain Gar dine?'.) ^f*ft ■J- -.:__ _..?_.&■ v.y 7 y^.-'A-'- ■"' A ZULU WARRIOR IN UNIFORM. (Sketch by Captain Gar dine?'.) He set himself the task not merely of conquering but of exterminating the tribes as far as he could reach. With this object he greatly improved the discipline of the army, and substituted for the light WARS AND DEVASTATIONS OF TSHAKA. 164 assagai a short-handled long-bladed spear formed either to cut or to stab. With this weapon in his hand, the highly trained Zulu soldier, proud of his fame and his ornaments, and knowing that death was the penalty of cowardice or disobedience, was really invincible. THE WARS AND DEVASTATIONS OF TSIIAKA. Tribe after tribe passed out of sight under the Zulu spear, none of the members remaining but a few of the handsomest girls and some boys reserved to carry burdens. These boys, with only the choice before them of abject slavery or becoming soldiers, always begged to be allowed to enter the army, and were soon known as the fiercest of the warriors. The territory that is now the colony of Natal was densely peopled before the time of Tshaka. But soon after the commencement of his career, various tribes that were trying to escape from his armies fell upon the inhabitants of that fair land, and drove before them those whom they did not destroy. As far as the Umzimvubu river the whole population was in motion, slaughtering and being slaughtered. One large horde of fugitives made its way as far as the river Umgwali, and was there attacked and beaten by a combined force of Tembus and Xosas. After the battle the horde dispersed, and its fragments settled down in a condition of vassalage among the clans between the Kei and the Umtata. So also at a little later date did other remnants of various tribes from the north, all of the refugees taking the common name of Fingos, or wanderers. By the beginning of 1824, between the rivers Tugela and Umzimvubu there were not left more than THE MAN TAT I HORD 1 65 five or six thousand wretched starvelings, who hid themselves in thickets, and some of whom became cannibals as the only means of sustaining life. cannibals as the only means of sustaining life. On the other side of the great mountain range known as the Kathlamba or Drakensberg, the de- struction of human beings was even greater. Before the rise of the Zulu power, Bantu tribes peopled densely the northern part of the territory now termed Basutoland, the north-eastern portion of the present Orange Free State, and the whole area of the Trans- vaal Province of our days. During the winter of the year 1822 a tribe fleeing from the Zulus crossed the mountains and fell upon the people residing about the sources of the Caledon. They, in their turn, fell upon others in advance, until the whole of the inhabitants of the country as far as the Vaal in one great horde crossed the river and began to devastate the region beyond. THE WARS AND DEVASTATIONS OF TSIIAKA. Among their leaders was a woman named Ma Xtatisi, from whom the horde received the name Mantatis. After crossing the Vaal, the Mantatis turned to the north-west, and created awful havoc with the tribes in their line of march. As each was overcome, its cattle and grain were devoured, and then the murderous host passed on to the next. Their strength was partly kept up by incorporating captives, but vast numbers of the invaders, especi- ally of women and children, left their bones mingled with those of the people they destroyed Twenty- eight distinct tribes are believed to have disappeared before the Mantatis received a check. Then Makaba, chief of the Bangwaketse, fell upon them unawares, WARS AND DEVASTATIONS Of TSHAKA. 1 66 defeated them, and compelled them to turn to the south. In June 1823 they sustained another defeat from a party of Griqua horsemen, and then the great horde broke into fragments. horde broke into fragments. One section — the Makololo — went northward, de- stroying the tribes in its course, and years afterwards was found by Dr. Livingstone on a branch of the Zambesi. Another section, under Ma Ntatisi, re- turned to its old home, and took part in the devastation of the country along the Caledon. And various little bands wandered about destroying until they were themselves destroyed. Several thousand refugees from the wasted country found their way into the Cape Colony, where they were apprenticed by the government to such persons as were not slaveholders. In the winter of 1828 a Zulu army penetrated the country as far south as the Bashee. Tshaka himself with a body-guard remained at the Umzimkulu, and sent one of his regiments to destroy the Fondos, while another division of his force proceeded to deal in the same manner with the Tembus and Xosas. The Pondos were plundered of everything they possessed, but the chief and most of his people managed to hide themselves until the Zulus retired. The Tembus and Xosas fared better. There was an Englishman, named Henry Fynn, with Tshaka, and he succeeded in inducing the chief to recall the army before there was much destruction of life or property. The Tembus and Xosas, however, were greatly alarmed. THE WARS AND DEVASTATIONS OF TSIIAKA. They sent to beg help from the Europeans, and to prevent them from being driven into the MURDER OF TSHA \6j colony a commando of a thousand men, under Lieutenant-Colonel Somerset, marched to their aid. This commando encountered a large body of fierce warriors, who were believed to be Zulus, and an engagement followed which lasted several hours. Afterwards it was discovered that the men whom the Europeans were fighting with were some of those who had fled from Tshaka, and whose course was marked by fire and blood. They were defeated with heavy loss, and as soon as they were scattered the Xosas and Tembus fell upon them and nearly exterminated them. In September 1828 Tshaka was murdered by two of his brothers, one of whom — Dingana by name — succeeded as chief of the Zulus. The new ruler was equally as cruel, but not so able as his predecessor. Under his government the military system was kept up, though the only people left within reach that he could exercise his arms upon were the Swazis. War with them was almost constant, but their country contained natural strongholds which enabled them to escape destruction. Various armies, however, that had been put in motion at an earlier date were still moving on, some at a great distance from their starting places. starting places. One of these was under a chief named Moselekatse, whose reputation as a shedder of human blood is second only to that of Tshaka himself. He was in command of a division of the Zulu army, and had acquired the devoted attachment of the soldiers, when a circumstance occurred which left him no choice but flight. After a successful onslaught upon WARS AND DEVASTATIONS OF TSHAKA. l68 a tribe which he was sent to exterminate, he neglected to forward the whole of the booty to his master, and Tshaka, enraged by such conduct, despatched a great army with orders to put him '</■ i I V» PORTRAIT OF DINGANA. {From a Sketch by Captain Gardiner.) V» PORTRAIT OF DINGANA. {From a Sketch by Captain Gardiner.) and all his adherents to death. THE WARS AND DEVASTATIONS OF TSIIAKA. These, receiving in- timation of their danger in time, at once crossed the mountains and began to lay waste the central zone of the country that is now the Transvaal Province, RISE OF THE MATABELE POWER 1 69 The numerous tribes whose remnants form the Bapedi of our times looked with dismay upon the athletic forms of the Matabele, as they termed the invaders. They had never before seen discipline so perfect as that of these naked braves, or weapon so deadly as the Zulu stabbing-spear. All who could not make their escape were exterminated, except the comeliest girls and some of the young men who were kept as carriers. These last were led to hope that by faithful service they might attain the position of soldiers, and from them Moselekatse filled up the gaps that occurred in his ranks. The country over which he marched was covered with skeletons, and literally no human beings were left in it, for his object was to place a desert between Tshaka and himself. When he considered himself at a safe distance from his old home he halted, erected military kraals after the Zulu pattern, and from them as a centre his regiments traversed the land north, south, and west in search of spoil. in search of spoil. It is impossible to give the number of Moselekatse's warriors, but it was probably not greater than ten thousand. Fifty of them were a match for more than five hundred Betshuana. They pursued these wretched creatures even when there was no plunder to be had, and slew many thousands in mere wanton- ness, in exactly the same spirit and with as little compunction as a sportsman shoots snipe. While the Matabele were engaged in their career of destruction, other bands were similarly employed farther north, so that by 1828 there was not a single Betshuana tribe left intact between the Mao'alisber"- WARS AND DEVASTATIONS OF TSIIAKA. I/O and the Limpopo. On the margin of the Kalahari desert several were still unbroken, though they had suffered severely. In 1830 Moselekatse moved against these tribes, and dispersed them. They were not exterminated, because they took refuge in the desert, where they found sustenance in places to which the Matabele could not pursue them ; but they were reduced to a very wretched state. they were reduced to a very wretched state. THE WARS AND DEVASTATIONS OF TSIIAKA. After this Moselekatse built his military kraals on the banks of the Marikwa, and was lord of the country far and wide. Only one tribe escaped, and that the weakest and most degraded of all the southern Betshuana. The principal Batlapin kraal was then at the source of the Kuruman river, where missionaries resided for a short time at the beginning of the century. The station was soon abandoned, but was occupied again in 18 17 by agents of the London society, and four years later the reverend Robert Moffat went to live there. Towards the close of 1829 Mr. Moffat visited Mose- lekatse, whose kraals were then about a hundred miles east of the Marikwa. The chief could not comprehend the character or the work of the mis- sionary, but he was flattered by the friendship of such a man, and conceived a great respect for one who could weld two thick pieces of iron. He believed Mr. Moffat to be lord of the people at the Kuruman, and, to show his regard, he abstained from sending his warriors there. Thus the Batlapin, who would have fled from the smallest division of the Matabele army, were saved by the presence among them of a courageous and able European. GENIUS OP MOSHE \/\ Meantime in one corner of the vast waste that had been created the process of reconstruction was going on. In the territory that is now called Basutoland a young man named Moshesh was collecting together dispersed people of various tribes, and forming them into a compact political body. He was only in rank the son of a petty captain, and his father was still living, so that under ordinary circumstances he would have had little chance of raising himself to power. But Moshesh possessed abilities of a very high order as a military strategist, a diplomatist, an organiser of society, and a ruler of men. His seat of government was Thaba Bosigo, an impregnable mountain stronghold. He prevented attacks of the Zulus by professing himself the humblest vassal of Tshaka and Dingana, and by frequently sending tribute of furs and feathers. All who submitted to him were treated alike, no matter to what tribe they originally belonged, and as much assistance as pos- sible was given to those who needed it. THE WARS AND DEVASTATIONS OF TSIIAKA. Even bands of cannibals were provided with grain and gardens, that they might become agriculturists once more. Men of tribes that had recently been destroying each other were induced to live side by side in friendship and peace. Thus a new community was forming under Moshesh, by far the ablest black ruler known in South Africa since the arrival of Europeans in the country. Moselekatse sent plundering parties against him, but his scouts gave warning in time, so that the raiders were not able to do much harm. In 1831 a Matabele army laid siege to Thaba Bosigo, but could WARS AND DEVASTATIONS OF TSHAKA. 1/2 not take the stronghold. When the besiegers were compelled by want of food to retreat, Moshesh provided them with provisions sufficient for their homeward journey, and a friendly message accom- panied the gift. He was never again attacked by them. In 1833 missionaries of the Paris evangelical society went to reside with Moshesh, from whom they received a hearty welcome, as he recognised that their assistance in temporal matters would be of great service. In the same year a number of wandering bands — Bantu, Hottentots, and half-breeds — were persuaded by Wesleyan missionaries to settle on the right bank of the Caledon, not very far from Thaba Bosigo. In 1836 a vast portion of the territory east and north of the Cape Colony was lying waste. Between the Keiskama and Umzimvubu rivers were the Xosa, Tembu, and Pondo tribes, with the Fingos, and various clans driven down from the north. Mission- ayes of the London, Glasgow, and Wesleyan societies were endeavouring to christianise and civilise these people. Between the Umzimvubu and Tugela rivers there were only five or six thousand inhabitants. North of the Tugela were the Zulus, under the chief Dingana, who had twenty-five or thirty thousand highly-trained soldiers at his command. highly-trained soldiers at his command. Within the western border of the present Trans- vaal Province, along the Marikwa river, were the Matabele military kraals ; but the greater portion of that vast territory was unoccupied, except in the most rugged places, where the broken remnants of former CONDITION OF THE TRIBES IN 1 836 1 73 tribes were lurking. THE WARS AND DEVASTATIONS OF TSIIAKA. The present Orange Free State contained a few hundred Griquas or people of mixed Hottentot, negro, and European blood, who had migrated from the Cape Colony, a few hundred Hottentots of the Korana tribe, the remnant of the horde under Ma Ntatisi around Lishuane, and some Bantu clans at Mekuatling, Thaba Xtshu, and Bethu- lie. In the territory now called British Betshuana- land the population consisted of the Batlapin tribe, some roving Koranas, and a few stragglers on the border of the desert. And in Basutoland there were the people collected by Moshesh. American mission- aries were attempting to settle in Natal and with the Matabele on the Marikwa, a clergyman of the church of England had just gone to reside with Dingana, and missionaries of the Paris, London, Berlin, and Wesleyan societies were busy wherever there were inhabitants between the Kathlamba mountains and the desert. XV. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. It is not a pleasant admission for an Englishman to make, but it is the truth, that it would be difficult to find in any part of the world a people with so much cause to be discontented as the old inhabitants of the Cape Colony for many years after the fall of the ministry of the earl of Liverpool. There was no sympathy whatever shown towards them by the authorities in England, in fact there was a decided antipathy, which was fostered by the so-called philan- thropic societies, then at the height of their power. The most outrageous stones concerning the colonists were circulated by men who bore the title of Christian teachers — and nothing was too gross to be believed in England, — until the word Boer (Dutch for Farmer) came to be regarded as a synonym for an ignorant and heartless oppressor of coloured people. It was useless for the governors to report differently, or for the courts of law to pronounce the stories libellous : the great societies condemned " the Boers," and the great societies represented and led public opinion in England. 174 COURTS OF JUSTICE REMODELLED 175 Something, however, must be said on the other side. The inhabitants of the Cape Colony were not all white people, and the British government tried to do what it held to be justice to the blacks. Then the whole number of Dutch colonists was only equal to the population of a third-class English town, and their sentiments must have been regarded as of little importance by those who were guiding the destinies of a mighty empire. To make them fall into line with the notions of the day in Great Britain seemed not only easy, but the correct policy to carry out. No one imagined that they were capable of making any effectual opposition. any effectual opposition. In 1828 the whole of the courts of justice in the Cape Colony were remodelled after the English pattern. In the country districts the landdrosts and heemraden were done away with, and in their stead civil commissioners, resident magistrates, and justices of the peace were created. A supreme court was established, with judges appointed by the crown and independent of the governor, and though the Dutch code of law was retained, the forms of procedure were assimilated to those customary in England. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. Since that time criminal cases have been tried by a single judge and a jury of nine men, whose verdict must be unanimous in order to convict. At the same time the burgher senate was abolished, and the government took upon itself the municipal and other duties previously performed by that body. As if these sweeping changes were not sufficient irritation for the old colonists, a notice was issued that all documents addressed to the government CAPE COLONY FROM 1826 TO 183$. 1/6 must be written in English or have a translation attached, otherwise they would be returned to those who sent them. A little later one of the judges removed the criminal cases from the circuit court at Worcester to Capetown for trial, on the ground that there was not a sufficient number of English-speaking men to- form a jury at Worcester, though the prisoners and the witnesses spoke Dutch only, and every word that they said had to be translated to the court. The judges were divided in opinion whether it was necessary for jurymen in every case to understand English, and the question remained open until 1 83 1, when an ordinance was issued defining their qualifi- cations, among which a knowledge of English was not included. In the interval, however, the burghers, who regarded their exclusion from the jury-box as an insult, were deeply incensed. But they sent in no memorials, because they would not be driven to have them written in English, and there was little hope of success had they even done so. And now was heard the first murmuring of a cry that a few years later resounded through the colony, and men and women began to talk of the regions laid waste by the Zulu wars, if it might not be possible to find there a refuge from British rule. One measure, however, was carried out at this time which gave general satisfaction. The salaries of the officials sent from England had been far beyond the means of the colony, and they were now greatly reduced. INJUDICIOUS MEASUR If? The condition of the Hottentots and other free coloured people had long been a subject of discussion in England, where it was commonly believed that they were treated with much injustice. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. In reality it was not so, though there certainly were instances of ill usage, just as there are outrages in all countries of the world. After November 1809 Hottentots were not allowed to wander over the country without passes, and after April 1812 Hottentot children born while their parents were in service, and maintained for eight years by the employers of their parents, were bound as apprentices for ten years longer. In the opinion of the governor who made this law it was better for the children that they should acquire industrious habits, even if restraint had to be used, than that they should become vagrants. The reverend Dr. Philip, however, who was superintendent of the London society's missions in South Africa, claimed for people of all colours and conditions exactly the same treatment ; and as the great philanthropical societies of England supported him, he was virtually master of the position. master of the position. In July 1828 an ordinance was issued which relieved the Hottentots and other free coloured people from the laws concerning passes and the apprenticeship of children, and placed them in all respects on a political level with Europeans. From that time the colony was overrun by idle wanderers to such an extent that farming could hardly be made to pay, and the coloured people were falling back in the scale of civilisation ; but when an attempt was CAPE COLONY FROM 1 826 TO 1 83 5. 1/8 made a few years later to get a vagrant act pro- claimed, Dr. Philip and his party opposed the measure so strongly that it had to be abandoned. measure so strongly that it had to be abandoned. Sir Lovvry Cole, who became governor in 1828, caused between two and three thousand Hottentots and people of mixed blood to be located at the Kat river, in the territory ceded by Gaika to the colony. Several small streams unite to form this river, and in their valleys the land is easily irrigated and is of great fertility. In the best places settlements were formed, each divided into plots of from four to six acres in extent, upon which a family was placed. The ground between the settlements was to remain as a commonage, each family having the right to graze cattle on it. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The settlers were to remain five years on trial, at the end of which period those who had built cottages and tilled the ground were to receive grants in freehold, but every plot not im- proved within that time was to revert to government. For a while the settlement appeared to flourish. The government supplied seed corn, furrows for leading out water were made, and a large extent of ground was brought under cultivation. In the course of a few years, however, it was seen that the pure Hottentots could not sustain such efforts beyond two or three seasons, but there were many half-breeds among those to whom plots of ground were assigned, and they formed a more stable class. Early in 1834 Sir Benjamin D'Urban arrived as governor, with instructions from the secretary of state to carry out several important measures. The first was retrenchment of expenditure on a very CHANGES IN GOVERNMEN 1 79 extensive scale, as the colonial revenue was less than the outlay, and the public debt was increasing. Various offices were now combined, so that one man had to do the work previously performed by two, and all salaries were greatly reduced. The outlay on roads, buildings, and, indeed, everything main- tained by the government, was cut down as much as possible. as possible. The second measure was a slight change in the form of government, caused by the creation of distinct legislative and executive councils. The colonists had often sent petitions to England to be allowed to have a representative assembly, but these had always failed. At length, however, the imperial authorities resolved to make the government of the Cape appear a little less despotic, and for this purpose a legislative council was created. It con- sisted of the governor, as president, five of the highest officials, and five colonists selected by the governor. Its power can be inferred from a remark of Sir George Napier to one of the unofficial members who was combating the government view of a question : " You may spare your breath in this matter, everything of importance is settled before it comes here.3' Still it was a step — though a very short one — in the right direction. short one — in the right direction. The council of advice previously existing now became an executive council, and was made to consist of four high officials. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The third special object which Sir Benjamin D' Urban was instructed to carry into effect was the emancipation of the slaves. CAPE COLONY FROM 1 826 TO 1835. I SO As long as the Dutch East India Company held the colony slaves were brought into it, but not in very large numbers, for their services were only needed to a limited extent. During the first British occupation a great many were imported, as the trade was then profitable, and English energy was em- ployed in it. The Batavian government, being opposed to the system, allowed very few to be landed, and had it lasted a couple of years longer, every child born thereafter would have been declared free. The suppression of the foreign slave trade by the British government followed so closely upon the second conquest of the colony, that there was only time in the interval for five hundred negroes to be imported. From that date the increase in the number of slaves was due to the large excess of births over deaths. There never was an attempt in South Africa to defend the system in theory. Indeed, it was a common remark that it was worse for the white man, who had all the care and anxiety, than for the negro, who had only manual labour to perform. But it is not easy to disturb any system, good or bad, upon which the habits of a people have been formed, and in the Cape Colony money to the amount of over three million pounds sterling was invested in slaves. The testimony of every one competent to form a correct opinion concurred that in no other part of the world was bondage so light. Except in planting and harvesting the labour of the negroes was easy, and they certainly did not feel themselves degraded CONDITION OF THE SLAVE I8l by compulsory service. They were the most light- hearted of mortals. The English governors were of opinion that they enjoyed more comfort than labourers in Great Britain, and that the Dutch laws gave them sufficient protection against ill-usage. To judge of their condition by imagining what a Euro- pean would feel in a similar state leads to a false conclusion, for their hereditary training and line of thought were entirely different. thought were entirely different. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. In 1816 laws began to be made for reducing the power of the masters and conferring rights upon the slaves, and almost every year they increased in stringency. For some time the colonists made no objection to them, but at length control over depen- dents was so limited that many negroes became insubordinate. Public meetings were then held, at which the opinion was maintained that the ties between master and slave were too weakened to beat- further straining. A resolution was passed with one voice at a meeting of slaveholders at Graaff-Reinet, and was generally agreed to in the other districts, that if the English government would stop irritating legislation they would consent that from the date of the arrangement all female children should be free at birth, so that slavery might gradually die out. birth, so that slavery might gradually die out. Another plan was adopted by a few well-meaning persons in Capetown, who formed a society for aiding deserving slaves and slave children to purchase their freedom. The society collected subscriptions, and turned its attention chiefly to the emancipation of young girls. It hoped to receive aid in money from the British treasury and from benevolent persons in CAPP. COLONY PROM 1 826 TO 1 83 5. 1 82 England, but was disappointed in both. With means limited almost entirely to colonial subscriptions, how- ever, it was able to purchase the freedom of about twenty-five girls yearly. twenty-five girls yearly. In England neither of these plans met with favour. twenty-five girls yearly. In England neither of these plans met with favour. In 1830 an order in council was issued prescribing the quantity and quality of food to be given to slaves, the clothing that should be provided for them, the hours during which they should not be obliged to work, and many other matters. It was followed in 1 83 1 by another order in council, limiting the hours of slave labour to nine daily, and nearly destroying the owners' authority. The excitement was now so great that the governor thought it necessary to pro- hibit public meetings and to threaten to banish any one who should attempt to disturb the peace. one who should attempt to disturb the peace. As soon as the clamour subsided, however, he gave his consent to a public meeting being held, and about two thousand slaveholders came together in Capetown. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The utmost order was observed, though resolutions were carried that the lately made laws were highly unjust. The whole assembly then marched to the open space in front of government house, when two gentlemen were deputed to inform the governor that the slaveholders were prepared to suffer the penalties of the orders in council, but could not obey them. The strain upon the colonists was so great that it was felt as a relief when in August 1833 an emanci- pation act, to have force in all the British posses- sions, was passed by the imperial parliament. Eor the Cape Colony it provided that on the 1st of EMANCIPATION OF THE SLAVE 1 83 December 1834 slavery was to cease, and after a short term of apprenticeship the negroes were to have exactly the same civil rights as white people. The sum of twenty million pounds sterling was voted to compensate the owners in the nineteen slave colonies of Great Britain, and the share of each colony was to be determined by the appraised value of its slaves. There was a general impression that this money would suffice to meet the whole, or nearly the whole, value of the slaves, and as most people believed that a vagrant act would be passed before the day of emancipation, they were disposed to accept the new condition of things without demur or heartburning. There were then in the colony thirty-nine thousand slaves, of whom between three and four thousand were aged or infirm, and the others were appraised at rather over three million pounds sterling. The year 1835 was well advanced when intelligence was received from England that the returns for all the colonies were complete, and that of the twenty millions sterling the share awarded to the Cape was a little less than one million and a quarter. The intelligence created a panic greater than any ever known before in South Africa. Many of the late slaves were mortgaged to the various institutions for lending money, and every bond contained a clause covering all other property. At once there was a demand for the redemption of the bonds, and goods and effects were sold at any price that could be obtained. In some instances slaves had been the sole property of widows, or minors, or aged people CAPE COLONY FROM 1 826 TO 1 83 5. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. 184 and the late owners were at once reduced to indigence. But the whole loss was not even yet known. Succeeding mails brought information that the imperial government would not send the money to South Africa, but that each claim would have to be proved before commissioners in London, when the amount allowed would be paid in stock, after certain charges were deducted. This decision brought into the country a swarm of petty agents, who purchased claims at perhaps half their real value, so that a colonist, instead of receiving two-fifths of the ap- praised value of his slaves, often received only one- fifth or one-sixth. It would be difficult to picture too darkly the misery caused by this confiscation of two millions' worth of the property of a small and not over flourishing community. Some families never re- covered from the blow. Aged men and women who had not before known want went down to the grave penniless, and in hundreds of the best households of the country the pinch of poverty was sorely felt. Emancipation in itself assuredly was a righteous act, for there can be nothing more abominable than one man holding another as property ; but a vast amount of distress might have been prevented by effecting it in the manner that the colonists proposed. In addition to the direct loss, the wheat and wine farmers for many years were unable to bring as much produce to market as before, owing to the scarcity of labour. One industry only — but that afterwards a very important one, — the breeding of merino sheep EFFECTS OF THE EMANCIPATIO I cS ^ for the sake of wool, received a great impetus from the emancipation of the slaves, for it could be carried on with fewer workmen than were required in agriculture. The liberated slaves mostly flocked into the towns and villages, where missionary and philanthropic energy in all conceivable forms was expended upon them. In general they could now exist by working perhaps half as many hours as before the emanci- pation, for much of their time was passed in idleness ; but they were neither more comfortably clothed nor better fed or housed, nor — with few exceptions — did they make any perceptible intellectual advancement. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. During recent years, however, there has been a great improvement in the character of many of the descendants of the slaves, who now earn a respectable living by manual labour. These have probably im- proved in morals ; very many of those who attend the mission chapels certainly have. But as free agents the pleasures of a large proportion of them continue to be of a low order. Given any noisy musical instrument, a bright sun, and a gaudy dress, and their mirth is unattainable by Europeans. Without energy, or ambition, or a thought of the responsi- bility of life, they manage to pass their days in an easy and joyous manner. Three of the special duties assigned to Sir Benjamin D'Urban have been mentioned : another was to enter into treaties of friendship with the independent chiefs beyond the colonial frontier. Both on the north and the east the border was then in a disturbed condition. There was a band CAPE COLONY FROM I 826 TO 1 83 5. I 86 of freebooters — mostly Hottentots — plundering the graziers of the northern districts, and as their haunts were on some islands in the Orange river, which were covered with jungle and very difficult of access to strangers, they were able to set their pursuers at defiance. Farther up the river a petty Griqua captain, named Andries Waterboer, was living, and through the agency of his missionary a treaty was entered into with him. He was to receive ,£100 a year as a subsidy for himself and ,£50 a year for a mission school, he was provided with two hundred muskets and a quantity of ammunition, and he engaged in return to be a faithful friend and ally of the colony and to preserve peace along the border from Kheis to Ramah. This was the first treaty of the kind ever made in South Africa, and it was the only one that answered its purpose. Waterboer kept his engagement, and rooted out the freebooters. Sir Benjamin D' Urban intended to visit the eastern frontier to make similar arrangements with the Xosa chiefs, but was detained in Capetown by pressing business, and before he had been a year in the colony the sixth Kaffir war commenced. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The plan of Lord Charles Somerset to keep the tract of land between the Fish and Keiskama rivers unoccupied soon proved a failure, as robbers made their way across it without difficulty. Then clans of Xosas supposed to be friendly — among others those under two sons of Gaika named Makoma and Tyali — were allowed to occupy the ground, in hope that they would prevent cattle-lifters passing through. But they proved to be as expert and unscrupulous SIXTH KAFFIR WA 1 87 robbers as ever the followers of Ndlambe had been, and it became necessary to make reprisals upon them just as upon the others. After a while Ndlambe and one of his sons were recognised by the government as chiefs of the people who through all their troubles had adhered to them, and from that moment the sons of Gaika regarded the Europeans as enemies. Their conduct now became so bad that the governor was obliged to remove first one and then the other from the ceded territory, and this, of course, created a strong feeling of resentment on their part. In 1829 Gaika died, leaving a young lad named Sandile as his principal heir, when Makoma's power was greatly increased, as he became regent for his half-brother. Ndlambe had died in the preceding year, and quarrels arose among his sons and grand- sons, some of whom allied themselves with Makoma to gain support. Thus it happened that the family whose position the government had done so much to build up was now both strong and hostile. build up was now both strong and hostile. A rupture had long been threatening, when some Xosas, by stealing the horses of the officers at Fort Beaufort, drew a party of soldiers into a quarrel in which a petty captain was slightly wounded and some cattle belonging to Tyali were seized. This was announced by the chiefs to be a declaration of war, and a few days later — 21st of December 1834 — between twelve and twenty thousand warriors made a sudden rush into the colony, swept off nearly all the cattle east of the Sunday river, burned the houses, and murdered every white man who could CAPE COLONY FROM I 826 TO I S3 5. 1 88 not escape. Among others, most of the British settlers of 1S20 who were living on farms were reduced to destitution. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The unfortunate people had bare!)' time to flee to Grahamstown, Bathurst, or some other place of refuge, and were compelled to abandon everything. abandon everything. When intelligence of the invasion reached Cape- town, Colonel — afterwards Sir Harry — Smith hastened to the frontier, and began to organise a force to operate against the Xosas. The governor followed as speedily as possible. The burghers all over the colony were called out, and as soon as they could muster, an advance was made into Kaffirland, the raiders having in the meantime retired to the fast- nesses of their own country; after sending the cattle over the Kei to be guarded by Hintsa. over the Kei to be guarded by Hintsa. The Xosas, as is their custom, refused to meet the Europeans on open ground, and it was no easy matter to deprive them of their strongholds. They simply retired from one jungle to another, after resisting as long as they could, and reoccupied every place that was not well guarded after being taken. To meet this difficulty, Sir Benjamin D'Urban formed several camps in commanding positions, from which patrols could be sent out frequently to scour the forests in their neighbourhood. It was the only plan open to him, but the country was too large to be held in subjection in this way by the force at his disposal. As soon as this arrangement was completed, the governor crossed the Kei with a considerable army to recover the cattle. Messages were sent to Hintsa offering peace if he would give them up, but for some SIXTH KAFFIR WAR, time he made no reply. Colonel Smith was then directed to scour his country, and met with such success that the chief himself came to the British camp and agreed to the terms demanded. He left his son Kreli and one of his brothers as hostages with Sir Benjamin D'Urban, and volunteered to guide a PORTRAIT OF HINTSA. {From a Sketch by Captain Mich ell.) PORTRAIT OF HINTSA. {From a Sketch by Captain Mich ell.) detachment of troops and burghers under Colonel Smith to the place where the cattle were kept ; but on the way he attempted to escape, and was shot dead by a colonist who pursued him. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. Kreli succeeded his father as paramount chief of the Xosa tribe, and peace was concluded with him CAPE COLONY FROM I 826 TO 1 83 5. I QO upon his undertaking to restore the cattle in instal- ments. The clans west of the Kei were still holding out, but in September 1835 they tendered their submission, and hostilities came to an end. The arrangements made by Sir Benjamin D'Urban for the preservation of peace were such as every one approves of at the present day. He brought some eighteen thousand Fingos from beyond the Kei, and gave them ground between the Keiskama and Fish rivers, where they would form a buffer for the colonists. They and the Xosas hated each other bitterly, and this feeling was deepened by their appropriating and taking with them twenty-two thousand head of cattle belonging to Kreli's people. It was thus to their interest to act honestly towards the Europeans, whose support alone could save them from destruction. Between the Keiskama and the Kei the western Xosa clans were located as British subjects, but a great deal of authority was left to the chiefs. The territory was named the Province of Queen Adelaide, and Colonel Smith was stationed at a place in it which was called King-Williamstown, to command the troops and control the chiefs. This plan of settlement commended itself to the great majority of the colonists and of the missionaries, who hoped that under it the Xosas would make rapid advances towards civilisation and that property on the border would be secure. There was, however, in Capetown — five hundred miles from the Kaffir frontier — a party under the leadership of the reverend Dr. Philip, that entirely disapproved of the governor's plans. It was composed. THE MISSIONARIES AND THE NATIVES. I9I of only a few individuals, but it had powerful support from abroad. This party desired the formation of states ruled by Bantu chiefs under the guidance of missionaries, and from which Europeans not favoured by missionaries should be excluded. It maintained the theory that the Xosas were an eminently docile and peaceably disposed people, who could easily be taught to do what was right, and who must therefore have been provoked to take up arms by great wrongs and cruelties. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. The utmost fear was expressed by its members that the Bantu tribes would perish if exposed to free intercourse with white people. exposed to free intercourse with white people. To push his views Dr. Philip visited England with a Xosa and a half-breed Hottentot who had been trained by missionaries. A committee of the house of commons was at the time collecting information upon the aborigines in British colonies, and Dr. Philip appeared before it. His evidence was received at great length, and though it consisted largely of opinion, it was allowed to outweigh that of the officers of greatest experience in South African affairs. Before the committee of the house of commons appeared also a disappointed retired official from the colony, Captain Andries Stockenstrom, who de- nounced the proceedings of the government on the eastern frontier as unjust and oppressive. He asserted his belief that there were civilised nations in which the proportion of thieves was greater than among the Xosas, and he was of opinion that treaties could be made with the chiefs by which cattle-lifting could be suppressed. . CAPE COLONY FROM I 826 TO 1 835. 1 92 No evidence could have been more gratifying to Lord Glenelg, who in April 1835 became secretary of state for the colonies. He held the same views as Dr. Philip, and here was Captain Stockenstrom, a South African by birth, in full accord with him. The secretary resolved at once to undo all that Sir Benjamin D'Urban had done. EVENTS IN THE CAPE COLONY FROM 1 826 TO 1835. In a despatch dated the 26th of December 1835, he announced that the sovereignty over the country between the Keiskama and the Kei must be with- drawn, because " it rested upon a war in which the original justice was on the side of the conquered, not of the victorious party." He asserted that " the Xosas had an ample justification of the war in the conduct which was pursued towards them by the colonists and the public authorities through a long series of years, they were urged to revenge and des- peration by the systematic injustice of which they had been the victims, and they had a perfect right to endeavour to extort by force that redress which they could not expect otherwise to obtain." He added that a lieutenant-governor would immediately be appointed for the eastern districts, who would be entrusted with authority to arrange border affairs in accordance with his views. The contents of this despatch spread consternation widely over South Africa. Outside of Dr. Philip's little party in Capetown there was but one opinion : that it destroyed all hope of the preservation of order, and placed life and whatever property was left in the eastern districts at the mercy of the Xosas. The next mail brought tidings that Captain Stocken- EFFECT OF LORD GLENELC's ACTION 1 93 strom had been appointed lieutenant-governor, and might shortly be expected. The British settlers at once sent an earnest protest to England, with an appeal for a close investigation of all occurrences on the border, but they could obtain no redress. Sir Benjamin D'Urban wrote, pointing out that the colonists at any rate were free of blame, as they had no voice in devising the various modes of dealing with the Xosas that had been in force, but the only result was his dismissal from office. To the Dutch colonists in the frontier districts who still possessed the means o{ moving there seemed to be but one course open : to flee from British rule, and to seek a new home somewhere in the vast wilderness leff unpeopled by the wars of Tshaka. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. To people in England one of the strangest events of the nineteenth century is the abandonment of their homes by thousands of Cape colonists after 1836, and their braving all the hardships of life in the wilderness for no other cause than to be free of British rule. Yet there is nothing to cause surprise in the matter, if the character of the Dutch people is considered. These colonists were of the same blood as the men who withstood the great power of Fhilip II of Spain, who laid the richest part of their country under water rather than surrender it to Louis XIV of France. They were not the men and women to submit to what they believed to be mis- rule, if there was a possibility of successful resistance or a chance of making their escape. Many of them, as we have seen, were accustomed to live in waggons and to subsist to a large extent upon game, so that moving deeper into the continent was in itself no great difficulty. Before them was a 194 CAUSES OF THE EMIGRATIO 1 95 vast waste swarming with wild animals, what wonder that they should move into it with such powerful motives to urge them on. Let us look again briefly at the grievances which determined their conduct. First, there was subjection by a foreign and unsympathetic government. Second, there was the prohibition of their language in the public offices and courts of law. Third, there was the superintendent of the London missionary society, their ablest and most relentless opponent, in posses- sion of boundless influence with the British authorities. Fourth, there were the slanderous statements made by the philanthropic societies in England concerning them. Fifth, there was the sudden emancipation of their slaves without adequate compensation. Sixth, there was the whole mass of the coloured people placed upon a political footing with them, and that without a vagrant act being put into force. Seventh, there was no security for life or property in the eastern districts, which were exposed to invasion by the Xosas, as the secretary of state took part with the barbarians. These were the chief causes of the great emigration, and there were many others of less importance. importance. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. And now all over the frontier districts the great waggons were laden with household goods and pro- visions and ammunition, and bands of people set out to seek a new home in the north. Each party was usually made up of families related to each other, and the man of greatest influence in it was elected its leader, with the title of commandant. The horned cattle, horses, sheep, and goats were driven slowly on, GREAT EMIGRATION FROM THE CAPE. I96 and often when the pasture was good the caravans would rest for weeks together. They went up from the grass-covered hills along the coast and the bare Karoo farther inland, till they came to one or other of the steep passes into the elevated basin drained by the Orange and its numerous tributaries. With twenty to thirty oxen before each waggon they struggled up, and then went on without difficulty down the long slope to the river and across the wide plains of the present Orange Free State. plains of the present Orange Free State. North of the Orange the emigrants regarded them- selves as beyond English authority, for over and over again it had been officially announced that Great Britain would not enlarge her possessions in South Africa. The first party that left the colony made its way northward to the Zoutpansberg, where it divided into two sections of about fifty individuals each. One of these sections was cut off by a band of blacks, and all its members, young and old, were murdered. The , other attempted to explore the country to Delagoa Bay, but lost its cattle by the tsetse, and was then attacked by fever, from which only one man and barely half the women and children recovered. The unfortunate survivors after almost incredible hardships reached Delagoa Bay, where they were very kindly treated by the Portuguese authorities, being provided with food and shelter until their friends could send a vessel from Natal to rescue them. The second party was much larger, and was under the leadership of a man of considerable ability, FATE OF THE FIRST AND SECOND PARTIES. 1 97 named Hendrik Potgieter. It moved slowly on until it reached the banks of the Vet river, a tributary of the Vaal, where it halted. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. Potgieter found here a Bantu captain in a very wretched condition, who claimed to be the descendant of chiefs that had once ruled over numerous followers in a wide expanse of country. Having lived long in fear of doing any- thing that might bring him to the knowledge of Moselekatse, he was delighted at the appearance of the white people, especially when he received from them a very liberal offer. Potgieter proposed that he should sell the country which he claimed, except a reserve of ample size for himself and his followers, and receive in exchange protection and a small herd of cattle. The captain at once consented, and then the emigrants took possession of the land between the Vet and Vaal rivers, some of them even moving beyond the Vaal. beyond the Vaal. After a while Commandant Potgieter and eleven others went out to explore- the country northward, and travelled as far as the Zoutpansberg, where they were much pleased with the fertility of the soil and the rich pasture. They believed also that communi- cation with the outer world could be opened through Delagoa Bay, so that the country seemed to offer every advantage that could be desired for a settle- ment. In high spirits they set out to return to their families, but on arriving at the place where they had left the last waggons they were struck with horror by finding that many of their friends had been massacred in a dreadful manner not Ions: before. A band of GREAT EMIGRATION FROM THE CAPE. I98 Moselekatse's warriors, while traversing the country to keep it from being occupied, had suddenly come upon a little party of white people, and murdered all who could not escape. Most of those along the Vaal, however, had notice in time to draw their waggons around them, and, when attacked, were able to beat off their assailants. The Matabele soldiers then returned to the Marikwa for reinforcements. Potgieter immediately selected a suitable hill, and formed a strong lager on it, by lashing fifty waggons together in a circle and filling all the open spaces except a narrow entrance with thorn-trees. He had not long to wait before the Matabele attacked him. They rushed upon the lager with loud hisses, but were received with a deadly fire from the forty men inside, and were obliged to fall back. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. Again they rushed on regardless of death, and strove to tear the waggons apart, but could not. The forty defenders of the lager were keeping up a rapid fire, for their wives and mothers were loading spare guns for their use. As a last resource the men of one of the Matabele regiments threw their spears over the waggons, where over eleven hundred were afterwards picked up, and when this failed they withdrew, but drove off the whole of the emigrants' cattle. They left a hundred and fifty-five corpses on the ground outside the lager. Potgieter's party was now in great distress. In- cluding servants, forty-six of its members had been murdered, and the survivors were in a solitary waste without the means of moving and with very little food. Fortunately, however, the third band of emigrants, ATTACK ON THE MATABEL 1 99 under Commandant Gerrit Maritz, had just encamped at Thaba Ntshu, and learning what had happened, sent oxen to bring away the unfortunate people and their effects. And now it was to be seen what metal the emi- grants were made of. It might be thought that with such experience they would have retreated at once, but the idea of abandoning their project never occurred to one of them. Instead of fleeing from Moselekatse, they resolved to attack him in his own kraal, and punish him severely for what he had done. One hundred and seven farmers mustered for this purpose, and with them went forty half-breeds and a few blacks to look after the horses. A deserter from the Matabele army volunteered to act as guide. the Matabele army volunteered to act as guide. So thoroughly depopulated was the country that not an individual was met between Thaba Ntshu and Mosega, and the commando under Potgieter and Maritz was able to surprise the southernmost military kraal of the Matabele one morning at break of day. Moselekatse himself was not there at the time, and the induna in command of the soldiers happened also to be absent. This was a fortunate circumstance for the farmers. The soldiers grasped their spears and shields, and rushed forward ; but volleys of slugs drove them back in confusion, and there was no one of sufficient authority to restore order. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. They took to flight, and were hunted by the farmers until the sun was high overhead, when it was computed that at least four hundred must have been slain. The com- mando then set fire to the kraal, and with nearly seven thousand head of cattle returned to Thaba Ntshu. GREAT EMIGRATION FRO}f THE CAPE. 200 After this Potgieter's party formed a camp on the Vet river, at a place to which the name Winburg was given in memory of the recent victory. There it was strengthened by the arrival of numerous families from the colony. At this time also a band reached Thaba Ntshu from the Winterberg with a very able man, named Pieter Retief, as its head. On the 6th of June 1837 a general assembly of the emigrants was held at Winburg, when a pro- visional constitution, consisting of nine articles, was adopted. The supreme legislative power was en- trusted to a single elective chamber termed the volksraad, the fundamental law was declared to be the Dutch, a court of landdrost and heemraden was created, and the chief executive authority was con- fided to Mr. Retief with the title of commandant- general. The strong feeling of antagonism that Dr. Philip had roused is shown in one of the articles of the constitution, which provided that every member of the community and all who should thereafter join them must take an oath to have no connection with the London missionary society. That body was regarded by them as purely a political institu- tion, advocating and spreading principles of anarchy; and they regarded it as something like blasphemy to speak of its superintendent in Capetown as a minister of the gospel. Fresh bands of emigrants were frequently arriving, and some of them thought it would be better to go down into Natal than to remain on the highlands of the interior. Pieter Uys, the leader of one of these SECOND DEFEAT OF THE MATABELE 201 bands, had visited Natal a couple of years before, and waxed eloquent when describing its beauty and fertility. Retief himself was inclined to favour a settlement near the sea, but before making up his mind finally, he and some others proceeded to inspect the country below the mountains and ascertain if Dingana would dispose of it. Dingana would dispose of it. While they were away the second expedition against the Matabele set out. GREAT EMIGRATION FROM THE CAPE COLONY. EXPULSION OF MOSELEKATSE FROM THE TERRITORY SOUTH OF THE LIMPOPO. It consisted of one hundred and thirty-five farmers in two divisions, under Hendrik Potgieter and Pieter Uys. Mosele- katse was found on the Marikwa, about fifty miles north of Mosega, and he had with him at least twelve thousand warriors, all splendidly trained and as brave as any troops that ever lived. But the advantage of the farmers in their guns and horses was so great that the hundred and thirty-five did not hesitate to attack a force which was to theirs as ninety to one. For nine days the Matabele tried to reach their opponents, but all their efforts were in vain. The farmers were more than once nearly surrounded, still their plans were so perfect that they were never quite entrapped. They had little else than dried meat to live upon, and they had no resting-place but the bare ground with a saddle for a pillow. Only the hardiest of men and horses could have carried on aggressive operations so long. The loss of the Matabele was great, so great that at the end of the nine days Moselekatse gave up the contest and sought only to escape. With his people and his cattle he fled to the north, and in the country beyond the Limpopo commenced to destroy the GREAT EMIGRATION FROM THE CAPE. 202 Mashona tribes as he had destroyed the southern Betshuana. The farmers were too wearied to follow him, and indeed they could not have continued in the field much longer under any circumstances, so they contented themselves by seizing six or seven thousand head of cattle, with which they returned to Winburg. After the flight of Moselekatse, Commandant Potgieter issued a proclamation, in which he declared that the whole of the territory which that chief had overrun and now abandoned was forfeited to the emi- grants. It included the greater part of the present Transvaal Province, fully half of the present Orange Free State, and the whole of Southern Betshuana- land to the Kalahari desert, except the district occupied by the Batlapin. This immense tract of country was then almost uninhabited, and must have remained so if the Matabele had not been driven out. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. In all the world there is not a fairer country than the pleasant land of Natal, stretching in steps from the Drakensberg, which bounds it like a mighty wall, downward to the shore of the Indian sea. The coast belt is covered with sub-tropical vegetation, for it is heated by the warm Mozambique current, which runs southward along it, and gives it a higher temperature than is due to its distance from the equator. Each terrace, as the traveller ascends, is cooler than the one below, though it is nowhere cold. It is a well-watered land. Numerous streams, issuing from the Drakens- berg and the fronts of the lower terraces, rush along in deep gorges to the sea, and carry off the super- abundant moisture, so that it is also well drained. Its soil is rich, its forests yield excellent timber, and the grass in its valleys resembles a meadow. Its climate everywhere is healthy for Europeans. When Pieter Retief and his companions went down into it by a pass they had discovered, there were between the Tugela and Umzimvubu rivers only NATAL AND ZULULAND IN 1837. 20 from five to ten thousand inhabitants. These acknowledged as their chiefs a few Englishmen whom Tshaka and Dingana permitted to reside at the port as semi-independent vassals, and who carried on trade with the Zulu despot. The tract of land between the Tugela and Buffalo rivers, which in later years formed part of the colony of Natal, was then occupied by Zulu subjects, of whose numbers it is impossible to give an estimate. Retief liked the appearance of the country, and the accounts which he received from the Englishmen at the port confirmed his impression. These Englishmen had in vain petitioned the imperial government to declare it a British possession, so now they were only too glad to welcome the emigrant farmers. Two of them, who had been in the country thirteen years and who spoke Zulu as well as English, accompanied Retief to Dingana's residence in the capacity of guides and interpreters. guides and interpreters. The Zulu capital was called Umkungunhlovu. It was in the shape of an enormous hollow circle formed by concentric rows of huts that served as barracks for the soldiers. The interior space was the drill ground. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. An English missionary clergyman, named Owen, was living there at the time, but he had not been able to get any one to listen to his teaching. There was also an English lad named William Wood — son of one of the residents at Port Natal — who was a favourite of Dingana and his confidential interpreter. Dingana and his confidential interpreter. The despot received Retief with every show of friendship. There was a grand parade, apparently o please him, and a big dance in which highly trained ARRAXGFMFXT WITH DINGAN 205 oxen took part with soldiers. Beef in huge pieces was sent to the visitors from the chiefs own eating-mat, which served as a dish does on a European table, and the best millet beer, such as Dingana himself used to drink huge draughts of, was supplied in calabashes of the largest size. When Retief spoke about Xatal, the chief said he might have it for his countrymen to live in, but first he must prove his friendship by recovering a herd of about seven hundred cattle that Sikonyela, son of Ma Xtatisi, had recently stolen from a Zulu outpost, Retief accepted the condition, and upon his return to Winburg sent for Sikonyela, whose residence was at Imparani, north of the Caledon, and required him to surrender Dingana's oxen and some horses he had stolen from the emigrants. They were given up without bloodshed, and then nearly a thousand white-tilted waggons in a long line went down the steep pass of the Drakensberg and halted on the banks of the Bluekrans and Bushman's rivers in the uplands of Xatal. Retief now prepared to proceed to Umkungunhlovu with the cattle. There were among the emigrants men who were suspicious of Dingana's good faith, and who thought their leader should not risk a life of such value to the community by going again himself, but he was so confident in the chiefs friendly disposi- tion that he would not listen to them. Sixty- six Europeans and about thirty Hottentot servants accompanied him. They were received, as before, with even* show of respect and hospitality, and Dingana expressed MASSACRE OF EMIGRANTS. 20 himself well pleased with the restoration of his cattle. He requested the reverend Mr. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. Owen to draw up a paper to show that he had given Natal to Retief, and when this was done in the English language and translated to him, he said it was correct and handed it to the emigrant leader. The farmers were so en- tirely thrown off their guard that when they were ready to leave and were invited to go into the centre of the kraal to partake of some beer and bid adieu to the chief, they left their guns outside. They were seated on the ground without a thought of danger, when Dingana suddenly exclaimed " Seize them ! " and immediately a regiment of soldiers rushed upon them, drew them to the place of execution, and broke their skulls with clubs. Not a single emigrant or a Hottentot, not even their English interpreter from Port Natal, was suffered to escape. Port Natal, was suffered to escape. A few hours later some ten thousand soldiers left Umkungunhlovu, and, after eleven days' march, at early dawn one morning fell upon the most advanced emigrant encampment, which was near the present village of Weenen. Who can describe in adequate language the horrors that followed ? Babes with their brains dashed - out against waggon wheels, women ripped up with Zulu spears, girls and boys with their necks twisted, were lying thick on the ground when the sun rose that morning. Forty-one white men, fifty-six white women, one hundred and eighty-five white children, and about two hundred and fifty coloured servants perished in the dreadful massacre. Needless to say, the waggons and their contents were utterly destroyed. DESPERATE El 'GETT 20? Every emigrant in Natal must have met a similar fate had not, providentially, a young man been awake and close to a cattle kraal when the assault was made. He managed to spring on an unsaddled horse, and rode with all speed to give warning to the parties farther on. These had barely time to draw their waggons around them when the Zulus appeared, but though the most desperate efforts were made by the savage warriors, the simple lagers proved effective, for not one of them was taken. The courage, and skill, and coolness in danger displayed that day by the emigrants, men and women alike, have never been excelled in the world's history. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. The Zulus, too, were brave, and literally heaps of their dead lay around the lagers before they turned to retreat. At dusk they set out for Umkungunhlovu with as many cattle as they could collect. cattle as they could collect. The night was spent by the emigrants in watch- fulness and prayer, each little party hardly daring to hope that any other had escaped. Next morning communication between the lagers was opened, and the full extent of their loss became known. Their first care now was to concentrate and strengthen an encampment, in case the Zulus should renew the attack, and then a general assembly was held to decide what was to be done. One or two men pro- posed that they should leave Natal, but they were put to shame by the women, who declared they would never abandon the country till the Zulus were punished for shedding so much innocent blood. This feeling was general, for it was regarded as a duty to bring the murderers to account. Then they put up INVASION OF ZULULAND. 20 an earnest prayer to the Gocl of heaven that He would not forsake His people, nor allow the heathen to triumph over them. From the bible — the only book with which they were thoroughly conversant — they drew consolation and hope, which enabled them to bear up against their trouble, and to take courage for the prosecution of the task before them. for the prosecution of the task before them. When these events took place, Fieter Uys was at the top of the Drakensberg in readiness to go down, and Hendrik Potgieter was at Winburg, as his party preferred to remain on the interior highland. Both of them now collected as many fighting men as possible, and hastened to the assistance of the dis- tressed people in Natal. The Englishmen at the port also, having lost two of their companions in the massacres, offered to help with all their followers, many of whom were trained soldiers who had deserted from the Zulu army. When it came to a question of who was to be the leader, however, jealousy of each other — the bane of the emigrants — showed itself at once. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. Even Pieter Retief could not preserve perfect concord among the various heads of parties, and after his death there was no one whom all the others would admit as a superior. At length it was arranged that the English chiefs with their people should attack Dingana on one side while Potgieter and Uys attacked him on another, and Maritz should remain at the camp to protect it. The two commandants, with three hundred and forty-seven men, rode directly towards the Zulu capital. After five days' march through an unin- habited country, they came in sight of a division DEATH OF PIETER UY 200, of the Zulu army, which they attacked impetuously, and were drawn into a skilfully planned ambuscade. Before them were two parallel ranges of hills, with a long defile between them, and into this the farmers were led by the Zulus apparently retreating before them. Uys's division was in advance. When in the narrowest part of the gorge they found themselves surrounded by an immense force which had been lying in ambush, and by which they were so hemmed in that they could not fall back rapidly after firing and again load and charge, as was their mode of fighting with Moselekatse. They therefore directed all their fire upon the mass of the enemy behind them, when, having cleared a path by shooting down hundreds, they rushed through and escaped. Ten of them were killed, and they were obliged to leave their led horses, baggage, and spare ammunition behind. Among the slain was Commandant Uys, who was assisting a wounded comrade when he received a stab from an assagai. His son, Dirk Cornelis Uys, a boy of fifteen years of age, was some distance off, but, looking about, he saw his father on the ground, and a Zulu in the act of stabbing him. The gallant youth turned his horse and rode to help his parent, but could only die at his side. A few days later seventeen Englishmen left Port Natal with about fifteen hundred blacks, of whom between three and four hundred were armed with muskets. A few miles south of the Tugela they came upon a Zulu regiment, which pretended to take to flight, left food cooking on fires, and even threw away a number of shields and assagais. The 15 DESTRUCTION OF THE NATAL ARMY. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. 210 Natal arm)- pursued with all haste, crossed the Ttigela, took possession of a kraal on the northern bank, and then found it had been drawn between the horns of a Zulu army fully seven thousand strong. The battle that was fought, on the 17th of April 1838, was one of the most desperate contests that ever took place on that bloodstained soil. Three times in succession the Natal army beat back the regiments that charged furiously upon it. Then a strong Zulu reinforcement came in sight, and renewed the enemy's courage. Another rush was made, which cut the Natal army in two, and all hope of successful resistance was over. One of the divisions tried to escape by the only open path, which was down a steep bank of the Tugela and across that river. A Zulu regiment hastened to cut off the retreat of the fugitives, and many were killed in the water ; but four Englishmen and about five hundred blacks managed to get through. The other division was entirely surrounded. But no lion at bay ever created such havoc among hounds that worried him as this little band caused among the warriors of Dingana before it perished. The young regiments were selected to charge upon it, while the veterans watched their prowess from a neighbouring hill. Whole masses went down before the withering fire, the survivors recoiled, but again they were directed to charge. At last a rush of a regiment, with another in reserve close behind, carried every- thing before it, and the stubborn fight was over. Thirteen Englishmen lay dead on the field of battle, ARRIVAL OF ANDRIES PRETORIUS 211 with a thousand Natal blacks and probably three times that number of Zulus. After these disasters the emigrants were unable to take the offensive again for some time. Owing to the excessive jealousy between the leaders, Potgieter and his adherents left Natal and went to reside along the Mooi river, where they founded the village of Potchef- stroom. Those who remained behind did not venture far from their fortified camp, and suffered much from sickness and insufficiency of food. While in this distress, however, they were strengthened by the arrival of many of their friends from the colony, and they never once gave up hope of ultimate success. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. During the winter Dingana sent an army to attack them, but they were careful not to be drawn out of the lager by stratagem, and all attempts to reach them behind their defences failed. In November a man of great natural ability, named Andries Pretorius, arrived in Natal, and was elected commandant-general. He at once assembled a force of four hundred and sixty-four men, all that could be spared from guarding the camp, and marched towards Umkungunhlovu. He took with him a sufficient number of waggons to form a lager, and at every halting-place these were drawn up in a circle and lashed together. While on the march scouts were patrolling in all directions to guard against sur- prise. The commando resembled an itinerant prayer meeting rather than a modern army, for the men were imbued with the same spirit as the Ironsides of Cromwell, and spoke and acted in pretty much the same manner. There was no song, no jest heard DEFEAT OF A ZULU ARMY. 21 in that camp, but prayers were poured forth and psalms were sung at every halting-place. The army made a vow that if God would give them victory over the cruel heathen, they would build a church and set apart a thanksgiving day in every year to com- memorate it. The church in Pietermaritzburg and the annual celebration of Dingana's day bear witness that they kept their pledge. They did not wish to fight merely for the sake of revenge. On three occasions the scouts brought in some captured Zulus, and Mr. Pretorius immediately sent these to Dingana to inform him that if he would restore the property taken from the emigrants they were prepared to enter into negotiations for peace. Dingana's reply came in the form of an army ten or twelve thousand strong, which attacked the camp at early dawn on Sunday the 16th of December 1838. For two full hours the soldiers persevered in the attempt to force a way in, notwithstanding the terrible havoc created among them by the fire from the farmers' guns and several small pieces of artillery. When at length they broke and fled, over three thousand corpses were lying on the ground, and a stream that flowed past the field of carnage was discoloured with gore. It has ever since been called the Blood river. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. Pretorius marched to Umkungunhlovu as rapidly as possible, but before he could reach the Zulu capital Dingana set it on fire, and fled. He was pursued, but escaped to a part of the country where cavalry could not act, so the commando returned to Natal with four or five thousand cattle, all that were seen. In the REVOLT OF PAN 21 3 campaign six white men were killed and three were wounded. Since the commencement of hostilities Dingana had lost about ten thousand warriors, but his army was still so large that he was by no means humbled. When the farmers retired he rebuilt his capital, and though he pretended to fall in with overtures which were made for peace, it soon became evident that he was only watching for an opportunity to destroy the emigrants. It was therefore not con- sidered advisable to scatter upon farms, so a town, named Pietermaritzburg, was laid out in such a manner that each family could have a large garden, and the cattle be kept under constant protection. In September 1839 a very important event took place in the Zulu country. Panda, a half-brother of Dingana, conspired to seize the chieftainship. A great number of the incorporated Zulus — the remnants of tribes that had come under Tshaka as the only means of saving themselves — were ready to rally round any leader who could give them reasonable hope of deliverance from incessant bloodshed, and when the induna Nongalaza, who was in command of the district along the northern bank of the Tugela, declared for Panda, they joined him. The rebel chief with a large following then crossed the Tugela, and sent to ask assistance from the Europeans. The emigrants at first regarded him with suspicion, as it was by no means certain that his flight was not merely a pretence to draw them to destruction. But he soon convinced them of his sincerity, and an DF.STRUCTIOX OF THE ZULU POWER. 2T4 arrangement was then entered into by which he became a vassal of the emigrants in consideration of receiving their support. He remained in Natal under their protection until January 1840, when a burgher force of four hundred men under Com- mandant - General Pretorius marched with him against Dingana. His own army was about five or six thousand strong, and was commanded by Nongalaza. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. It marched in a parallel line, but at a distance of twelve or fifteen miles from the burgher commando. Dingana now realised the danger of his position, and attempted to come to terms with the emigrants. There were two officers immediately under him, whose advice he frequently sought, and through whom he carried on his government. Their names were Tambusa and Umthlela. The first named he sent to the European camp to negotiate for peace. Upon Tambusa's arrival, he and his servant Kombazana were made prisoners, and contrary to all law and justice were brought to trial before a court-martial for having taken part in the massacre at Umkungunhlovu, were sentenced to death, and were executed. A few hours after this great crime was committed a messenger from Nongalaza brought word to the burgher column that on the preceding day, 30th of January 1840, he had fought a great battle with Dingana's army led by Umthlela, and had won a complete victory. This battle proved a decisive one. At its com- mencement Dingana's army was superior in number F1XAI. DEFEAT OF DIXGAX 21 5 but during the action a body of his troops went over to Panda's side, and turned the scale. Those who were faithful stood their ground, and fell as became Zulu warriors. The slaughter on each side was enormous. The two best regiments of Dingana perished, for the veterans who had won their plumes under Tshaka chose to die rather than show their backs to the traitors who had deserted their cause. Umthlela placed himself at the head of the reserve, and went into the hottest part of the field, where he was pierced through the heart with an assagai. Still the issue of the day was doubtful, when the cry echoed along Nongalaza's ranks : " The farmers are coming !" It was not so, but the belief that it was answered Nongalaza's purpose. The remnant of Dingana's army, the men who could not flee from a foe armed with spear and shield, gave way in their fear of those dreaded horsemen who had power to deal out death without meeting it them- selves. A bushy country spread out before them, and favoured their escape. The battle was over, and the terror which the Zulu name had inspired was a thing of the past. DESTRUCTION OF THE ZULU POWER AND FOUNDA- TION OF THE REPUBLIC OF NATAL. Dingana fled northward to the border of the Swazi country, where he was soon afterwards assassinated by a man who stole upon him unawares. Those who had adhered to him in his misfortunes then tendered their submission to Panda, by whom they were received with every mark of favour. After the decisive engagement an enormous booty in cattle fell into the hands of the conquerors. About forty thousand head were delivered to Mr. DESTRUCTION OF THE ZULU POWER. 2l6 Pretorius, and were subsequently distributed among the emigrants in proportion to their losses, which enabled them to follow again their ordinary occu- pation of cattle breeders, though the stock now acquired was greatly inferior in quality to that they had brought with them from the Cape Colony and that the Zulus had driven off and slaughtered for food. Before returning to Natal, Mr. Pretorius formally installed Panda as chief of the Zulus, but in vassalage to the volksraad, to which he promised fidelity. This meant that he could continue to rule his people in their own way, without any interference by the Europeans, but that he should do nothing detri- mental to the interests of the white ^people or such as the volksraad might not approve of. The republic of Natal was declared to extend from the Umzim- vubu to the Tugela, and the land between the Tugela and Black Umvolosi was proclaimed an appanage of that republic, set apart for the use of the Zulu people. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. The emigrant farmers had now freed South Africa of the destructive Zulu power, and had driven the ferocious Matabele into regions unknown to Euro- peans. Seldom have such great events been accom- plished by means apparently so feeble. Yet they took no credit to themselves for what they had done, because in their view it was God who had wrought the great deliverance, and they were merely humble instruments in His hands. No Israelite of old ever held a belief of this kind more firmly than did these wanderers who had suffered so much and acted so bravely. It was, however, soon evident that they were less qualified for self-rule than for war, as the government which they established was the weakest and most imprudent that ever existed. It could not be carried on efficiently without a suitable revenue, and they refused to pay any but the most trifling taxes. Every measure of importance after adoption by the volks- 217 SEIZURE OE NAT AE BY BRITISH FORCES. 2l8 raad had to be referred to the burghers in primary assembly, and nothing but confusion was the result. The public offices from the highest to the lowest — with a solitary exception — were held by uneducated men, who could barely write an ordinary letter, and who were of course ignorant of routine duties. Above all, the utmost prudence was needed to avoid irritating the British government, and they acted as if they could afford to be indifferent to English opinion. opinion. The elevation of the coloured races was then a leading — and surely a praiseworthy — idea in England, but, unfortunately, the great philanthropic and mis- sionary societies had made up their minds as to the precise manner in which this should be effected, and condemned as unchristian all views that differed from their own. Applying their principles to South Africa, the formation of large Bantu states under missionary guidance and British protection was what they desired, and the reverend Dr. Philip, the exponent of their views, was urging this scheme upon the Cape govern- ment. Time has shown how faulty it was, but no one even in South Africa could foresee the full extent of the harm it would cause to the black people as well as to the white. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. The devastations which the Zulus and Matabele had wrought were unknown in Europe, and therefore when intelligence reached England that many thousands of the men of those tribes had fallen before the farmers' guns, public opinion was shocked. No one suspected that the destruction of those fierce warriors meant life to all other black people in the country. The great CONDUCT OF THE NATAL GOVERNMENT. 210, societies brought their influence to bear upon the government, in order — as they believed — to stop further bloodshed by compelling the emigrants to return to the Cape Colony. Hardly any one con- sidered it advisable that the British dominions in South Africa should be enlarged by the annexation of the territory which they occupied. of the territory which they occupied. While this was the feeling in England, the repub- lican government resolved not to allow Bantu from beyond the borders to settle in Natal, and to confine those who were already there to certain locations. A commando was also sent against a marauding chief who lived between Natal and the Cape Colony, and he was severely dealt with. If the emigrants had sought to provoke the British government, they could hardly have devised a surer plan. As soon as the intelligence reached Sir George Napier, who was then governor of the Cape Colony, a body of troops was sent to protect the Bantu, and a military camp was formed within a short distance of the southern boundary of the republic. For some time after the arrival of the emigrants in Natal, every possible effort had been made by the authorities in Capetown to cut off their supply of ammunition, but all attempts to do so had failed. They had now a port of their own, and foreign vessels were beginning to find their way to it. This natu- rally caused English merchants engaged in the South African trade to feel irritated, for it was supposed that the harbour of Natal might become the principal gateway to the interior. SEIZURE OF NATAL BY BRITISH FORCES. 220 A resolution of the volksraad to compel some recent Bantu immigrants to retire to a location on the southern side of the republic brought matters to a crisis. The troops on the border were reinforced, and were ordered by Sir George Napier to move on and take possession of Port Natal. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. Accordingly two hundred and sixty-three soldiers of all ranks and of different arms, under Captain Thomas Smith, marched forward, meeting with no molestation on the way, and formed a camp at Durban. The volksraad sent a protest, but no notice was taken of it. Commandant-General Pre- torius then assembled a number of farmers, and formed a camp at the head of the inlet, from which he sent a demand that the English troops should leave without delay. He claimed for the emigrant farmers perfect independence, but Captain Smith maintained the English view, that they had not ceased to be British subjects and could not by any act of their own throw off their allegiance to the crown of England. crown of England. A contest was now inevitable. Captain Smith who altogether underrated the vigilance and courage of his opponent, thought to crush out opposition by a single blow, and left his camp one evening at the head of a hundred and thirty-seven soldiers with the intention of falling by surprise upon Pretorius, who had then with him two hundred and sixty-four men. No military operation could have been worse planned. It was clear moonlight, yet it was thought that the troops would not be noticed. The distance was a march of three miles, and the road was along the SIEGE OF THE BRITISH CAMP. 221 shore of the inlet, which was bordered at one place by dense scrub. by dense scrub. The troops were marching fully exposed past the thicket, with two field-pieces drawn by bullocks, when a sharp fire was opened upon them. They returned the volley, but without doing the slightest damage to the farmers, who were well protected and thoroughly concealed. Another discharge from the thicket wounded some of the oxen, which broke loose from the yokes and rushed furiously about, adding to the confusion. There was no remedy but retreat. Six- teen killed and thirty-one wounded were found by the farmers next day, and three others were drowned. The two guns, the oxen, and indeed everything that could be left behind, fell into the hands of the farmers. Mr. Pretorius now again demanded that the troops should leave Natal, and to gain time to strengthen his camp, Captain Smith agreed to a truce of a few days, under pretence of considering the matter. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. A messenger, Richard King by name, provided with two good horses, was directed to ride with all speed through Kaffirland to Grahamstown with a request for help, and he managed to get safely away. for help, and he managed to get safely away. When the truce expired the English camp was invested, and fire was opened upon it from the farmers' batteries, on which three small cannons were mounted. Captain Smith caused deep trenches to be dug, in which the soldiers could remain in security, and he increased his stock of provisions by slaughter- ing his horses and drying their flesh. The men were put upon short allowance, which, as the siege SEIZURE OF NATAL BY BRITISH FORCES. 2 22 advanced, became less and less, until they had nothing more than a few ounces of biscuit-dust and dried horseflesh daily. Fortunately for them there was no want of water, which was obtained from wells sunk within the camp. The force under Pretorius increased by fresh arrivals until it amounted to six hundred men. They fortified the entrance to the inner harbour, and pressed the siege with vigour. Their cannon-balls having become exhausted, they manufactured others by casting leaden ones over links cut from a chain cable. But so well were the soldiers protected that the fire against them was almost harmless, only eight men being killed and eight wounded on the British side during the twenty-six days that the siege lasted, though six hundred and fifty-one cannon-shot were fired at the camp. On the other side four men were killed, and eight or ten — the exact number cannot be given — were wounded. given — were wounded. The messenger sent by Captain Smith overland, who was familiar with the language and customs of the Bantu tribes on his way, reached Grahamstown in safety, and informed the military authorities of what had happened. A hundred soldiers were thereupon embarked in a schooner at Algoa Bay, and sailed for Port Natal. A wing of a regiment was also taken on board a frigate at Simon's Bay, and proceeded to the same destination. On Sunday, the 25th of June 1842, the schooner sailed into the inner harbour with a fair wind, having as many soldiers on board as could find room, and towing a number of boats containing others. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. The RELIEF OF THE BRITISH CAMP 223 frigate at the same time opened fire with her heavy guns upon the high land commanding the entrance. Three men were killed and five were wounded when passing under the farmers' batteries, but no further resistance was offered, for as soon as the fresh troops landed and Captain Smith was relieved, the burgher force under Pretorius dispersed. force under Pretorius dispersed. Natal thus became a British possession. Some of the farmers remained in it, but most of them packed their effects in their waggons, and moved over the Drakensberg into the interior. More than three years elapsed, however, before a government under English officials was established, and during that time great numbers of Bantu — chiefly refugees from Zululand — moved into the nearly vacant territory. An arrangement was made with Panda, by which he ceded to the British government the ground between the Buffalo and the upper Tugela river, so that the boundary was extended on the north beyond the passes through the mountain range. Thereafter the Zulu chief was treated as an independent sovereign, and immediately the process commenced of building up again that great military power which cost so much English blood in later years to overthrow. On the south all the land beyond the Umzimkulu river was given to the Pondo chief Faku, and thus Natal was much reduced in size in that direction. The farmers who went back over the Drakensberg settled in the territory between the Magalisberg and the Vaal river, that had previously been occupied by Commandant Potgieter's adherents. These now moved away to the north-east, in hope of being able - ,3 z -S ? -2 PROJECT OF TREATY STATE 225 to open communication with the outer world through Delagoa Bay, which, as it belonged to the Portuguese, they thought would be safe against attack by Great Britain. They halted on the headwaters of some streams flowing into that bay, and built a village which they named Ohrigstad. There, however, they suffered very severely from fever, so that they were obliged to move again. They then divided into two parties, one of which founded the village of Lyden- burg, and the other, under Potgieter himself, went away north to the Zoutpansberg and settled there. away north to the Zoutpansberg and settled there. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. In England the conduct of the emigrants in thus persistently retiring from British authority was regarded as very objectionable. The opinion was general that something should be done not only to compel the wanderers in the interior of the continent to return to their old homes, but to prevent others from abandoning the colony and joining them. The project of forming a barrier along the colonial border, by means of the creation of a chain of large native states, had for some time been advocated by the great societies, and was now determined upon by the govern- ment. Such a barrier, it was imagined, would cut off commercial communication with the emigrants, and leave them no alternative but to retrace their steps. In carrying this scheme into execution, Sir George Napier followed the method suggested by the reverend Dr. Philip, who made the preliminary arrangements. His plan was to select in a given area the most competent chief, that is the one supposed to be most amenable to missionary guidance, to enter into treaty with him as a sove- treat CREATION OF TREATY STATES. 2 26 reign, and to support him with all the influence of the British government. the British government. At Thaba Bosigo one such chief was found in the person of the wise and able Moshesh, the friend and patron of missionaries. He had already built up a considerable power, which it was the great object of his life to increase and solidify. Nothing, there- fore, could have been more in accordance with his desires than the scheme which was proposed: alliance with the British government, a subsidy in money, a vast extension of territory, and supremacy over all other chiefs within the area assigned to him. In 1843 a treaty was concluded, in which he was acknowledged to be the sovereign of a large vacant tract of land north of the Orange river, of the basin of the lower Caledon, where European farmers were settled, of the territory along the western bank of the Caledon higher up, which was occupied by various clans brought there by Wesleyan missionaries, and of all the land on which his own people lived. He was to have a subsidy of £7$ a year, payable either in money or in arms and ammunition, as he might choose. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. It will be seen in future chapters that no other document ever signed in South Africa cost so much blood and treasure as this, or was so productive of evil in various ways. ways. West of the territory assigned to Moshesh there were no Bantu, but at and around a mission station of the London society, named Philippolis, there were some fifteen hundred or two thousand Griquas, under a captain named Adam Kok. These people were of mixed European, Hottentot, and negro blood, and THE GRIQUAS. most of them had recently migrated from the Cape Colony. They were supposed to be under missionary guidance and to be partly civilised, but the men lived chiefly by hunting, and their character was far from stable. It was not then known that they were a ■« GRIQUA MAN AND WOMEN. {From a Sketch by Mr. Thos. Baincs.) GRIQUA MAN AND WOMEN. {From a Sketch by Mr. Thos. Baincs.) perishing race. For one or two generations the hybrid offspring of Europeans and coloured people possess a fair amount of fertility, but they must then intermix with one of the pure original stocks, or. die out. Within -fifty years the Griquas, by attempting perishing race. For one or two generations the hybrid offspring of Europeans and coloured people possess a fair amount of fertility, but they must then intermix with one of the pure original stocks, or. die out. Within -fifty years the Griquas, by attempting CREATION OF TREATY STATES. 22 to live as a separate people, decreased to little more than one-fourth of their original number. than one-fourth of their original number. There were more white people than Griquas living in the territory between the Modder river and the Orange, but at the same time that the treaty was made with Moshesh a similar one was made with Adam Kok, and thereafter this petty captain was officially regarded by the British government as the sovereign of all the land from the new Basuto boundary to the territory claimed by Andries Waterboer under the treaty of 1834. He was to receive a subsidy of ^100 a year in money and the use of a hundred stand of arms with a reasonable quantity of ammunition. The London society was to receive £50 a year for the maintenance of a mission school. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. Thus, as far as paper treaties could make states there was now a barrier along the whole northern border of the Cape Colony from the Kalahari desert upward. A little later, by another treaty, the Pondo chief Faku became the nominal ruler of all the terri- tory between the Umtata and Umzimkulu rivers, the Drakensberg and the sea, and thus the girdle was made complete. But it was soon found that for the purpose intended the treaty states were useless. The emigrant farmers ridiculed the idea either of their removal or of their subjection to the puppet sovereigns thus set up, and matters went on pretty much as before, so far as they were concerned. In the territory assigned to Moshesh trouble of an unexpected kind immediately arose. The chiefs of the clans along the Caledon indignantly EFFECTS OF THE TREATIE 229 refused to acknowledge him as a superior, and the Wesleyan missionaries took part with them in doing so. The French missionaries, on the other hand, did their utmost to support and build up the Basuto power. Thus jealousies and quarrels were fomented, and the clans were kept in perpetual disturbance. and the clans were kept in perpetual disturbance. In the territory assigned to Adam Kok many of the white people had come to fear that such anarchy as had prevailed in Natal was inseparable from a republican form of government, and they were not only willing but anxious to see the country annexed to the British dominions. There were circumstances in their condition and in the manner of their removal from the colony that made them the least disaffected of all the Dutch-speaking people of South Africa. But they, too, repudiated the sovereignty of Adam Kok, and refused to acknowledge him as anything but a Griqua captain. Besides these people there were two large parties of emigrants in the country bitterly hostile to England, and they at once declared that if Kok attempted to interfere with them in any way whatever they would resist with arms. The treaty states were thus no barrier to commer- cial intercourse with the emigrants in the interior, they did not prevent further emigration, nor did they cause a single individual to retrace his steps. SEIZURE OF NATAL BY BRITISH FORCES. CREATION OF TREATY STATES ALONG THE FRONTIER OF THE CAPE COLONY. They provoked disputes and quarrels among people who were before friendly, and they enabled Moshesh to build up a power antagonistic to the interests and welfare of South Africa. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. Sir Peregrine Maitland, who succeeded Sir George Napier as governor of the Cape Colony, determined to support Adam Kok with a military force if he should be attacked, and a promise to that effect was made to him. Thereupon he assumed a very haughty tone towards the white people, and shortly afterwards sent a band of his followers to arrest a farmer who ignored his government. The farmer was not at home when the Griquas arrived at his house, so they poured a storm of abuse upon his wife, and took possession of his guns and am- munition. Upon this the burghers formed a lager about thirty miles from Philippolis, and having placed their families in safety within it, they left a guard for its defence and took the field. There was at the time a body of British troops stationed at Colesberg, on the colonial side of the Orange river, two hundred of whom now marched to Philippolis to aid the Griquas. Adam Kok was also supplied with muskets and 230 EXPEDITION TO AID ADAM KOK 23 1 ammunition from the military stores. He was thus able to defend himself until a regiment of dragoons, with some artillery and a company of light cavalry, could march to his support from Grahamstown. As soon as the force reached Philippolis, Colonel Richardson, who was in command, issued a pro- clamation calling upon the farmers to surrender un- conditionally; but they took no notice of it. He then marched towards their lager, and by a stratagem drew two hundred and fifty men out of it, who were nearly surrounded before they were aware that British troops were acting against them. Taken by surprise, they did not attempt to make a stand, but in their efforts to escape three were killed. A little later in the same day possession was taken of the lager with- out resistance, when all the arms found there were confiscated. Colonel Richardson next called upon the emi- grants to take an oath of allegiance to the queen, when all those— three hundred and sixteen in number — who have been mentioned as not ill-affected towards the British government did so. The others were not arrested, as there were no means of supporting them in detention, so they moved away to Winburg, beyond the territory claimed by Adam Kok. the territory claimed by Adam Kok. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. By this time Sir Peregrine Maitland had become convinced that the Griqua treaty state, as originally planned, could not be maintained without the constant presence of a considerable military force, and in that case to regard Kok as a sovereign would be an absurdity. But he did not know what change to make, and so he visited the country in order to learn TO CLOSE OF SEVENTH KAFFIR WAR. 232 its condition by personal intercourse with the different people there, and to devise some plan of action. At Touwfontein, where the emigrant lager had been, he met a great number of farmers, all the chiefs between the Orange and Vaal rivers, and most of the mission- aries. During several days matters were discussed, and the views of the different parties were laid before the governor. the governor. Adam Kok contended that he was a sovereign in alliance with Great Britain, that every one within his dominions who did not implicitly obey his orders was a rebel, and he requested that all the white people should be removed. The farmers contended that as there was no one living in the territory claimed by Kok whose parents had been born there, all being recent immigrants, their right was equal to that of the Griquas. Much of the land they occupied was vacant when they took possession of it, and the remainder had been purchased or leased from individual Griquas who by an earlier selection had prior claims. They could not return to the colony, where they had no ground, nor could they submit to such a government as that of Adam Kok and his missionary. Moshesh contended that as he was acknowledged to be the sovereign of the territory assigned to him by treaty, no one within it should be communicated with except through him. The chiefs along the Caledon contended that their independence of Moshesh ought to be recognised, and declared that they would rather die with arms in their hands than submit to him. ARRANGEMENT WITH ADAM KOK 233 Each of the missionaries supported the claims of the particular chief with whom he was living, so that their opinions differed greatly. that their opinions differed greatly. Out of this confusion Sir Peregrine Maitland saw but one way of establishing order. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. He gave up all idea of the return of the emigrants to the Cape Colony, and endeavoured to arrange for the proper govern- ment of those who were living in the treaty states. He proposed to Adam Kok that the land between the Modder and Riet rivers should be allotted to Euro- peans, and that between the Riet and Orange rivers to Griquas. The Europeans were to be governed by an English officer whom he would nominate, and to whom Kok was to give a commission. Quitrent was to be levied on their farms, one half of which was to be devoted to the payment of the English officer and his assistants, and the other half was to be handed over to Kok, whose sovereignty over the whole terri- tory was in this manner still to be recognised. The Griqua captain at once closed with the offer, for it relieved him of a great difficulty and gave him an addition to his income. The Europeans also accepted the proposal, though some of them grumbled at having to pay tribute to a man whose right to the ground was no better than their own. Major Warden was selected by the governor to rule the European community between the Riet and the Modder, and fixed his residence at a place named Bloemfontein. A few soldiers of the Hottentot regi- ment were stationed there to support his authority, and he received all his instructions from Capetown, so that practically the territory was a British depen- TO CLOSE OF SEVENTH KAFFIR WAR. 234 dency, though writs and other public documents ran in the name of Adam Kok. This arrangement worked fairly well, and a short period of peace and prosperity followed in that part of South Africa. prosperity followed in that part of South Africa. A similar proposal was made by Sir Peregrine Maitland to Moshesh, but that chief was in a very different position from the Griqua captain, and was loth to exchange power for money. He wanted to keep on good terms with the governor, however, and so he made an appearance of consenting to the plan while really thwarting it. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. He offered for the use of Europeans a tract of land in the angle of the Orange and Caledon rivers, so far away from the possessions of his own people that there was no likelihood of its ever being of value to him, and so small that no revenue derived from it could cover the salary of a British official. This offer he could not be induced to enlarge, and though negotiations were carried on with him after Sir Peregrine Maitland's return to Cape- town, nothing came of them. Thus within the Basuto treaty state matters remained in a most unsatisfac- tory condition. tory condition. At this time the eastern frontier of the Cape Colony was exposed to depredations as it had never — even in the worst times — been exposed before. Lord Glenelg, through his agent Lieutenant-Governor Stockenstrom, had given up to the Xosas the whole country east of the Kat and Fish rivers, and had entered into treaties with the chiefs as sovereign rulers. This action they considered a proof of weakness, and in consequence they laid aside all respect for the British authorities. Within the next ten vears over a hundred murders MARKS OF PROGRE 2$$ were committed by their people on colonial ground, and the country as far west as the Sunday river was harried and wasted almost as in a time of war. The unfortunate British settlers of 1820 were the principal sufferers, but their prayers for relief were altogether disregarded in England. Modifications of the treaties were made by Sir George Napier and Sir Peregrine Maitland, but the position was not improved, for it was the system itself that was the cause of the evil. In other respects this period was marked by many beneficial changes. The cost of government was greatly reduced, so that every year a surplus could be applied to the reduction of the public debt. By the sale of the old drostdies, the conversion of a number of quitrent farms into freehold, and licenses for the removal of a quantity of guano from some small islands off the western coast, by 1847 the debt was entirely paid off. Magistrates were increased, and churches of various denominations were multi- plied throughout the colony. Municipal government of the towns and villages was introduced. An ex- cellent system of schools was brought into operation. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. Good waggon roads were made — principally by con- vict labour — through many mountain passes, where previously produce could only be transported with extreme difficulty. The value of the exports was rapidly rising by increase in the production of wool. More than all, four or five thousand English, Scotch, and Irish agriculturists and mechanics were brought into the colony by a system of aided immi- gration, and partly filled the places of those who had SEVENTH KAFFIR WA 237 moved into the interior. No people in any country have thriven better than these. About seven hundred destitute children sent out from London by a bene- volent society were apprenticed to carefully selected persons, and though a few turned out badly, most of them became useful and prosperous members of the community. Unfortunately, however, these were not the only immigrants. The Cape was made the station where all slave ships captured by British cruisers south of the equator were brought, and the negroes were apprenticed here for short periods, after which they became merged in the general coloured population. The farmers and townspeople, who were without a sufficient supply of labour, were very glad to get them, and the missionary societies welcomed them as material to work with ; but they were not a class to add permanently to the prosperity of the country. After the frontier colonists had been exposed for ten years to the depredations of the Xosas, an event took place which brought on open war. A Xosa, who was detected in an act of theft at Fort Beaufort, was arrested, and was being conveyed to the nearest magistrate's office for trial, when a party of his clans- men crossed the border, and after overpowering the constables and murdering a Hottentot, released their friend. Sandile, the legal heir of Gaika, was then the principal chief of Western Kaffirland. The governor applied to him to surrender the raiders for trial, as their crime had been committed on colonial ground, and he had bound himself by treaty to give up offenders of this kind. But he TO CLOSE OF SEVENTH KAFFIR WAR. 238 made light of the matter, and refused to carry out his engagement. his engagement. A military force was then directed to enter Kaffir- land and occupy Sandile's kraal, so as to bring him to terms. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. A very long waggon train accompanied it, conveying provisions, tents, baggage, and ammuni- tion ; and, as if to invite attack, this tempting prize was almost unguarded. The movements of the expedition were, of course, closely watched by the keen eyes of Xosa scouts, and when in a spot con- venient for the purpose, while the main body of the English troops was some miles distant, a strong band of warriors rushed upon the train, and without any difficulty made themselves masters of a great portion of it. By this disaster the British force was compelled to retreat precipitately. After considerable loss it reached the Lovedale mission station, just within the colonial border, and hastily fortified a large stone building used as a boarding school, which enabled it to stay there in safety. to stay there in safety. At once a great body of Xosa warriors poured into the colony, swept off all the cattle east of Uitenhage, burned many dwelling-houses, and murdered several individuals who had not time to escape to villages or lagers. Their success encouraged a large portion of the Tembu tribe to join them, and these people laid waste the country north of the Winterberg just as the Xosas had done south of that range. Thus the European settlement in the eastern districts was reduced to the towns and villages, which were crowded with helpless and destitute people. The COURSE OF THE WA 239 farms — except a few where there were lagers — were abandoned. Another disaster followed. The garrison of the most advanced fort on the frontier was in urgent need of supplies of food and ammunition, and a train of waggons, under military escort, left Grahamstown for its relief. In a thicket the train was attacked, the guard was obliged to retire, and the supplies fell into the hands of the Xosas. The whole burgher force of the colony was called out, and every soldier that could be spared from duty in Capetown was sent to the front. Hottentots and other coloured people were enrolled, waggons and oxen were everywhere impressed, and in a short time a mass of combatants sufficiently large for offensive operations was assembled on the frontier. A greater difficulty than that of collecting men, however, was that of collecting provisions. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. It was not only the army that the government had to feed, but the unfortunate European women and children whose property had been destroyed, and the families of all the Hottentots of the frontier. Some successes were gained, but operations were stayed by the collapse of the transport service, and the army was obliged at one time to encamp on the coast, where supplies could be obtained by sea, in order to escape starvation. After a while several regiments of soldiers arrived from abroad, the trans- port service was organised on a better plan, and provisions were sent from the western districts. Then a kind of lull took place, in consequence of a declaration of submission by most of the" hostile TO CLOSE OF SEVENTH KAFFIR WAR. 24O clans, whose object was to get a crop of maize and then renew the war. During this lull Sir Peregrine Maitland was re- called, as every governor since Lord Charles Somerset has been in whose term of office war has broken out. He was succeeded by Sir Henry Pottinger, who was also appointed high commissioner for the purpose of dealing with matters beyond the colonial border. All succeeding governors of the Cape Colony have been high commissioners also. The crops of maize were gathered, and the war was resumed. But soon afterwards Sandile found himself hardly pressed, and surrendered, upon which there was another general profession of submission. In later years the Xosas laughed at the ease with which the white people were deceived, and ridiculed the idea of their being beaten in this war, But the governor, judging them by a European or an Indian standard, believed that they were subdued, and was about to proclaim peace when he received news of his transfer to Madras. The enormous expense of the war had brought home to the imperial government the folly of the treaty system, of which it was the result, in a manner that the prayers of the colonists had never done. The ruined eastern farmers were clamouring for com- pensation from the British treasury for their losses, on the ground that they had protested against the measures which led to the war, and Lord Glenelg had accepted the responsibility of carrying them out. They did not get what they asked for, but the whole military and commissariat charges were of necessity RESULTS OF THE WA 2\\ borne by England. EVENTS TO THE CLOSE OF THE SEVENTH KAFFIR WAR. Both Sir Peregrine Maitland and Sir Henry Pottinger had come to the conclusion that Sir Benjamin D'Urban's system of dealing with the Xosas should be reverted to, and even Sir George Napier was of the same opinion. Napier was of the same opinion. The imperial authorities then resolved upon another complete change, and to carry it out they selected as governor and high commissioner Sir Harry Smith, who, as Colonel Smith, had been Sir Benjamin D'Urban's most able lieutenant in South Africa, and who had recently won high military renown in India. But while making this resolution they were beginning to comprehend that it was impossible at a distance of six thousand miles to direct the affairs of a country either safely or satisfactorily, especially under such pressure as could be brought by the great societies to bear upon a government in England; and they were already impressed with a belief that the best course they could adopt would be to let the affairs of the Cape Colony be settled by its own people. To the Kaffir war of 1846-7 more than to any other event is due the liberal constitution that was granted a few years later. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. No governor has ever been more heartily welcomed in South Africa than Sir Harry Smith. Every section of the inhabitants of the colony hailed him as an old and tried friend, and there was a general hope that better days had now set in. He was not long in making known the details of the changes which he came to effect. Hurrying to the eastern frontier, he issued a proclamation, extending the Cape Colony on the north to the Orange river from its mouth to the junction of the Kraai, and on the east to the Keiskama and the Tyumie. Keiskama and the Tyumie. The territory between the Keiskama and Tyumie on one side, and the Kei on the other, he then pro- claimed a British possession, but to be kept entirely for the use of the western clans of the Xosa tribe, just as Sir Benjamin D'Urban intended when he annexed it under the name of the province of Queen Adelaide. Colonel Mackinnon was appointed a commissioner to exercise general authority over the clans, an office which the governor himself had once 242 242 PORTRAIT OF SIR HARRY SMITH. PORTRAIT OF SIR HARRY SMITH. ADMINISTRATION OF SIR HARRY SMITH. 244 held. The chiefs remained the rulers of their people in many matters, but vicious customs were no longer to be tolerated, and punishment of persons accused of dealing in witchcraft was to be suppressed. A strong body of troops was to garrison various forts in the territory, and to support the authority of the commissioner and his assistants. The new province was named British Kaffraria. The whole of the chiefs who had been in arms agreed to this arrangement, and those who resided within the province took an oath to maintain it. The others, who lived east of the Kei, were left perfectly independent. The governor, the colonists, and the missionaries — whose views were greatly modified by the late war — alike considered this settlement satisfactory, and to all outward appear- ance the Xosas were pleased with it ; but within three years the chiefs declared that they had only agreed to it as a truce, in order to get material together for another trial of strength with the Europeans. Europeans. As soon as these arrangements were made, Sir Harry Smith proceeded to the territory north of the Orange river. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. Treaties between the British govern- ment and Bantu chiefs he regarded as agreements between a full-grown man and little children, and he repeatedly and emphatically declared that there should be no more of them. As for the treaty states on the northern border, he looked upon them as the creations of supreme folly, and he therefore intended to destroy them. But as neither Adam Kok nor Moshesh had violated any of the conditions of the END OF THE TREATY STATE 2\$ treaties, he could not declare the documents annulled, and it was thus his object by some means to induce those persons to consent to their own effacement as sovereign rulers. sovereign rulers. The emigrant farmers between the Riet and Modder rivers gave him an enthusiastic reception, for many of them had fought under his command thirteen years before, and they had always liked him as he had liked them. They had no complaint to make against Major Warden, but they had a griev- ance, in that half of the land tax which they paid went into the pocket of Adam Kok, and they had no return for it. Was it not scandalous, too, they asked, that they should be officially termed subjects of that petty captain of a mongrel band ? of that petty captain of a mongrel band ? By the governor's desire, Kok went from Philip- polis to Bloemfontein to meet him. At the conference Sir Harry stated that he was about to place the white people in the territory under the direct rule of the queen of England, but he would not interfere with Kok's government of his Griquas. The whole of the land between the Riet and Orange rivers — which was ten times as much as the Griquas could make use of — would be regarded as their reserve, the captain should have a perpetual pension of £200 a year, and as some of his people had leased farms north of the Riet river, which would now be lost to them, they should have among them ^"ioo a year in perpetuity as compensation. The captain demurred to these terms, and spoke of his dignity in such a way that the governor lost all patience and threatened him with speedy punishment. He then submitted, and ADMINISTRATION OF SIR HARRY SMITH. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. 246 affixed his name to a document which put an end to the Griqua treaty state, but left him far more than he had any reasonable claim to. than he had any reasonable claim to. A little later the governor had an interview with Moshesh, to whom he made some valuable presents, at the same time professing his friendship in the warmest language. He then announced his intention as regarded the white people, and asked for the co-operation of the chief. Probably Moshesh felt somewhat overawed in the presence of the impetuous governor, and it is improbable that he fully compre- hended what the proposals laid before him would lead to, but he attached his mark to a document which destroyed the Basuto treaty state. which destroyed the Basuto treaty state. These preliminaries having been settled, on the 3rd of February 1848 Sir Harry Smith issued a proclamation, adding to the British dominions the whole territory between the Vaal river, the Orange river, and the Kathlamba mountains, under the name of the Orange River Sovereignty. The Europeans in it were placed under the immediate rule of the queen, and a staff of officials was appointed to administer justice and collect taxes in her Majesty's name. Major Warden was appointed head of the new administration. The coloured people were left under the government of their chiefs, and the land then actually in their occupation was to be reserved for their use and secured against encroachment. What might be termed their foreign relations, that is everything affecting the dealings of one head chief with another, or of any chief with Europeans, were to be under the control of the British authorities. BATTLE OF BOOMPLAA 2\J Here, at last, was a policy such as nearly every man in the Cape Colony approved of. Unfortunately, however, it came too late. The vast majority of the white people living between the Modeler and Vaal rivers were indisposed to submit to British rule in any form, and prepared to fight for the independent government they had lived under for twelve years. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. Moshesh, who had by this time built up a power far greater than Sir Harry Smith was aware of, began to devise schemes for the destruction of the Sovereignty government as soon as he found that he was to be confined to a reserve covering only the actual ground on which his people lived, that his practice of incor- porating members of other tribes with his own was likely to be severely checked, and that the clans along the Caledon were treated as independent of him. the Caledon were treated as independent of him. Sir Harry Smith had not long returned to Capetown when he received intelligence that the farmers in the north of the Sovereignty had elected Mr. Andries Pretorius to be their commandant and had risen in arms, that Major Warden with the little garrison of Bloemfontein had been obliged to capitulate, and that the whole of the English officials had been driven over the Orange river and were then in a camp near Colesberg. The energetic governor at once directed a strong body of troops to march to the Orange, and followed himself to take command in person. Commandant Pretorius did not attempt to defend the passage of the river, but made a stand at a strong position called Boomplaats, where on the 29th of August 1848 a severe engagement took place, which ended in the defeat of the farmers. ADMINISTRATION OF SIR HARRY SMITH. 248 All who were inveterately opposed to British rule now made their way in haste across the Vaal river, and there was no attempt to follow them. The Sovereignty government was re-established, and a much larger garrison than before was left in Bloem- fontein. The places of those white people who had moved away were filled by fresh emigrants from the Cape Colony, many of whom were Englishmen, so that from this time forward the European population of the territory consisted of people either well affected to the British government or not bitterly opposed to it. British government or not bitterly opposed to it. Leaving now the region beyond the Cape Colony for a short time, an event must be related which caused intense excitement throughout South Africa. This was a project of the imperial ministry of the day to make of the Cape a convict settlement. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. The tidings caused a feeling somewhat akin to what a proposal would have done to introduce a dreadful disease. Men and women of respectability every- where raised their voices against it, for if a class of people that had either by choice or of necessity become criminal, and whose self-respect was destroyed by conviction, were once allowed to mix with the coloured races, the country would no longer be fit to live in. Petitions and protests against the measure were sent to England in great number, and when the ship Neptune with convicts on board arrived in Simon's Bay, the people of the Cape peninsula — with few exceptions — bound themselves together under a pledge not to supply anything whatever to persons who had dealings with her, nor to have any inter- course with them. ANTI-CONVICT AGITATI 249 This pledge was so strictly carried out that not a particle of food could be obtained for the convicts, and it was with much difficulty that supplies for the troops in garrison were procured. Any one who opposed the popular will in the matter did so on peril of being assaulted and having his property destroyed. Sir Harry Smith was very much opposed to the scheme of making the country a convict settlement, but he was obliged to carry out the instructions which he received from England, and so he could not send the ship away, though the colonists were very anxious that he should. colonists were very anxious that he should. Five months the Neptune lay at anchor in Simon's Bay. Her crew and the convicts on board could get nothing to eat but provisions out of ships of war. If the plague had been in her she could not have been more carefully avoided. All this time the greatest excitement prevailed in the colony, and great caution had to be used by the government to prevent a collision with the people. At length, to the joy of every one, instructions were received from England that the convicts should proceed to Tasmania, as the secretary of state had changed his mind, owing to the numerous petitions of the colonists. to the numerous petitions of the colonists. The anti-convict agitation had hardly died out when the country became involved again in war with the Xosa and Tembu tribes. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. The principal chiefs of these people had never regarded the cessation of hostilities at the close of 1847 as anything but a truce, though they were crafty enough to conceal their views from even those Europeans who were most intimately acquainted with them, and it was only at a later date ADMINISTRATION OF SIR HARRY SMITH. 250 that this became known. The common people were ready to support their chiefs with their lives as well as their substance, and, from their point of view, they had at least one very serious grievance against the European authorities. European authorities. The Bantu believe most implicitly that diseases and disasters of all kinds are caused by wizards and witches, and in every clan there is a recognised witch- finder who, whenever any trouble occurs, goes through certain forms called " smelling out," and then points to an individual whom he pronounces guilty of having caused it. The individual thus accused is, without further investigation, subjected to torture of different kinds, often resulting in death, and may consider himself fortunate if he escapes with a few scars and the loss of all his property. The British authorities suppressed the practice of "smelling out," and punished the witchfinders. They believed that by so doing they were conferring a benefit upon the people, who would be grateful for relief from the danger of being despoiled and tortured without cause or guilt. But the people supposed to be relieved looked at the matter in a different light. The English, they said, are giving us over to the wizards and witches to do as they like with us. Their view was what ours would be if a government were to suppress punishment for murder and imprison the constables who arrested a man for committing it. Only a slight pretext was therefore needed for a renewal of the war, and any accident might have precipitated it, but, as it happened, the frontier colonists received timely warning of what was EIGHTH KAFFIR WA 25 1 coming. It became known that a man named Umlanjeni, who was credited by his people with great magical knowledge, was issuing charms which he asserted would turn bullets fired at their wearers into water, and the Xosa warriors were repairing to him in hundreds at a time to procure them. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. On receiving a report to this effect, Sir Harry Smith proceeded to King-Williamstown, and con- vened a meeting of all the chiefs in British Kaffraria, in order to discuss matters with them. Sandile, the most powerful among them, did not appear. Still, as the others made no complaints of any kind, and seemed to be prosperous and happy, the governor thought they could not have war in their minds. He returned to Capetown, but reports followed him that there would surely be a speedy outbreak. that there would surely be a speedy outbreak. With all the soldiers that could be mustered, Sir Harry was soon back in King-Williamstown, and as Sandile was known to be in one of the forests at the sources of the Keiskama, a body of troops was sent to arrest him. On the way the troops were attacked in the Boomah pass by thousands of Xosas, and lost twenty-three men killed and as many wounded in fighting their way through. A few hours later in another part of the country a patrol of fifteen soldiers was met by some of Sandile's people, and all were put to death. On the following morning — Christmas 1850 — three villages named Auckland, Woburn, and Juanasburg, close to the colonial side of the border, were surprised by Xosas, when forty-six men were murdered in cold blood, and the houses were burned to the ground. In this manner the eighth Kaffir war commenced, and it was the longest and most costly in blood and treasure that the Cape Colony had ever been engaged in. The frontier districts were ravaged once more, and the burghers of all parts of the country were obliged to leave their homes and take up arms. The Xosas were joined by a great part of the Tembu tribe and by several hundreds of Hottentots from the settlement at the Kat river and other places. Even some of the soldiers of the Hottentot regiment deserted and went over to them, as the colonists had always feared would some day happen. had always feared would some day happen. A very sad event was the loss of the transport steamship Birkenhead, which was sent from England with troops to assist in the war. She was proceeding to Algoa Bay, when in the middle of the night she struck on a reef running out from Danger Point. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. The women, children, and sick people were put into the boats, while the soldiers were drawn up on the deck as on a parade ground. The sea was swarming with sharks, the shore was so far distant that the strongest swimmer could not hope to reach it, and the wreck was breaking up fast. Yet those brave men stood calmly there till the boats with the help- less ones got away. Then, just as the ship fell to pieces and sank, they leaped into the sea, and a few, by clinging to floating wreckage, got to land. Over four hundred perished. There had never before been so strong a force in South Africa as there was in KafTraria at this time. For more than two years the soldiers, burghers, and auxiliaries of various kinds were employed against EIGHTH KAFFIR WA 2 53 an enemy that could not be brought to a decisive action, but that seemed to go from one forest to another with the facility of birds, and that carried on war by doubling upon pursuers, cutting off stragglers, and seizing everything that was not strongly guarded. At length, however, the food of the hostile clans was completely exhausted, and the chiefs then asked for peace, which was gladly granted. granted. It was not Sir Harry Smith's fault, but his mis- fortune, that the war had taken place. It would have been beyond the power of any man to have staved it off permanently, for even the settlement made at its close, as will hereafter be seen, was only regarded by the Xosas as a truce. But, following the invariable custom in such cases, the secretary of state recalled the governor. Sir George Cathcart, who was sent out as his successor, took over the duty on the 31st of March 1852, and thereafter directed operations in person until the conclusion of peace. of peace. He located the Tembus — who were really subdued — in the district that is now called Glen Grey, and gave much of the remainder of the land they had occupied for the last quarter of a century to colonists to be held under military tenure. It was for a time called North Victoria, but subsequently became known as the district of Queenstown, from the neat and flourishing village that was built near its centre. EVENTS DURING THE ADMINISTRATION OF SIR HARRY SMITH. The Fingos, who had fought well on the European side, received the best of the land along the foot of the Amatola mountains and some ex- SETTLEMENT OF BRITISH KAFFRARIA. 255 tensive tracts forfeited by the Tembus. The inde- pendent section of the Xosa tribe, under the chief Kreli, was left in possession of the territory between the Bashee and the Kei ; and the western clans of this tribe, who had been British subjects since 1847, had all the open ground from the Kei to the Keiskama assigned to them. Keiskama assigned to them. The government of British Kaffraria was re- established with a strong and reliable force to support it. Before the war there had been a large body of Kaffir police, but at the commencement of hostilities the men composing it had gone over to their own people. Their place was now taken by a corps composed chiefly of young colonists, with a few Fingos attached to it as detectives. The men, who were armed with the best weapons, were mounted, and proved a most useful body for either military or police purposes. Several regiments of British troops were also stationed in the province. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. FOR a short time after the restoration of British rule in the Orange River Sovereignty, everything went on smoothly, and the people appeared to be prosperous. But this satisfactory state of affairs did not continue long. Moshesh was unwilling that the growth of his power should be restricted, and as he did not wish openly to break with the British government, he endeavoured secretly to foment such disturbances as would destroy the arrange- ments then existing. He — a self-made Bantu ruler — cannot be blamed for doing this ; but what can be said of the treaty system which enabled him to build up sufficient power to do it? It was easy for him to bring about a collision between one of his vassal captains and the chief Sikonyela, while all the time he was professing to be an advocate of peace and apparently making sacrifices to secure it. Presently other clans became 256 256 FIRST BASUTO WA 2$? nvolved in the quarrel, and Major Warden, who had done all that was possible to restore order by advice and expostulation, then tried to quell the disturbance by force. by force. This course of action was regarded by the Euro- peans in the Sovereignty as a mistake. They main- tained that the government ought not to meddle with matters affecting only Bantu clans, as it had no spare strength to squander, and should reserve its interference for occasions when Europeans were threatened with damage. But Sir Harry Smith thought differently. He had no idea that the Basuto power was as great as it afterwards proved to be, nor indeed had any other European in South Africa- He was of opinion that by adding the English soldiers at Bloemfontein and an equal number of farmers to any side in a quarrel in the Sovereignty, he could turn the scale against the other side. And so it was by his instructions that Major Warden attempted to punish the disturbers of the peace. the peace. With one hundred and sixty-two soldiers, one hundred and twenty farmers, and from a thousand to fifteen hundred blacks, the major marched against Molitsane, the vassal of Moshesh who was held to be the cause of the disturbance, and at Viervoet was drawn into a trap and suffered a crushing defeat. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. No one was more surprised than Moshesh himself at the issue of the battle, for he had not believed that the troops and farmers under Major Warden's command could have been driven from the field so easily. He at once threw off the mask he had 18 INDEPENDENCE OF SOUTH AFRICAN REPUBLIC, 2$S hitherto worn, and laid aside his assumed respect for British authority. It has before been stated that the majority of the farmers in the Sovereignty were well affected towards England, but a strong minority were at heart opposed to English rule, though up to this time not inclined to offer open resistance. These last were hardly less surprised than Moshesh at the decisive success of the Basuto in the battle of Viervoet. They knew that no aid could be sent to Major Warden from the Cape Colony, which was then involved in a war of its own, and so, as a matter of self-protection, they set aside their duty to the Sovereignty govern- ment and entered into an engagement with Moshesh. They promised not to take part in hostilities against him, and he engaged not to allow his people to molest them. On both sides this agreement was faithfully kept. The Europeans who were loyal to the British government, on the contrary, were sought out by bands of Basuto and plundered mercilessly. The clans along the Caledon were dispersed, and were reduced to great distress. Major Warden was perfectly helpless, for without a strong military force order could not be restored, and he had only men enough to guard the fort in Bloemfontein. enough to guard the fort in Bloemfontein. Some of the farmers now sent a request to Com- mandant Pretorius to come and devise some plan to put an end to the prevailing anarchy, and Moshesh joined in the invitation. Since the battle of Boomplaats Pretorius had been living north of the Vaal, with a reward of ^2000 for his apprehen- THE SAND RIVER CONVENTIO 259 sion hanging over his head all the time. When urged to interfere in matters in the Sovereignty, he wrote to Major Warden announcing his intention to do so, but intimating that he would prefer to make a treaty of peace with the British govern- ment, in which the independence of his adherents should be acknowledged. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. Major Warden hereupon reported to Sir Harry Smith that the fate of the Sovereignty depended upon the movements of a proscribed man. He had been instructed to act strictly on the defensive until troops could be spared from the Kaffir war to aid him, but if Pretorius and the emigrants north of the Vaal united with the Europeans who ignored his authority and with Moshesh, he would be entirely at their mercy. Under these circumstances the governor decided to acknowledge the independence of the Transvaal emigrants, as the imperial ministers had announced their determination not to add another square inch of ground in South Africa to the queen's dominions, and advantages which could be obtained by a con- vention were not to be had in any other way. Two assistant commissioners — Major Hogg and Mr. Owen — -were therefore sent to make the necessary arrange- ments with Commandant Pretorius and a number of delegates from the Transvaal people. The con- ference took place on a farm in the Sovereignty, and there, on the 17th of January 1852, a document — known ever since as the Sand River convention — was signed, in which the British government guaranteed to the emigrants north of the Vaal the INDEPENDENCE OF SOUTH AFRICAN REPUBLIC. 260 right to manage their own affairs without inter- ference. The convention was confirmed by the secretary of state for the colonies, and was ratified by the volksraad, so that thereafter the South African Republic — as the country was named — had a legal as well as an actual existence in the eyes of the British government. The Sovereignty was thus preserved from inter- ference by Mr. Pretorius, and its government became somewhat stronger than before, because a good many of those who had ignored Major Warden moved over the Vaal. But Moshesh's people still continued to plunder and harass the loyal farmers, and the clans that had opposed him remained in great distress. that had opposed him remained in great distress. This was the state of matters until Sir George Cathcart was able to spare a strong body of troops from British Kaffraria, with which he marched north- ward to restore order. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. Me reached Platberg on the Caledon with a splendidly equipped force, consisting of nearly two thousand infantry, five hundred cavalry, and some artillerymen with two field-guns, hoping that the mere presence of such a body of troops would enable him to settle everything to his satis- faction, without having recourse to hostilities. From Platberg, after a minute investigation of affairs, he sent an ultimatum to Moshesh, demanding that chiefs compliance with certain conditions and the delivery within three days of ten thousand head of horned cattle and one thousand horses, as compensa- tion for the robberies committed by the Basuto. Moshesh personally was willing to accede to the high commissioner's terms, for he dreaded a struggle CnXDITfOX OF THE BASUTO TRIBE 26 1 with the British power now that the Tembus had been subdued, the Xosas were ceasing to fight, and the Transvaal farmers were pacified. He knew that the army at Platberg was only a small portion of the force at Sir George Cathcart's disposal, and he was in that condition that any serious reverse might ruin him. The great tribe that called him master was composed of the fragments of many others that had not yet thoroughly blended, and disaster would cause its disintegration. There were numerous indi- viduals in it of higher rank by birth than he, so that elements of discord were present, though they did not show themselves in times of prosperity. In short, to save his dynasty it was necessary for Moshesh to avoid defeat. But the Basuto people, preferred a trial of strength to the surrender of so many cattle and horses as the high commissioner demanded, and the great chief could not afford to act in opposition to their wishes, as a ruler by hereditary right could have done. The result was a kind of compromise. Moshesh sent in three thousand five hundred head of cattle, with a faint hope that they would be accepted as sufficient, and then assembled his warriors at Thaba Bosigo to resist the British army if it should advance. resist the British army if it should advance. The country of the Basuto is an exceedingly difficult one to penetrate. It is the Switzerland of South Africa. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. Resting on the interior plateau of the continent, five thousand feet above sea-level, it rises like a gigantic billow in successive waves of moun- tains until the summit of the Drakensberg is reached, the highest peaks of which are over eleven thousand S BATTLE OF BER 263 feet above the ocean. The lower valleys are remark- ably fertile, so that the country can support a large population, though no other use is made of the higher lands than to pasture cattle in summer. There are many hills with flat tops and precipitous sides that can easily be defended against an enemy, and of all these Thaba Bosigo, the seat of Moshesh's government, is the strongest. government, is the strongest. On the 20th of December 1852 Sir George Cath- cart crossed the Caledon from Platberg, and entered Basutoland, with the intention of occupying Moshesh's mountain. He made the great mistake of under- estimating the strength and courage of his opponent, and not giving him any credit for ability as a strate- gist. His officers took no thought about the matter, but looked upon their occupation of Thaba Bosigo as a certainty, and their march as a pleasant excursion. The army entered the Basuto territory in three divisions. By a simple stratagem — that of exposing an immense herd of cattle in a position on the Berea mountain where their capture appeared easy — one of the British divisions was drawn into an ambush, and after suffering considerable loss was obliged to retreat to the camp at Platberg. It drove before it, however, some four thousand horned cattle, with a few horses and sheep, which the enemy was unable to recover. Another of the divisions, under Sir George Cath- cart in person, suddenly found itself face to face with about six thousand Basuto horsemen armed with European weapons, and though the discipline of the soldiers enabled them to keep the untrained mass W1SB. ACTION <>/■ MOSHF.S/L 26 from breaking their ranks, no further advance was now thought of. A little before dusk the third division managed to join the commander-in-chief, and a defensible position among rocks was then taken for the night. At daybreak next morning the army commenced its retreat to the camp at Platberg. It had lost thirty-seven men killed, fifteen wounded, and one made prisoner, who was murdered by his captors. captors. ACKNOWLEDGMENT BY GREAT BRITAIN OF THE INDEPENDENCE OF THE SOUTH AFRICAN REPUBLIC, AND ABANDONMENT OF THE ORANGE RIVER SOVEREIGNTY. Though he had gained such a success, the wise Basuto chief's first thought after the battle was to obtain peace. He sent for the reverend Mr. Casalis, one of his missionaries, and after consulting with him, the most politic document that has ever been penned in South xAirica was written. It was as follows : — "Thaba Bosigo, "Midnight, 20th December 1852. "Thaba Bosigo, go, "Midnight, 20th December 1852. "Midnight, 20th December 1852. "Your Excellency, -This day you have fought against my people, and taken much cattle. As the object for which you have come is to have a com- pensation for Boers, I beg you will be satisfied with what you have taken. I entreat peace from you — you have chastised — let it be enough, I pray you ; and let me be no longer considered an enemy to the Queen. I will try all I can to keep my people in order in the future. " Your humble servant, servant, " MOSHESH." It was some time before a messenger could be found who would venture near the English sentries, ARRIVAL OF SIR GEORGE CLERK 26$ and when at length one left Thaba Bosigo with a flag of truce, Sir George Cathcart was retiring to his camp at Platberg. The messenger followed and delivered the letter. The English general, on his part, was not less anxious for peace. In his opinion there was every- thing to lose in a war with a tribe so strong as he had found the Basuto to be, and so he eagerly availed himself of the opening for escape from a grave diffi- culty which Moshesh's letter afforded. He announced that he was satisfied with the number of cattle cap- tured, that he considered past obligations fulfilled, and that he would at once retire. There was much murmuring in the English camp when this announce- ment was made, but the general shut his ears to it all, and before the end of the month the army reached the Orange on its return march. the Orange on its return march. For some time the imperial government had been undecided whether to retain the Sovereignty as a British possession or not, but as soon as intelligence of the engagement with the Basuto reached England a decision was formed. The next mail brought a despatch from the secretary of state for the colonies that the territory was to be abandoned. that the territory was to be abandoned. To carry this resolution into effect, Sir George Clerk was sent out as special commissioner. go, "Midnight, 20th December 1852. He called upon the European inhabitants to elect a body of representatives to take over the government ; but when the representatives assembled, they objected in the strongest terms to be abandoned by Great Britain, for even while they were debating, Moshesh was crushing Sikonyela and another of his opponents, ABANDONMENT OF THE SOVEREIGNTY. 266 and adding their territory to his own. In effect, the representative assembly said to Sir George Clerk that the>- held England in honour bound to reduce the great barbaric power she had done so much to build up. When that was done, they would not need military assistance, and would be prepared to take over the government of the country, though they wished to remain permanently connected with the British empire. The special commissioner, however, was prevented by his instructions from paying any attention to language of this kind, and was obliged to term those who used it " obstructionists." The assembly then sent two delegates to England to implore the queen's government and the parliament not to abandon them, but those gentlemen met with no success in their mission. Sir George Clerk now encouraged the remnant of the party that was at heart opposed to British rule to assert itself openly. With his concurrence, one of its ablest leaders returned from beyond the Vaal, and went about the country addressing the people and arguing that connection with England meant nothing but restraint, for no protection whatever was received. In the special commissioner's phraseology, Air. Stander and those of his way of thinking, who used language to that effect, were " well-disposed." used language to that effect, were " well-disposed." This party elected a body of delegates, who met in Bloemfontein, and opened negotiations with Sir George Clerk. The " obstructionist " assembly pro- tested, and was thereupon dissolved by the special commissioner, when most of its members and sup- porters, finding resistance to the will of the British SOUTH AFRICA AFTER 1 8 54 267 government useless, went over to the " well-disposed " side, and tried to get as good terms as possible. Geld was freely used to suppress complaints — it was termed part compensation for losses, — and nothing that was possible to be done was neglected to make the abandonment acceptable to the people generally. go, "Midnight, 20th December 1852. The result was that on the 23rd of February 1854 a convention was signed at Bloemfontein by Sir George Clerk and the members of the " well-disposed " assembly, by which the government of the territory previously termed the Orange River Sovereignty, thereafter the Orange Free State, was transferred, and its future independence was guaranteed. and its future independence was guaranteed. There were now in South Africa five distinct European governments, namely of — 1. The Cape Colom, ) „ . . , , r J British 2. Natal, \ 3. British Kaffraria, ) ^sessions. 4. The South i\frican Republic, 1 Independent 5. The Orange Free State. j Republics. 1. The Cape Colo , r J 1. The Cape Colo , r J 2. Natal tal 3. British Kaffra al 3. British Kaffra 5. The Orange Free State In 1854 the Cape Colony comprised all the land between the Orange river on the north, the Indian ocean on the south, the Atlantic ocean on the west, and British Kaffraria and the rivers Indwe and Tees on the east. Xatal comprised the territory between the Buffalo and Tugela rivers on the north-east, the Umzimkulu river on the south-west, the Kathlamba mountains or Drakensberg on the west, and the Indian ocean on the east. 26 SOUTH AFRICA AFTER 1S54. British Kafifraria comprised the territory between the rivers Klipplaats, Tyumie, and Keiskama on the west, the river Kei from the junction of the Klip- plaats to the sea on the north-east, and the Indian ocean on the south-east. The boundaries of the South African Republic were undefined, but, roughly speaking, they were the Limpopo river on the north, the Vaal river and a line a little above Kuruman on the south, the Kalahari desert on the west, and the mountainous country corresponding with the Drakensberg on the east. corresponding with the Drakensberg on the east. The Orange Free State comprised the territory between the Vaal river, the Orange river, and the Drakensberg, except Basutoland and the reserves set apart for coloured people. THE CONSTITUTION OF THE CAPE COLONY. The story of the Cape Colony from this time onward is very different from that of the preceding period. Arbitrary rule is henceforth a thing of the past, and a constitution of a liberal nature, granted by Great Britain, gradually removes the memory of old grievances, and creates a strong feeling of loyalty to the throne and the empire in all sections of the civilised inhabitants. The imperial authorities having resolved to confer upon the Cape people the privilege of parliamentary institutions, the details were referred for arrangement to the legislative council, and when everything was settled, on the- iith of March 1853 the constitution was promulgated by an order in council. By it two chambers — termed the legislative council and the house of assembly — were created, both of which were elective. The upper chamber for some years consisted of fifteen members, but in course of time this number was increased to twenty-six, beside the chief justice as president. For the purpose of elect- ing the members, the colony was divided into two 269 THE CONSTITUTION OF THE CAPE COLONY. 2/G provinces, more recently into nine circles. The members held their seats for seven years. The house of assembly on its creation consisted of forty- six members, elected by twenty-two divisions, Cape- town having two more representatives than any of the others. Later it consisted of one hundred and seven members, elected by forty-seven divisions, and holding their seats for five years. Parliament was summoned to meet by the governor, but a period of twelve months was not to elapse between the sessions. The right to vote for members of both chambers was conferred upon every male British subject over twenty-one years of age, who occupied a house or land worth £2$, or was in receipt of a salary of £2$ a year with board and lodging or £$0 without. There was no distinction as regards race, or colour, or religion, or manner of living. In course of time, however, it was found expedient to alter these qualifi- cations, as in the Cape Colony there was a large class of people unable to comprehend the nature of repre- sentative institutions, and yet in possession of sufficient property to bring them within one of the conditions specified above. THE CONSTITUTION OF THE CAPE COLONY. In IcSq2 the right to vote was re- stricted to such adult male subjects as were able to sign their names and write down their addresses and employment, and who either occupied property worth £75, or received £50 a year as salary or wages. £75, or received £50 a year as salary or wages. Parliament met for the first time in June 1854. After that date no law could be made without the approval of both houses and the sanction of the governor. The right was reserved to the queen to THE CAPE PARLIAMENT. 271 disallow any law so made within two years of its reaching England, but in practice this right was very rarely used. The ordinary yearly sessions of the parliament usually lasted about three months, from early in June to the end of August. early in June to the end of August. Naturally the colonists were gratified with the PARLIAMENT HOUSE, CAPETOWN. PARLIAMENT HOUSE, CAPETOWN. change from arbitrary to representative government, but there was still much to wish for. The officials of highest rank, who formed the executive council and were therefore the governor's advisers, continued to be sent out from England, and held their appoint- ments during the pleasure of the secretary of state change from arbitrary to representative government, but there was still much to wish for. The officials of highest rank, who formed the executive council and were therefore the governor's advisers, continued to be sent out from England, and held their appoint- ments during the pleasure of the secretary of state THE CONSTITUTION OF THE (APT. COLONY. •2; 2 for the colonies, no matter whether parliament liked them or not. They framed all government bills, and no measure involving the expenditure of money could be brought before the house of assembly unless introduced by them. They possessed the right of discussion, though not of voting, in both chambers. This condition of things lasted eighteen years. In 1872 responsible government was introduced, and the ministers — as the high officials were termed — after that time were the leaders of the party in parliament that could command the largest number of votes in support of their measures. They were the colonial secretary, the treasurer, the attorney-general, the commissioner of public works, and the secretary for native affairs. At a later date a secretary for agriculture was substituted for the last named. THE CONSTITUTION OF THE CAPE COLONY. There was also the premier, or prime minister, who might hold any of these appointments, or none of them; but who, in any case, after 1893 na<^ a^ matters connected with the coloured races directly under his care. When any important measure, in- troduced by the government, failed to secure the support of a majority of the members of parliament, the ministers resigned, and the leader of the opposi- tion was entrusted by the governor with the task of forming a new cabinet. In practice this system gives to the men who are chosen by the people the power of making and altering laws, of levying taxes and controlling the manner of using the public money, and of creating and doing away with offices. But it is a system THE DUTCH LANGUA 2JI adapted only for races of high civilisation. The majority of the day possesses supreme power, and if it came to consist of men whose constituents were incapable of acting with moderation, the minority could be more grievously oppressed than under the purest autocratic rule. There were many thinking people in the colony who regarded the franchise as still too low for perfect safety, with this form of government and the political equality of the various races that composed the population. races that composed the population. Until 1882 the English language only could be used in debate in parliament, just as in the pro- ceedings of courts of justice or in transactions in the public offices. This was decidedly unfair, for Dutch was habitually spoken by fully three-fifths of the white people in the colony, and by a still larger proportion of the coloured inhabitants, exclusive of Bantu. It will be remembered that its suppression as the official language was one of the chief grievances that rankled in the breasts of the old colonists. The parliament could not be said in truth to represent the people as long as the language of the majority was pro- scribed, and in point of fact comparatively few of the old stock sought admission into it. It was some time before they realised the full significance of responsible government, but when they did, one of their first acts was to secure the same rights for their own tongue as for the English. THE CONSTITUTION OF THE CAPE COLONY. Either could then be used in parlia- ment, courts of law, and public offices, at the choice of the speaker, and no one was admitted into the ordinary branches of the civil service without a perfect knowledge of both. 19 THE CONSTITUTION OF THE CAPE COLONY. 274 It would be incorrect to say that this measure raised the tone of debate in parliament or improved the administration of justice in the slightest degree, but it certainly made the parliament more truly repre- sentative of the people, and it removed a serious obstacle to the perfect co-operation in public matters of the colonists of Dutch and British blood. It did not of course make one people of the two nationalities, whose ideas are in some respects dif- ferent, and each of which clings to its own mother tongue with the utmost tenacity. But it did away with a real grievance felt by a decided majority of the electors, who were now for the first time raised to an equality with their English neighbours, not only in the opinion of the later colonists, but in their own. All acts of parliament and other public docu- ments were thereafter printed in both languages, and correspondence with government departments could be carried on in either at the option of the writer. English is, and bids fair to remain, however, exclu- sively the language of commerce throughout the country. THE PROVINCE OF BRITISH KAFFRARIA. BEFORE 1857 there were hardly any Bantu in the Cape Colony except the Fingos who had been in- troduced by Sir Benjamin D' Urban and the Tembus of Glen Grey, while British Kaffraria — the territory- bet ween the Keiskama and the Kei — had very few white inhabitants except soldiers, as the land there was reserved for the section of the Xosa tribe that was under English rule. After that date many thousands of Xosas were scattered over the country as far west as Port Elizabeth, and a population of European blood occupied a considerable portion of the land eastward to the Kei. This change in the position of the two races was caused by an event more astounding than anything in the pages of the wildest romance. The chiefs had accepted the terms imposed upon them at the close of the last war, but resolved to renew the struggle with the white people as soon as circumstances would permit it. Shortly after the conclusion of peace, Sir George Cathcart was suc- ceeded as governor and high commissioner by Sir POLICY OF SIR GEORGE GREY 2JJ George Grey, one of the ablest administrators the country has ever had, and he immediately took steps to prevent, as he hoped,. another outbreak of hostilities. As high commissioner he exercised supreme con- trol in British Kaffraria. Provided with a large amount of money from the imperial treasury, he attempted to pacify the chiefs by giving them pensions, payable monthly, as compensation for the power they had apparently lost, and he tried to break the belief in witchcraft by building a large and beautiful hospital in King-Williamstown, where any sick black person was attended by skilful medical men and provided for free of charge. Further, he commenced to make roads in the province, and to build a great sea wall at the mouth of the Buffalo river — called the port of East London — with the express object of teaching the Xosas the advantage of earning money by labour. In the same spirit he encouraged the Wesleyan and Free Church mis- sionary societies to establish industrial institutions, where young Fingos and Xosas could be trained as gardeners, carpenters, blacksmiths, and waggon- makers, and where a number of the most intelligent boys could be educated as interpreters, schoolmasters, and evangelists. THE PROVINCE OF BRITISH KAFFRARIA. One of the institutions which he thus assisted with funds is still in existence. This is Lovedale, an establishment of the Free Church, where an exceedingly s^ood training has ever since been given, and where at the present time some five or six hundred youths of both sexes are living as As high commissioner he exercised supreme con- trol in British Kaffraria. Provided with a large amount of money from the imperial treasury, he attempted to pacify the chiefs by giving them pensions, payable monthly, as compensation for the power they had apparently lost, and he tried to break the belief in witchcraft by building a large and beautiful hospital in King-Williamstown, where any sick black person was attended by skilful medical men and provided for free of charge. Further, he commenced to make roads in the province, and to build a great sea wall at the mouth of the Buffalo river — called the port of East London — with the express object of teaching the Xosas the advantage of earning money by labour. In the same spirit he encouraged the Wesleyan and Free Church mis- sionary societies to establish industrial institutions, where young Fingos and Xosas could be trained as gardeners, carpenters, blacksmiths, and waggon- makers, and where a number of the most intelligent boys could be educated as interpreters, schoolmasters, and evangelists. One of the institutions which he thus assisted with funds is still in existence. This is Lovedale, an establishment of the Free Church, where an exceedingly s^ood training has ever since been given, and where at the present time some five or six hundred youths of both sexes are living as pupils. 2?S THE PROVINCE OF BRITISH KAFFRARIA. These truly philanthropic measures, however, re- quired many years to produce a good effect, and even then a very small proportion of the people would be benefited by them. They had not well been taken in hand when tidings reached the high commissioner in Capetown that cattle in unusual numbers were being slaughtered in and beyond British Kaffraria, and that the Xosas were assuming a defiant attitude. Colonel Maclean, who had succeeded Colonel Mackin- non as head of the local government, was not long in finding out and reporting the cause. in finding out and reporting the cause. THE PROVINCE OF BRITISH KAFFRARIA. One morning in May 1856 a girl named Nong- kause went to draw water from a little stream that flowed past her home. On her return, she stated that she had seen by the river some men who differed greatly in appearance from those she was accustomed to meet. Her uncle, whose name was Umhlakaza, went to see the strangers, and found them at the place indicated. They told him to return home and go through certain ceremonies, after which he was to offer an ox in sacrifice to the spirits of the dead, and to come back to them on the fourth day. There was that in their appearance which commanded obedience, and so the man did as they bade him. On the fourth day he went to the river again. The strange people were there as before, and to his astonishment he recognised amon£ them his brother who had been many years dead. Then, for the first time, he learned who and what they were. The eternal enemies of the white man, they announced themselves as having come from battlefields beyond the sea to aid the Xosas with their invincible power in driving the SELF-DESTRUCTION OF THE XOSAS 2JQ English from the land. Between them and the chiefs Umhlakaza was to be the medium of communication, the channel through which instruction would be given. For strange things were to be done, stranger than any that had ever been done before, if the proffered assistance was welcomed. And first, he must tell the people to abandon dealing in witchcraft, to kill fat cattle and eat. Such is the tale which the Xosas told each other of the manner in which Umhlakaza and Nongkause became acquainted with the secrets of the spirit world. Umhlakaza and Nongkause ! What terrible visions of suffering and death are called forth in Kaffirland now at the mention of those two names ! Kreli, the paramount chief of the tribe, hailed the message with joy, and indeed it is generally believed — though it cannot be proved — that he was the instigator of the scheme. His word went forth that the command of the spirits was to be obeyed, that the best of all the cattle were to be killed and eaten. THE PROVINCE OF BRITISH KAFFRARIA. Messengers from him hastened to the chiefs in British Kaffraria to inform them of what had taken place, and to require their co-operation. Instantly the clans were in a state of commotion. Most of the chiefs commenced to kill, but one, Sandile, timid and hesitating, for a time held back. The high com- missioner sent word to Kreli that though in his own territory he could do as he pleased, he must cease from instigating those who were British subjects to destroy their property, or it would become necessary to punish him. But he cared little for such a threat. THE PROVINCE OF BRITISH KAFFRARIA. 280 as the time was at hand when it would be for him to talk of punishing. talk of punishing. The revelations communicated through Umhlakaza grew apace. The girl, standing in the river in pre- sence of a multitude of deluded people, heard strange unearthly sounds beneath her feet, which Umhlakaza pronounced to be the voices of spirits holding council over the affairs of men. The first order was to slay cattle, but the greedy ghosts seemed insatiable in their demands. More and more were killed, but still never enough. And thus the delusion continued month after month, every day spreading wider and embracing fresh victims in its grasp. After a while Sandile gave way to the urgent applications of his brother Makoma, who asserted that he had himself seen and conversed with the spirits of two of his father's dead counsellors, and that they commanded Sandile to kill his cattle if he would not perish with the white man. Before this time the last order of Umhlakaza had been given, that order whose fulfilment was to be the final preparation of the Xosas, after which they would be worthy of the aid of a spirit host. Not an animal out of all their herds must be left living, every grain of corn in their granaries must be destroyed. But what a future of glory and wealth was predicted for the faithful and obedient ! On a certain day myriads of cattle, more beautiful than those they were called upon to kill, were to issue from the earth and cover the pastures far and wide. Great fields of millet, ripe and ready for eating, were in an instant to spring into existence. THE PROVINCE OF BRITISH KAFFRARIA. The ancient heroes of the race, the SELF-DESTRUCTION OF THE XOSAS 28 1 great and the wise of days gone by, restored to life on that happy day, would appear and take part in the joys of the faithful. Trouble and sickness would be known no more, nor would the frailties of old age oppress them, for youth and beauty were to return alike to the risen dead and the feeble living. Such was the picture of Paradise painted by the Xosa prophet, and held before the eyes of the infatuated people. And dreadful was to be the fate of those who opposed the will of the spirits, or neglected to obey their commands. The day that was to bring so much joy to the loyal would bring nothing but ruin for them. The sky itself would fall and crush them together with the Fingos and the white people. Missionaries and agents of the government tried in vain to stay the mad proceedings. A delirious frenzy possessed the minds of the Xosas, and they would listen to no argument, brook no opposition. White men who attempted to interfere with them in any way were scowled upon and warned to take care of themselves. Yet these fanatics, with their imagina- tions fixed on boundless wealth, were eagerly pur- chasing trifles from English traders, bartering away the hides of two hundred thousand slaughtered cattle. Most of them acted under the influence of supersti- tion alone, though there is no doubt that some of the leaders viewed the proceeding as calculated solely for purposes of war. To throw the whole Xosa tribe, fully armed and in a famishing state, upon the colony, was the end kept steadily in view by these. The terrible odds against the success of such a venture they were too blind to see or too excited to calculate. THE PROVINCE OF BRITISH KAFFRARIA. 282 Some there were who neither believed the predic- tions of Umhlakaza nor looked for success in war, and who yet destroyed the last particle of their food. Bukhu, Kreli's uncle, was one of these. " It is the chiefs command," said he, and then, when nothing more was left, the old man and his favourite wife sat down in their empty kraal and died. Kreli's prin- cipal counsellor opposed the scheme till he saw that words were useless. THE PROVINCE OF BRITISH KAFFRARIA. Then, observing that all he had was his chief's, he gave the order to kill and waste, and fled from the place a raving lunatic. Thus it was with thousands. The chief commanded, and they obeyed. they obeyed. In the early months of 1857 an unwonted activity reigned throughout the country from the Keiskama to the Bashee. Great kraals were being prepared for the reception of the cattle, so soon to appear like stars of the sky in multitude. Enormous skin bags were being made to contain the milk shortly to be like water in plenty. And even as they worked some were starving. East of the Kei the prophet's command had been obeyed to the letter, but the resurrection day was still postponed. It was in mercy to the Gaikas, said Umhlakaza, for Sandile had not finished killing yet. Nothing surely was ever more clumsily arranged, more blindly carried out than this mad act of the Xosas. One section of the tribe was literally starving, while another section was still engaged in destroying its resources. still engaged in destroying its resources. The government did all that was possible to protect the frontier. Every post was strengthened, and every available soldier was sent forward, The SELF-DESTRUCTION OF THE XOSAS 283 colonists, too, were prepared to meet the expected shock, come when it would. And then, after defence was provided for, stores of food were accumulated for the purpose of saving life. For there could be no heart so cold as not to feel pity for those misguided beings who were rushing into certain destruction. who were rushing into certain destruction. At length the morning dawned of the day so long and so ardently looked for. All night long the Xosas had watched with feelings stretched to the utmost tension of excitement, expecting to see two blood-red suns rise over the eastern hills, when the heavens would fall and crush the races they hated. Famished with hunger, half-dying as they were, that night was yet a time of fierce, delirious joy. The morn, that but a few short hours, slowly becoming minutes, would usher in, was to see all their sorrows ended, all their misery past. And so they waited and watched. At length the sun approached the horizon, throwing first a silver sheen upon the moun- tain peaks, and then bathing hillside and valley in a flood of light. THE PROVINCE OF BRITISH KAFFRARIA. The hearts of the watchers sank within them. " What," said they, " will become of us if Umhlakaza's predictions turn out untrue?" But perhaps, after all, it might be midday that was meant, and when the shadows began to lengthen towards the east, perhaps, they thought, the setting of the sun is the time. The sun went down — as it often does in that fair land — behind clouds of crimson and gold, and the Xosas awoke to the reality of their dreadful position. A blunder, such as a child would hardly have A blunder, such as a child would hardly have made, had been committed by the managers of this THE PROVINCE OF BRITISH KAFFRARIA. 284 horrible tragedy. Under pretence of witnessing the resurrection, they should have assembled the warriors of the whole tribe at some point from which they could have burst in a body upon the colony. This had not been done, and now it was too late to collect them together. An attempt was made to rectify the blunder, and the day of resurrection was again post- poned, but fierce excitement had given place to deepest despair. The only chance of life that re- mained was to reach the colony, but it was as suppliants, not as warriors, that the famished people must now go. The horrors that succeeded can only be partly told. There are intelligent men living now, then wild naked fugitives, who cannot recount the events of those days. The whole scene comes home to them as a hideous nightmare, or as the remem- brances of one in a state of delirium. In many instances all the ties were broken that bind human beings to each other in every condition of society. Brother fought with brother, father with son, for scraps and shreds of those great milk sacks so care- fully made in the days when hope was high. The aged, the sick, the feeble, were abandoned by the young and vigorous. All kinds of wild plants, and even the roots of trees, were collected for food. Many of those who were near the sea coast endea- voured to support life upon the shellfish found there. Being unaccustomed to such diet, they were attacked by dysentery, which completed the work of famine. In other instances whole families sat down and died together. THE PROVINCE OF BRITISH KAFFRARIA. From fifteen to twenty skeletons were SELF-DESTRUCTION OF THE XOSAS 285 afterwards often found under a single tree, showing where parents and children met their fate when the last ray of hope had fled. A continuous stream of emaciated beings poured into the colony, young men and women mostly, but sometimes fathers and mothers bearing on their backs half-dying children. Before the farmhouses they would sit down, and ask in piteous tones for food, nor did they ask in vain. piteous tones for food, nor did they ask in vain. Between the first and last days of 1857 the official returns of British KafTraria showed a decrease in the population from one hundred and five thousand to thirty-eight thousand of both sexes and all ages. Sixty-seven thousand had perished or dispersed. In the centre of this territory was King-Williamstown, where the government had provided a quantity of corn, by which the lives of thousands were saved. Between the Kei and the Bashee there was no such storehouse, and flight, except to rival and unfriendly tribes, was next to impossible. The death-rate there was consequently higher than in British KafTraria. The lowest computation fixes the number of those who perished on both sides of the Kei at twenty- five thousand, ordinary calculations give double that number. The power of the Xosa tribe was for the time completely broken. time completely broken. Large tracts of land in British KafTraria having become waste by this mad act of the Xosas, Sir George Grey allotted farms of about fifteen hundred acres in size to a considerable number of selected individuals from the Cape Colony, to be held under tenure of military service and a small quitrent. A strong body of European settlers was thus stationed the. rkon.vcr Of British kaffraAia. 286 in advance of the most formidable Kaffir strongholds. Some regiments of the German legion, raised by Great Britain during the Crimean war, were sent out, and were disbanded in the province, where plots of land were assigned to the officers and soldiers on a military village system. Many of these men prospered, and they were undoubtedly of great service to the country, but on the whole the villages were failures. The proportion of women was too small to give reasonable hope of permanency to the settlements, and the men were better adapted for life in towns than as tillers of the soil. THE PROVINCE OF BRITISH KAFFRARIA. Most of them dispersed as soon as the issue of rations ceased. dispersed as soon as the issue of rations ceased. A body of agricultural labourers selected from the hardy peasantry of Northern Germany was intro- duced shortly afterwards; The men were accom- panied by their wives and children, and were inured to toil and accustomed to rough living. In 1858 and 1859 these people, in number rather over two thou- sand, landed at East London. They were sent out under a contract between Sir George Grey and a merchant in, Hamburg, and were bound to refund within a certain period the cost of their transport and to pay twenty shillings an acre for the ground allotted to them. They were located in different parts of the province, but chiefly in the valley of the Buffalo river. No better settlers could have been introduced. By their industry, in the course of a few years they became possessed of a considerable amount of stock and brought their little farms to a high state of cultivation. As market gardeners they were unrivalled in South Africa. Frugal, temperate, ANNEXATION TO THE CAPE COLONY 2%J industrious, and religious, they contributed very largely to the prosperity of the province. largely to the prosperity of the province. King-Williamstown soon grew to be a place of importance. It was garrisoned by a strong body of British troops, and was the centre of a large trade, besides being the seat of the local government. besides being the seat of the local government. In theory the Cape parliament had no power to legislate for British Kaffraria, but in practice as soon as an act was passed in Capetown the high com- missioner proclaimed it of force in the province, and thus secured uniformity in the laws. The revenue was small, and required to be supplemented by grants in aid from the imperial treasury. But now that the territory had ceased to be occupied exclu- sively by Bantu, it seemed to the queen's ministers that it might with advantage be incorporated with the Cape Colon}', and this burden be removed from the British taxpayer. Proposals to that effect were therefore brought before the Cape parliament on several occasions, but were always rejected. THE PROVINCE OF BRITISH KAFFRARIA. At length the imperial parliament passed an act of union, which was, however, only to take effect after the lapse of a certain period, and provided the Cape parliament did not in the meantime annex the province. Armed with this document, Sir Philip Wodehouse, Sir George Grey's successor as governor and high commissioner, introduced a bill which provided for the incorporation of British Kaffraria as two electoral divisions — King-Williamstown and East London — and after much opposition it was passed by a majority of both houses of the Cape legislature, and in 1865 was carried into effect. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. TlIE introduction of responsible government had the effect of causing the people to take much greater interest in the public life of the colony than had previously been the case. One of the first things attended to was the opening up of the interior by means of railways, as communication with places on the upper terraces was often almost impossible in times of drought, and even in the best seasons was tedious and expensive. In 1874 the first large extensions were resolved upon by parliament, and were taken in hand without any delay. From Cape- town the existing line to Wellington was to be continued to Beaufort West, from Port Elizabeth a line was to be constructed to GraafT-Reinet, with a branch to the Bushman's river, and from East London one was to be constructed to Oueenstown. The gauge was to be forty-two inches, and the lines were to be made and worked by the government. Harbour works, which have made of Table Bay and East London safe and convenient ports, were INSECT PES 2%g pushed on, and telegraph lines were laid down in every direction. By means of a subvention to a company in 1879 connection with Aden was made by cable, which put South Africa in communication with Europe and India. An act was passed to encourage irrigation, a matter of the highest im- portance to the country. The system of public schools was greatly extended and improved. In 1873 the university of the Cape of Good Hope was established, as an examining body with power to confer degrees. Here certainly was a big bound forward towards equality with more densely popu- lated dependencies of the empire. lated dependencies of the empire. Unfortunately at this time two insect pests made their appearance, which caused great loss to large sections of the community. One of these was the dorthesia, that entirely destroyed the orange groves before it was discovered that a species of foreign beetle was its natural enemy. This was introduced, with the excellent result that the dorthesia was soon practically exterminated, and all varieties of the orange tree could again be cultivated with success. The other pest was the phylloxera, that attacked the roots of the grape-bearing vines, and threatened absolute ruin to the wine farmers of the west. But for it a remedy was found after it had caused very extensive damage. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. The roots of an American vine are immune from its attacks, and these were planted and grape-producing stocks grafted upon them. grafted upon them. Since that time the production of many varieties of fruit has gone on increasing, and a profitable 20 20 20 90 Progress of cafe colon v under home rule 290 market has been found in England, where it arrives when European fruit is out of season. Ostrich farming was rapidly extending, and in favourable situations was more profitable than any other industry. Naturally, with the great increase in the production of feathers their value diminished, but not to such an extent as to discourage the breeders of the birds. In 1879 an association termed the Afrikander Bond was founded, which afterwards had immense influence in the colony. It was founded at a time when great irritation existed owing to the annexa- tion of the Transvaal republic to the British empire against the wishes of its people, and consequently its first constitution favoured independence; but very shortly it came largely under the guidance of Mr. J. H. Hofmeyr, one of the ablest statesmen that South Africa has yet produced, who realised the advantages of connection with Great Britain and the absurdity of this country attempting to stand alone, and it then became perfectly loyal, confining its exertions to guarding the interests of the Dutch- speaking section of the inhabitants. In 1877 hostilities with the Bantu on the eastern border broke out, but as this matter led to the incorporation in the colony of the whole territory up to Natal, it will be dealt with in a separate chapter. On the northern border at a little later date there were two punitive expeditions that require notice. The Orange river in its central course for some eighty miles expands to a great width, abounds with PKESENT CONDITI 29 1 falls and rapids, and contains many islands of con- siderable size, covered with dense thickets. To the north the country is a waterless desert, and to the south it is not much better, so that approach to the islands was always difficult, and in addition to this the fords to them were unknown to people at a distance. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. They formed thus ideal strongholds for robber bands, and in 1879 they were occupied by a few hundred Korana, Griqua, Bantu, and Bushman depredators, the very scum of South x-\frica, who from them as a base made raids to places hundreds of miles distant. Volunteers and police were sent against the marauders, but month after month passed away, and the northern border war, as the operations were termed, still went on. The difficulty was to find the vagabonds. At length, however, they were tracked out, when most of them were killed in action, others were made prisoners, and the wretched affair was over. A greater contrast than this wild country, the haunt of robbers in 1879, and the same tract of land in 1916, is nowhere to be found in the world. Here at present is the Kakamas labour colony of the Dutch reformed church, where hundreds of poor white families are residing in comfort and leading honest and useful lives. There are large tracts of rich land along the banks of the river, and the falls and rapids offer facilities for leading water to them. Canals many miles in length have been constructed, and irrigation has converted this desert waste into gardens, orchards, and corn- fields. A railway conveys the surplus produce to PROGRESS OF CAPE COLONY UNDER HOME RULE. 292 distant markets, so that a thriving settlement has arisen. Near the source of the Orange there was another disturbance in 1879, which caused much trouble and expense. A petty Bantu chief named Morosi, who was a subject of the Cape Colony, rose in rebellion, and took refuge on an almost impregnable mountain. Two attempts to storm the stronghold failed, and then it was invested, in hope of starving the rebels out. Months later, as that did not succeed, an attempt was made to scale the mountain at night from ledge to ledge by means of long ladders, and this succeeded. The summit was gained by a few daring men, and in the combat that followed Morosi and most of his retainers were killed. At this time the Cape Colony was rapidly ex- panding. Along the western coast between Walvis Bay and the mouth of the Orange river there are several rocky islets upon which seabirds congregate in vast flocks, and as there is hardly any rainfall in that region the guano is of considerable value. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. In 1866 these islets, twelve in number, were proclaimed dependencies of the Cape Colony. The most valu- able of them is the one named Ichaboe, which is less than a mile in circumference. The only inhabitants are the men employed by the government to gather the guano, which is sold at a very low rate to colonial farmers. Missionaries and traders were living on the ad- joining mainland, but no civilised power claimed any authority there. That far-seeing governor, Sir ACQUISITION OF GRIQUALAND WEST. 293 Bartle Frere, on more than one occasion urged upo the colonial office in England the desirability of proclaiming it British territory as far north as the Kunene river, but his advice was unheeded, as the expense of following it was feared. If the little inlet named Angra Pequena be excepted, there is but one safe harbour along the whole coast. This is Walvis Bay, and on account of its strategical importance, in March 1878 it with a small tract of land behind it was taken possession of as part of the British empire, and a magistrate was sent to reside there. The Cape Colony bore the expense, as there was no revenue, and in August 1884 it was formally annexed and made subject to colonial rule and law. The inhabitants were the magistrate with his staff of police, a missionary family, a couple of traders, and about seven hundred Hottentots of a very low type. type. An important acquisition was that of the sterile territory then termed Griqualand West, which con- tained diamond mines of enormous value. One day in 1867 a child on a farm in the north of the Cape Colony was observed to be playing with a remarkably brilliant pebble, which a trader, to whom it was shown as a curiosity, suspected to be a gem of value. He obtained it and sent it for examination to a qualified person in Grahamstown, who reported that it was a diamond of twenty-one carats weight, and that its value was ^"500. Search was immediately commenced in the neighbourhood by several persons in odd hours, and soon another, though much smaller, was found. Then a third was PROGRESS OF CAPE COLONY UNDER HOME RULE. 294 picked up on the bank of the Vaal river, and atten- tion was directed to that locality. tion was directed to that locality. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. During 1868 several were found, though as yet no one was applying himself solely to looking for them. In March 1869 the Star of South Africa was obtained from a Korana Hottentot, who had been in possession of it for a long time without the least idea of its value except as a powerful charm. It was a magnificent brilliant of eighty-three carats weight when uncut, and was readily sold for ;£ 11,000. From all parts of South Africa men now began to make their way to the banks of the lower Vaal to search for diamonds, and trains of waggons conveying pro- visions and goods were to be seen on every highway to the interior. Some of the diggers were fortunate in amassing wealth, but this was by no means the case with all. Diamond digging, in fact, was like a great lottery, with a few prizes and many blanks. But it had a powerful attraction, and shortly many hundreds of adventurers from Europe and America were also engaged in it. were also engaged in it. The quiet, simple, homely life of the South African farm and village in olden times — rarely disturbed except by wars with Bantu tribes — had passed away for ever, and a bustling, struggling, restless mode of existence was rapidly taking its place. The wealth of the country was enormously increased, for dia- monds soon attained a high place in the exports ; but it may be questioned if the people are on the whole as happy as they were before. whole as happy as they were before. The southern bank of the lower Vaal was Free State territory, but the ownership of the northern THE DIAMOND MIXE 295 bank was disputed. Before the discovery of diamonds it was regarded as of so little value that no actual government existed there, though the South African Republic, the Orange Free State, the Batlapin tribe, and the Griqua captain Nicholas Waterboer all claimed the ground. The consequence was that each mining camp on that side of the stream formed a kind of government for itself, and a great deal of confusion and lawlessness was the result. After a while much richer diamond mines than those along the Vaal were discovered on some farms to the southward, and most of the diggers removed to them. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. The public offices of the district in which they were situated were at a considerable distance, but as soon as arrangements could be made by the government, a resident landdrost was appointed, a post-office was established, and some policemen were engaged. engaged. In the minds of people at a distance the various camps were confused with each other, and all were supposed to be in the lawless condition of those north of the Vaal. Most of the diggers were British subjects, so that her [Majesty's high commissioner considered it his duty to interfere in the interests of order. At that time one of the shrewdest men in South Africa was agent for the Griqua captain Nicholas Waterboer, and on behalf of his client had laid claim to a large part of the Orange Free State, including the locality in which the diamond mines were situated. No pretension could be more shadowy, but when Mr. Arnot, on behalf of Waterboer, offered the territory to the British government, it came to be PROGRESS OF CAPE COLONY UNDER HOME RULE. 296 regarded, on one side at least, as having some real foundation. The high commissioner proposed arbitration, which President Brand declined. The territory which Mr. Arnot claimed south of the Vaal, he said, had been part of the Free State ever since the convention of 1854. Before that date it had been part of the Orange River Sovereignty, and some of the farms in it were held under British titles issued at that time. Nicholas Waterboer and his people lived far away, and, as well as could be ascertained, had never occupied ground there. Under these circumstances he would not admit that there could be any question of ownership. The right of the state to land beyond the Vaal, however, he was willing to submit to arbitration, as it had been acquired by purchase, and the seller's title might be open to doubt. the seller's title might be open to doubt. While the high commissioner and the president were corresponding on this subject, Mr. Marthinus Wessel Pretorius, who was then president of the South African Republic, agreed to submit some disputes between that country and the Barolong, Batlapin, and Griquas, to arbitration, in consequence of which a court was appointed, with Mr. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. Keate, governor of Natal, as final umpire, and proceedings were opened at the little village of Bloemhof, on the northern bank of the Vaal. The Free State govern- ment was not represented in the court. The interests involved were greater than were recognised at the time. It was supposed that the sovereignty of some of the diamond mines was the great question at issue ; now it is seen that access by GRIQUALAND WEST A CROWN COLONY. 2QJ Great Britain to the distant interior was also involved. On one side the proceedings were a perfect farce. President Pretorius and his attorney did nothing whatever to work up their case; they did not attempt to meet evidence that might have been disproved with the greatest ease ; they even put in a spurious document given to them by one of their opponents purposely to befool them. On the other side was Mr. Arnot, who knew exactly what to withhold as well as what to bring forward. The result was that Mr. Keate, acting solely on the evidence before him, gave judgment against the South African Republic, and in defining the territories of the disputants included within Nicholas Waterboer's boundary the part of the Free State which that captain claimed. part of the Free State which that captain claimed. As soon as the Keate award was issued — October 1 87 1 — Sir Henry Barkly, who was then high commissioner, proclaimed Waterboer's country a British dependency, with boundaries enclosing the mines along the Vaal, and at Dutoitspan, De Beer's, and Kimberley. An armed force was sent to take possession of it, and the Free State officials with- drew under protest. The territory, which was named Griqualand West, then became a crown colony. It had a stormy existence, for many of the diggers, when unsuccessful, were difficult men to rule, and there was once a rebellion that required British troops to suppress. Then the Batlapin north of the Vaal resented their ground having been de- clared to be Nicholas Waterboer's, and rose in arms. The Griquas, too, in the west, finding they had gained nothing by the cession, gave endless PROGRESS OF CAPE COLONY UNDER HOME RULE. 298 trouble, and finally went into rebellion. The govern- ment, however, was strong enough to overcome all opposition, and even furnished a corps of volunteers for service in other parts of South Africa. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. As different potentates had made grants of the same ground, a special court was created to decide upon conflicting claims. During many weeks evi- dence was taken, and the most minute research was made into the history of the land and its people. When at length judgment was given, all claims within the diamond mining area that rested on grants by Waterboer were thrown out, because that captain was proved never to have had any rights there. President Brand then went to England and laid his case before the imperial authorities. In brief it was this : that Great Britain had taken the land from the Free State under pretence that it belonged to Waterboer, and that a British court, after a careful examination, had since decided that Waterboer had no right to it. The reply that he received was to the effect that it was a necessity for the paramount power in South Africa to be in possession of the diamond fields, but he would receive £90,000 from Griqualand West as a solatium. Griqualand West as a solatium. The president accepted the offer, and thus the long controversy between him and the secretary of state came to an end. The settlement removed the only objection the Cape parliament had to annexing the territory — the desire to do nothing to affect the Free State claims — and as the British government was desirous of being relieved of the responsibility of QUARRELS OF THE CLAN 299 directly ruling it, in 1880 Griqualand West was incorporated in the Cape Colony as the magisterial districts of Barkly West, Kimberley, Hay, and Herbert. The Keate award was hardly delivered when the clans cut off by it from the Transvaal republic began to quarrel with each other, and their feuds continued with hardly any intermission until 1877, when the British flag was hoisted at Pretoria. Then for a time there was comparative order, because the Transvaal authorities favoured the strongest chiefs, and the military force in the country commanded respect. military force in the country commanded respect. When the republic was restored in 1881 the old quarrels began afresh, and soon became more bitter than ever. Some of the chiefs now professed strong attachment to Great Britain, and as a matter of course their rivals professed equally strong attach- ment to the government of the farmers. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. At this stage a European renegade suggested to the chief with whom he was living that white men should be enlisted to fight for him, and as a result volunteers were called for, each to receive a farm in payment when the war was over. The other side adopted the same course, so that bodies of Europeans — decidedly of the vagabond type, however — were apparently pitted against each other in a cause that did not concern them in the least. In reality there was no fear of such combatants shedding each other's blood, except when an individual made himself particularly obnoxious ; but it soon became a certainty that unless some power intervened the volunteers would divide the best part of the country PROGRESS 0E CAPE COLONY UNDER HOME RULE. 300 among them and leave little worth having for their employers. employers. The western border of the Transvaal republic was the base of operations on one side, and President Kruger's government made little or no effort to pre- vent its being so used. The burghers of that state would not put themselves to trouble to protect clans that had thrown off their authority, and they had also an excuse that volunteers for the other side were enlisted at the diamond fields — on British soil — with hardly a show of secrecy. This implication in the disturbances caused people in England to regard the republic as being at the bottom of the whole matter, and public opinion supported the government in sending out a strong military force under Sir Charles Warren to protect the clans from being despoiled. being despoiled. Meanwhile the volunteers — or freebooters — had taken possession of two considerable tracts of ground and set up an independent government on each. One was termed the republic of Stellaland, the other the land of Goshen. When Sir Charles Warren approached, the people of Goshen, instead of pre- paring to resist, dispersed to other parts of South Africa, and the people of Stellaland submitted, so that there was no necessity to fire a shot in anger. The expedition, though it had not to fight, was of the utmost service to British interests in the country, as it secured an open highway to the interior of the continent. In September 1885 the territory which had been the scene of the disturbances was taken under British sovereignty. RAPID PROGRESS OF THE CAPE COLONY UNDER HOME RULE. It was formed into a BRITISH BETSHUANALAND INCORPORATED. 30I crown colony, governed by an administrator under her Majesty's high commissioner, and was divided into five magisterial districts — Mafeking, Vryburg, Taung, Kuruman, and Gordonia. The best of the land was set apart as reserves for the Betshuana clans, but considerable tracts were allotted to European farmers. It is well adapted for cattle runs, though agriculture only succeeds in limited localities, and in general there is a scarcity of surface water. No minerals of any importance except salt have yet been discovered. The climate is exceedingly healthy, and though the days in mid- summer are sometimes unpleasantly warm, the nights are invariably cool and enjoyable. are invariably cool and enjoyable. After the establishment of British authority order was observed as well as in any part of the world. The railway from Capetown, Port Elizabeth, and East London, passing through Kimberley, was ex- tended through the province, and continued to the far distant north. Vryburg and Mafeking, the largest centres of European population in the territory, are served by this line. In 1895 the crown colony of British Betshuanaland ceased to exist, by being incorporated in the Cape Colony. The boundary on the north now became the Molopo river and Ramathlabama Spruit, and the great block of land between the Transvaal republic and the twentieth meridian eastward from Greenwich was within the colonial border. FURTHER ENLARGEMENTS OF THE CAPE COLONY. SOON after the introduction of responsible govern- ment, as the colony had now taken upon itself the entire burden of its defence, the attention of the ministers was drawn to the country between the Kei and Indwe rivers on one side and Natal on the other,- the greater part of which was occupied by Bantu tribes almost constantly at war with each other. This state of things was a perpetual danger to the colony, because clans of two of the most important of the tribes — the Xosa and the Tembu — were British subjects living within its border, and could not be kept quiet while their friends and relations were engaged in strife. SOON after the introduction of responsible govern- ment, as the colony had now taken upon itself the entire burden of its defence, the attention of the ministers was drawn to the country between the Kei and Indwe rivers on one side and Natal on the other,- the greater part of which was occupied by Bantu tribes almost constantly at war with each other. This state of things was a perpetual danger to the colony, because clans of two of the most important of the tribes — the Xosa and the Tembu — were British subjects living within its border, and could not be kept quiet while their friends and relations were engaged in strife. The territory between the Kei and Indwe rivers and Natal is a very beautiful and fertile tract of land, resembling Natal in appearance, though the tempera- ture along the coast is not quite so high. On the elevated belt just below the Drakensberg the winter nights are too cold to be pleasant to Bantu, and consequently no one except Bushmen resided there permanently until quite recently. There are some SOUTH AFRICAN BOULDER. FURTHER ENLARGEMENTS OF THE CAPE COLONY. 304 fine forests on the lower terraces. The rainfall is abundant, and the drainage perfect, the rivers on account of their great slope speedily carrying off all superfluous moisture. The climate is therefore healthy, though the grass is so rich and other vege- tation so luxuriant that had the country been nearly level fever certainly would be endemic. level fever certainly would be endemic. South Africa abounds in cascades. FURTHER ENLARGEMENTS OF THE CAPE COLONY. The most celebrated of these, next to the Victoria falls of the Zambesi, are the great falls of the Orange, the Tugela's leap of sixteen hundred feet over the face of the Drakensberg, and the fall of the Umgeni a few miles from Maritzburg in Natal. But one of the most picturesque is in the Tsitsa river, in the territory now being treated of. Ordinarily the stream tumbles over the precipice in three or four rills, but in times of flood a volume of water from four to five hundred feet wide drops nearly four hundred feet into a narrow chasm. When white men first visited this territory, more than three hundred years ago, there were no Bantu tribes of any importance living in it. A number of little clans, each independent of all the others, occupied the land north of the Umzimvubu river, and south of that stream the people were of mixed Bantu and Hottentot blood. They had the same customs and language as the present inhabi- tants, but the tribes now existing were either not yet formed or were too small to attract the attention of travellers. Towards the close of the seventeenth century some shipwrecked sailors found four tribes occupying the SETTLEMENT OF IDUTYW 305 territory : the Pondos, the Pondomsis, the Tembus, and the Xosas. All are there at the present day, and the fragments of many others as well. At that time they had advanced southward as far as the river Kei, and one little offshoot was even found at the mouth of the Buffalo. They had partly incorporated and partly exterminated the Hottentots to the north, and were continuing that process with others in their way as they advanced. way as they advanced. In preceding chapters sufficient has been related of these people from that date until the destruction of their cattle and grain by the Xosas in 1856 and 1857. As punishment for the part taken by them in that occurrence, in 1858 Kreli and his people were driven over the Bashee by a body of the colonial armed and mounted police. A few hundred Fingos and some Xosas who professed to be attached to the British government were then located in the district of Idutywa, part of the vacant territory, and an English official was stationed there to preserve order among them. FURTHER ENLARGEMENTS OF THE CAPE COLONY. The rest of the old Xosa country between the Kei and Bashee rivers continued to be without inhabitants until 1864, when Sir Philip Wodehouse believed it necessary to strengthen the European element west of the Kei, as the military force on the border was being reduced by the imperial authorities. His first plan was to give out the vacant ground as farms to a selected body of young men from dif- ferent parts of the colony, but as the British govern- ment emphatically refused to extend its dominion in South Africa he was obliged to relinquish that 21 21 21 306 FURTHER ENLARGEMENTS OF THE CAPE COLONY. project. He then resolved to make room for more farmers within the established border by inducing some of the Bantu clans there to move eastward. In execution of this design it was necessary to attempt to turn Kreli from an enemy into a friend, and with this view he was offered the districts of Kentani and Willowvale, with a pension of ^iooa year as long as he should behave himself to the satis- faction of the governor. The chief joyfully accepted the offer, and immediately moved in with his people. This of course was no relief to the colony, and the Gaikas under Sandile, who were next offered land over the Kei in exchange for their location on the western side, refused to move. They professed that they wished to remain British subjects, but in reality their reason for declining the offer was attachment to Kreli, their tribal head, whom they thought they would be wronging if they took possession of land that had once been his. The Tembus of Glen Grey, however, and some of the Fingos west of the Keis- kama had no such scruples, and they accepted the governor's proposal. The former received the dis- tricts of Xalanga and Saint Mark's, the latter the districts of Tsomo, Nqamakwe, and Butterworth. Thus the whole of the land east of the Indwe and the Kei was again occupied by a Bantu population. British officials were stationed with all these people, but as the imperial government would not permit the territory to be formally annexed, they were really only diplomatic agents. only diplomatic agents. Sir Philip Wodehouse's object in removing these Bantu was entirely frustrated, for not a rood of ANNEXATION OF TEMBULAN 307 ground was obtained by the measure for Europeans. FURTHER ENLARGEMENTS OF THE CAPE COLONY. The Tembus and the Fingos in the colony simply threw off swarms, but took care not to abandon any part of their locations, and it was not possible for the government to use force to drive them away. The main branch of the Tembu tribe lived be- tween the Bashee and Umtata rivers. After 1863 its chief was a man named Gangelizwe, ordinarily a gentle-looking and soft-speaking individual, but subject to outbursts of violent passion. His great wife was a daughter of the Xosa chief Kreli, and his treatment of her was at times so inhuman that her father was exasperated to the last degree. There was an old feud between the Tembus and the Pondos, so that Gangelizwe had an enemy on the other side also. In 1875, in a mad fit of rage, he murdered one of his concubines, who was an illegitimate niece of Kreli and an attendant upon the great wife. His enemies on all sides were now ready to fall upon him, and as his tribe contained many fragments of others whose fidelity could not be depended upon in such a quarrel, the chief himself and his counsellors requested British protection and offered to become subjects of the queen. To prevent a disturbance on the border the high commissioner consented, and in 1875 the districts of Elliot, Engcobo, Umtata, and Mqanduli were added to the empire in the same loose way as those previously mentioned. loose way as those previously mentioned. Living on the eastern bank of the Bashee river, close to the coast, was a clan called the Bomvana, the fugitive remnant of a tribe destroyed in the early Zulu wars. This clan was nominally attached to the FURTHER ENLARGEMENTS OF THE CAPE COLONY. 308 Xosa tribe, but it did not destroy its substance in 1856 and 1857, and was consequently able to give shelter to Kreli when he was driven over the Bashee in the following year. From that time it was really independent, and remained so until 1878. Then the country around was involved in a war yet to be referred to, and Moni, the chief of the Bomvanas, who was too weak to maintain neutrality, applied to be received as a British subject. FURTHER ENLARGEMENTS OF THE CAPE COLONY. The high commissioner accepted his offer, and took possession of the district of Elliotdale, thus bringing the whole country between the colonial border and the Umtata river more or less authoritatively under the British flag. It was regarded as being under the protection and control of the high commissioner as representing the empire, and no part of it was made subject to the Cape Colony until 1879, when the districts of Idutywa, Tsomo, Nqamakwe, and Butterworth were formally annexed. The districts of Kentani, Willowvale, Xalanga, Saint Mark's, Elliot, Engcobo, Umtata, Mqanduli, and Elliotdale were annexed in 1885. The whole of the territory between the Umtata and Umzimkulu rivers was allotted to the chief Faku, when in 1844 an attempt was made to form a power- ful Pondo state. But that attempt was a failure, for the upper portion of the territory was nearly un- inhabited, and the lower portion was filled with refugee clans from the north, some of whom were almost as strong as the Pondos themselves. The country therefore continued to be convulsed with feuds and wars, and Faku became at length only POLICY OF FAK 3O9 too glad to part with his nominal right to a portion of it that he might have a chance of conquering and holding the rest. holding the rest. He first ceded the land along the coast between the Umtamvuna and Umzimkulu rivers to Natal, thereby getting rid of a number of his opponents, and then in 1861 he offered to the Cape government nearly two-thirds of the remainder. The line which he proposed cut off the Pondomsis — a tribe as old as his own and that had never been subject to him or his ancestors, — the Bacas, the Hlangwenis, and some others of less note, whom he desired to place under such control that they could not molest him while he subjugated the enemies that would be left on his side. His offer was not at once accepted, but thereafter Sir George Grey and succeeding governors looked upon the territory along the base of the Drakensberg, north of the proposed line, as waste land at their disposal as the highest authority in South Africa. Sir George Grey offered a portion of it to Adam Kok and his Griquas, if they would remove from the Orange Free State, and they accepted it. FURTHER ENLARGEMENTS OF THE CAPE COLONY. In 1862 Sir Philip Wodehouse located them in the districts of Kokstad and Umzimkulu, and from them the whole territory north of that which Faku retained for himself became known as Griqualand East. The object of placing the Griquas there was to establish a power, acting under British prestige, believed to be sufficiently civilised to set a good example and sufficiently strong to maintain order. 310 0 FURTHER ENLARGEMENTS OF THE CAPE COLONY. But the scheme was an utter failure, and in a few- years Adam Kok was obliged to ask that a British officer should be stationed in the country to en- deavour to keep the different sections of the in- habitants from exterminating each other. habitants from exterminating each other. During the last war between the Orange Free State and Moshesh, some Basuto, Batlokua, and Fingo clans moved in, and were located by Sir Philip Wodehouse in the districts of Mount Fletcher and Matatiele. Some freed slaves and colonial blacks had previously settled in the district of Maclear. had previously settled in the district of Maclear. Constant quarrels among the various clans at length induced the high commissioner to interfere, as nearly all the chiefs declared their readiness to place themselves under British authority. In 1873 an officer with the title of resident was sent into the country, and the districts of Maclear, Mount Fletcher, Tsolo, and Qumbu — the last two occupied by the Pondomsi tribe — were taken over. In the following year the districts of Matatiele, Kokstad, and Umzim- kulu, with Adam Kok's consent, were also taken over, as was in 1876 the district of Mount Frere, with the consent of the Baca chief Makaula, whose people occupied it. These eight districts comprise the whole of the territory north of the line proposed by Faku. In 1879 they were formally annexed to the Cape Colony. In 1886 the district of Mount Ayliff, occu- pied by the Xesibes under the chief Jojo, was also annexed. It was south of Faku's line, but it became a necessity either to take over the Xesibes, or to stand by and see a brave little clan exterminated by the Pondos. Port st. FURTHER ENLARGEMENTS OF THE CAPE COLONY. John 311 A small tract of land, less than sixteen square miles in extent, on the western side of the Umzim- vubu river at its mouth, together with the navigable part of the stream and the eastern bank from the lower ford to the sea, is called the district of Port St. John's. The mouth of the Umzimvubu, in common with all the streams along the coast, is nearly closed by a shifting bar of sand. When heavy rains fall in the uplands, a channel is sometimes opened across the bar thirty feet and upwards in depth at low water of spring tides, but on other occasions the channel is often not more than three feet deep. Above the bar a sheet of water from two hundred to two hundred and fifty yards across, and from twenty to thirty feet in depth, extends some eleven or twelve miles, when a ford is reached. There are other places along the coast where boats can effect a landing, but the mouth of the Umzim- vubu is the only place worthy the name of harbour between East London and Port Natal. The outer anchorage is fairly good, and the river is usually accessible to boats, coasting vessels, and small steamers. The greatest drawback to its use is not the difficulty of landing and shipping cargoes, but the difficulty of communicating with the back country, owing to the mggedness of the land near the coast and the want of a good road. the coast and the want of a good road. In July 1878 the colonial government purchased the ground at the mouth of the river from the Pondo chief Nquiliso, and in the following month British sove- reignty was proclaimed over the port. In September 1884 the district was annexed to the Cape Colony. 3 12 FL?RTHFR ENLARGEMENTS OF THE CAPE COLONY. The remainder of Pondoland continued under the government of its chiefs ten years longer. Constant quarrels between different clans, when the defeated parties often took refuge in colonial territory and caused endless difficulties there, at length made inter- ference a matter of necessity. In March 1894 a strong body of police was massed on the border, and a commissioner was sent into the country, who required the chiefs to place themselves and their people under British control. FURTHER ENLARGEMENTS OF THE CAPE COLONY. The destructive nature of maxim guns, as proved in the recent Matabele campaign, was just at that time a matter of discus- sion by the Pondos, and the chiefs shrank from coming into contact with such weapons. They agreed to receive pensions in exchange for their independence, and the territory was proclaimed part of the British dominions. In September 1894 it was formally annexed to the Cape Colony, and was divided into five districts named Libode, Ngqeleni, Umsikaba, Tabankulu, and Bizana. Thus all the territory from the Indwe and the Kei on one side to the Natal border on the other was brought under colonial rule. The Bantu in these dependencies have given very little trouble during recent years, but there were some serious disturbances in several of the dis- tricts before they became accustomed to European guidance. guidance. On the 3rd of August 1877 there was a marriage at a Fingo kraal just within the Butterworth border, and two petty Xosa captains, with a small party of attendants, crossed over from Kreli's district of Ken- NINTH KAFFIR WA 3l3 tani to partake in the festivities. On such occasions custom demands that all who attend are to be made welcome. In the evening, when the guests were excited with dancing and drinking millet beer, a quarrel arose, no one afterwards was able to tell exactly how or why. At any rate the Xosas were ranged on one side and the Fingos on the other, and they used their sticks so freely that the two captains were badly bruised and one of their attendants was killed. The visitors were then driven over the border. Strong bands of Xosas immediately mustered to avenge the insult offered to their friends, and began to sweep off the Fingos' cattle. The excitement on both sides soon became so great that the efforts of the officials to restore order were unavailing. The police were sent to the front, the colonial volunteers were called out, and an imperial regiment of the line marched to the border. Several sharp actions were fought with Kreli's people, who lost some seven hundred men, and then fled into Pondoland. In the belief that the disturbance was over, the volunteers were now permitted to go to their homes ; but they were hardly disbanded when the Xosas returned and attacked the police. FURTHER ENLARGEMENTS OF THE CAPE COLONY. In December Kiva, a relative of Kreli, crossed the Kei, and appealed to the Xosas in the colony to support the head of their tribe. Most of them, with the chief Sandile, responded to the appeal, and the country was involved in the ninth Kaffir war. In February 1878 the colonial camp at Kentani was attacked by about five thousand Xosas, who charged 3t4 FURTHER ENLARGEMENTS OF THE CAPE COLONY. 3t in dense masses, but were mown down by a fire from heavy guns. Both Kreli and Sandile were present in the engagement. The principal column was led by the tribal priest, who had performed certain cere- monies which caused the warriors to believe that they were invulnerable ; but this feeling of confidence being destroyed, they gave way to despair. When they broke and fled, the volunteer cavalry and the Fingos pursued and prevented them from rallying. As far as Kreli was concerned the battle of Ken- tani was a decisive one. He did not attempt any further resistance, but with his adherents at once crossed the Bashee. After the conclusion of peace a small location was assigned to him in Elliotdale, and there he spent the remainder of his life. there he spent the remainder of his life. West of the Kei the Xosas held out for many months, but at length Sandile was killed in action, and they then submitted. In October 1880, just after the Basuto tribe rose in rebellion, the Pondomsis in the districts of Tsolo and Qumbu, the Basuto in the districts of Mount Fletcher and Matatiele, and the Tembus in the districts of Saint Mark's, Xalanga, and Engcobo rose also against the Europeans. They committed some atrocious murders — notable among which was the treacherous massacre of the magistrate Hamilton Hope and the clerks Henman and Warrene by the Pondomsi chief Umhlonhlo, — and they destroyed much property, but within four months they were completely subdued by a combined force of burghers and rival Bantu. TWO SYSTEMS OF LAW 31$ Bantu law is in force in all civil cases between black people throughout this territory, so that disputes regarding marriage customs and inheritance of pro- perty are decided according to it. FURTHER ENLARGEMENTS OF THE CAPE COLONY. In each district there is a resident magistrate, and over the whole terri- tory there is a chief magistrate, to whose court, in which assessors sit, there is an appeal in all but trifling civil cases. The chiefs are at liberty to try any civil cases the people choose to bring before them, and they are allowed pensions to enable them to live comfortably. Police cases are decided by the magistrates, and criminal cases are tried before a judge of the supreme court on circuit. On the uplands, where there was vacant space, several thousand European farmers are living, and they and other white people are subject to colonial law alone. This operation of two different systems of law — one, for instance, recognising poly- gamy and the other punishing it severely — at the same time and in the same place, might seem con- fusing, but in practice it works smoothly and well. fusing, but in practice it works smoothly and well. Another difference between the laws of this terri- tory and those of the country west of the Kei is that no intoxicating liquor of any kind is allowed to be sold or supplied to any coloured person in it under very severe penalties. These people are unable to resist the temptation to drink to excess, so that this law is necessary for their protection. They are in- creasing in number rapidly, but since the destruction of their cattle by disease during the closing years of the nineteenth century the death-rate of little children has been higher than formerly, owing to the want of milk for food. 3 l6 FURTHER ENLARGEMENTS OF THE CAPE COLONY. There are two towns of importance, Umtata and Kokstad, in the territory, both centres of commerce and seats of mission enterprise on a very large scale. Mission stations and schools are dotted thickly over the land, and the Blythswood industrial institu- tion is doing excellent work. On the lower terraces Europeans are not permitted to acquire land, except in the towns, all of it being needed for the use of the Bantu, but good roads have been made, and even railroads have been constructed in some places. Everything that is possible is thus being done to lead the people onward in civilisation. CONDITION OF THE CAPE COLONY IN I9IO. The Cape Colony after the enlargements that have been mentioned covered an area of 276,565 square miles, equal to a square block of land five hundred and twenty-six miles each way. In consequence of the increase of territory and population, its legisla- ture had been enlarged, and the house of assembly now consisted of one hundred and seven members and the legislative council of twenty-six. and the legislative council of twenty-six. The principal industries of the European inhabit tants were still pastoral and agricultural, owing to the nature of the greater part of the country. the nature of the greater part of the country. The Karoo plains which occupy so much of its surface, thinly speckled with succulent plants, were the grazing ground of flocks of merino sheep and Angora goats, that were kept up to a high standard by the importation of the best foreign blood. From the Karoo to the Orange river and thence northward to the Molopo, the surface, carpeted in good seasons with long grass, supported large herds of horned cattle and considerable flocks of sheep. In these parts agriculture, except in certain limited localities 317 317 317 317 CONDITION OF THE CAPE COLONY IN I9IO. 3 1 S such as the basin of the Hart river, was precarious where there were no running streams, but a great deal of labour had been expended in making reser- voirs from which gardens and orchards could be irri- gated, and much success had attended the sinking of artesian wells. Along the Zak river and at Kakamas on the banks of the Orange large irrigation works had converted extensive areas of desert into rich cornlands and gardens. The cultivation of lucerne in such places had enabled horses, cows, and ostriches to be kept to advantage. to be kept to advantage. On the first and second terraces from the southern coast the rainfall is ample, and agriculture was carried on conjointly with cattle-breeding. Wheat and maize were the principal crops, but oats and barley were extensively grown for horses, and almost every kind of vegetable and fruit was abundant. In the south- western angle of the colony — the part settled in the seventeenth century, — the cultivation of wheat, fruit, and especially the vine were the chief industries. and especially the vine were the chief industries. CONDITION OF THE CAPE COLONY IN I9IO. Skins of wild animals had quite disappeared from the exports, for the large game with which the country once teemed had been exterminated. The same may be said of ivory. same may be said of ivory. The diamond mines, except those along the banks of the Vaal river, had been amalgamated and were controlled by one powerful company, that regulated the production and so kept the value from falling below a wide margin of profit. In the exports from the colony diamonds occupied the first place, though the area in which they were found was very small. They were contributing greatly to the public revenue, DRAWBACKS TO AGRICULTUR 319 were giving employment to large numbers of men, were furnishing an excellent market for farm produce, and in many ways were promoting trade. and in many ways were promoting trade. In 1852 rich copper mines were opened in the barren district of Little Namaqualand, and the part of the colony previously considered the least valuable began to contribute its quota to the exports. It has continued to do so ever since, and bids fair to main- tain its position for a long time to come. The only other mineral productions of the Cape Colony that were of any note in 1910 were salt and coal. Salt, ample in quantity for home use, was gathered from pans in several localities, and coal, not of the best quality, however, was found at Molteno and Indwe and used on the railways within a certain radius of the mines and for fuel in private houses. radius of the mines and for fuel in private houses. In dealing with the Cape Colony, it must be borne in mind that it, in common with all other parts of South Africa, is subject to many drawbacks. Long and severe droughts sometimes occur over very ex- tensive portions of its surface, when everything green perishes, and animals of all kinds die off in great numbers. Sometimes, though rarely, these droughts produce actual famine, especially among the coloured people, who are too improvident to lay by anything for a day of distress. Floods too sometimes cause great damage over large areas, sweeping away cultivated land, buildings, and animals, turning rivulets iato rushing torrents, and destroying massive bridges as if they were matchwood. Fortunately such terrible storms do not often occur. 320 CONDITION OF THE CAPE COLONY IN I9IO. CONDITION OF THE CAPE COLONY IN I9IO. The mild climate favours insect life, so that the farmers and fruitgrowers have to contend with diffi- culties unknown in Central and Northern Europe. Myriads of caterpillars sometimes devastate the cultivated land, and vast swarms of locusts appear, when before them the country may be as the garden of the Lord, but behind them it is a naked desert. Means to cope with these pests, to a considerable extent at least, have been discovered, however, and their ravages are now much less dreaded than formerly. Cattle are subject to various diseases. In the summer months, except in a few localities, horses require to be stabled at night, or they contract a disease which nearly always ends in death. Lung- sickness and redwater sometimes create havoc among horned cattle, and other diseases of a less deadly nature are common. During the last years of the nineteenth century the rinderpest swept down from the north, and in some parts carried off nearly all the cattle. Investigations into the nature of these and other diseases have, however, resulted in remedies or palliatives having been discovered for most of them. Scab in sheep, once the most dreaded of all cattle plagues, is now completely under control. plagues, is now completely under control. The agricultural and pastoral products of the colony were in 1910 about ten times as great in value as in 1872. Its mineral productions had increased to almost the same extent. Among the Bantu residents in the colony mis- sionaries of almost every Christian society had been labouring for nearly a century, and since the first MISSION A R Y LABO URS. 321 converts were made three or four generations had grown up under their care. The result upon the whole was disappointing to those who anticipated great improvement in a short space of time, although a considerable advance had certainly been made by a section of these people. Where youths had been separated from the surroundings of the kraals, and been trained in habits of order, cleanliness, and — as far as practicable — industry, the most good had THE BOYS' SCHOOL, LOVEDALE MISSIONARY INSTITUTION. THE BOYS' SCHOOL, LOVEDALE MISSIONARY INSTITUTION. been effected. In this direction the Free church of Scotland had led the way, and its noble institutions at Lovedale, on the western bank of the Tyumie river, and Blythsvvood, in Fingoland, were models which other Christian bodies were copying. CONDITION OF THE CAPE COLONY IN I9IO. It had become generally recognised that the system of edu- cation carried out by most of the missionary societies in earlier years was faulty. Their idea was to teach the children of barbarians to read and write, to give them a knowledge of grammar and geography, of arithmetic and English history, and especially to been effected. In this direction the Free church of Scotland had led the way, and its noble institutions at Lovedale, on the western bank of the Tyumie river, and Blythsvvood, in Fingoland, were models which other Christian bodies were copying. It had become generally recognised that the system of edu- cation carried out by most of the missionary societies in earlier years was faulty. Their idea was to teach the children of barbarians to read and write, to give them a knowledge of grammar and geography, of arithmetic and English history, and especially to 22 CONDITION OF THE CAPE COLONY /N I9IO. 32 2 instruct them in Christian doctrine and cause them to read the holy scriptures. The government sub- sidised the schools, and the rivalry among the different denominations was so keen that no loca- tion of any importance was lost sight of. But, after a vast expenditure of money and energy, it came to be seen that education of this kind was by itself of little value, and industrial training had come to be regarded as a necessity. The mixed breeds, descendants of slaves, and Hottentots had also been the objects of missionary solicitude, and as they had long been in close contact with white people, their mode of living was based upon the European model. At Genadendal the Moravian society, whose work is above all praise, had been training these people for a century and a quarter. Most of them professed Christianity, and in many places they formed congregations and sup- ported their own clergymen. They did the rough work of the farms and the towns, but some of them were averse to steady labour, and in general they were thriftless. The instances were rare of people of this class accumulating property, though they often had excellent opportunities for doing so. had excellent opportunities for doing so. The Asiatics in the colony were chiefly descendants of people from the Spice islands, who were sent here in the seventeenth and eighteenth centuries, and were commonly termed Malays. CONDITION OF THE CAPE COLONY IN I9IO. Those of pure blood were almost — if not wholly — without exception Mo- hamedans. But many Africans and people of mixed blood had adopted that creed, and as they had inter- married, the Malays presented every variety of PUBLIC SCHO 323 appearance between the pure Asiatic, the pure European, and the pure African, while some of the mixed breeds fluctuated between Christianity and Islam. They were certainly of a higher type than the ordinary Cape coloured labourer. Most of the men were mechanics, greengrocers, or shopkeepers, and the amount of property that they held was con- siderable. A large part of the fishing industry was in their hands. They were confined entirely to towns, far the greater number of them to the Cape peninsula. A few Indians from Hindostan had of late years found their way to the colony, but immi- gration of others was then prohibited. gration of others was then prohibited. Owing to the zeal of the missionaries, for many years greater efforts were made to give a good school education to coloured children than to white, and it was a lamentable fact that there was a large section of the European rural population without any know- ledge of the use of books. This matter had been brought to the attention of the government and parliament, and before 1910 was rectified, so that at this time white children were receiving as good an education throughout the Cape Colony as in any other country in the world. In the towns and vil- lages there were excellent public schools, separate for boys and girls, each under the joint control of a local board of management and the educational department of the government, half the cost being defrayed by the treasury. Those of the first and second class were attended exclusively by white children. Then there were several colleges in which higher education was imparted, such as the South CONDITION OF THE CAPE COLONY IN I9IO. 324 African college in Capetown, the Rhodes university college in Grahamstown, the Victoria college in Stellenbosch, the Dale college in King-Williams- town, the Diocesan college at Rondebosch, and the Huguenot college for girls at Wellington, where students were prepared for matriculation and degrees in the university. CONDITION OF THE CAPE COLONY IN I9IO. An excellent normal college in Capetown was devoted to the special training of teachers for ordinary schools, but the professors in the colleges and nearly all the teachers in the high- class public schools had been educated in European universities. In the thinly populated parts of the colony a difficulty existed in the distance between the residences on farms, but that too had been over- come by giving assistance if necessary to boarders where the children were too few for the employment of a qualified teacher. of a qualified teacher. No expense was spared in bringing justice within reach of every one. There were nearly a hundred stipendiary magistrates, holding courts in as many districts or sub-districts into which the colony was divided. All petty civil and criminal cases were tried by them. The supreme court consisted of the chief justice and eight puisne judges, but as two formed a quorum, in practice three of the judges sat in Gra- hamstown, three in Kimberley, and the others in Capetown. Twice a year a judge of the supreme court visited each district town, and tried cases which were beyond the jurisdiction of the magis- trates. The towns and villages were supplied with public libraries aided by government, hardly any were HARBO 32 5 without two or three churches of different denomi- nations, and banks, insurance offices, newspapers, and benevolent institutions were found in nearly all. Municipal government — in very rare instances abused — was in force in every community of the slightest note. Each district had a divisional council, with powers over a large area somewhat similar to those of municipal councils in villages. of municipal councils in villages. Good roads had been made even in the wildest parts of the colony, and the rivers on the principal routes had been bridged. There was scarcely a hamlet that was not connected with all parts of South Africa by the post and the telegraph wire. There was a network of railways over the colony, which were of the greatest use not only for transport pur- poses, but for promoting progress of all kinds. poses, but for promoting progress of all kinds. Very great improvements had been made in the harbours, especially in Table Bay and East London. CONDITION OF THE CAPE COLONY IN I9IO. In Table Bay in olden times the beach after storms in winter was frequently strewn with the wrecks of costly fleets ; in 19 10 ships of the largest size lay beside wharves in a dock in perfect safety, and received cargo from or discharged it into railway trucks. If damaged, they could be repaired in a graving dock, and when lying at the outer anchor- age they were protected by a magnificent break- water. At East London the bar at the mouth of the Buffalo had been removed, and large steamers and sailing ships could enter the river, where every facility was provided for loading and unloading them. was provided for loading and unloading them. On the coast numerous lighthouses stood as senti- nels to warn seamen of danger by night, and the EFFECTS OF THE WAR OF 1899-1902. 327 ancient terror of stormy seas off the Cape of Good Hope had long since been forgotten. It arose more from distance from home in the early days of circum- navigating Africa than from the real violence of the sea, for that is commonly trifling compared with the fury of the North Atlantic. fury of the North Atlantic. The colony was connected with Europe by three submarine cables, so that anything of importance that occurred there one day was known here through the newspapers on the next. Splendid steamships carrying mails and passengers arrived from and left for England weekly, often making the run of six thousand sea miles in less than sixteen days, and the passage was certainly one of the pleasantest in the world. The deplorable war with the two republics in 1 899-1902 did an immense amount of harm tempo- rarily to the Cape Colony. British gold was expended in the greatest profusion, and people lost their reason- ing power and seemed to think it would last for ever. Expensive habits of living were introduced, and the towns — especially those on the coast — were altogether overbuilt, while borrowed money was expended by the government in a most lavish manner. Crowds of immigrants, many of them British artisans, arrived, and for a short time it seemed as if the colony was really making a great bound forward. Then came peace, and with it the withdrawal of the great army employed in South Africa, and the cessation of the influx of British gold. CONDITION OF THE CAPE COLONY IN I9IO. Everything came at once to a standstill. The recent immigrants abandoned the country, as they saw no means of CONDITION OP THE CAPE COLONY IN I9IO. 328 obtaining a living in it, and half the houses in many of the streets in the towns and their suburbs were left empty. Most of the slaughter cattle and sheep in the country had been consumed by the soldiers and temporary settlers, and frozen meat was being imported in very large quantities for common use. Nearly everything that was needed for home con- sumption was brought from abroad, so that the cost of living was very much greater than it had been before the war. The railroads, which had cost ^32,000,000, ceased to bring in the amount required for their maintenance and the interest on the capital employed in their construction, which had to be made up from the general revenue. The interest on the public debt, which had been enormously increased, became a burden almost too great to be borne. Nor was this all, for the various municipalities had also incurred heavy debts, and the interest had now to be paid by a dwindling population. be paid by a dwindling population. Retrenchment in every branch of the public service and in every commercial establishment was unavoid- able, and was carried out to an extent that created an enormous amount of distress, and drove thou- sands of useful people from our shores. Taxation heavier than any previously thought of was of neces- sity imposed, the operation of the sinking fund was suspended, and yet the revenue could not be made to meet the expenditure, greatly reduced as this was, so that further debt had to be incurred. Never before in South Africa had there been such a period of depression, or such faint hopes of a speedy return of prosperity. AGRICULTURAL PROSPERI 329 But all the time the cows and sheep and goats were increasing on the farms, until it became no longer necessary to import frozen meat and dairy produce in large quantities, agriculture was greatly extended, and very fortunately the seasons were favourable, new industries were opened up, and the exports were rising steadily and showing year after year a more decided balance in favour of the country. The rapid increase in the production of gold in the Transvaal also aided the commerce of the colony. CONDITION OF THE CAPE COLONY IN I9IO. And so by 19 10 the period of great depression had wellnigh passed away, and there was a good prospect of matters resuming the same steady progress as before the war. THE COLONY OF NATAL FROM 1 842 TO 1 873. Natal became a British possession at a very un- fortunate time for the good of the country. Sentiment in England was then running so strong in favour of black people that this beautiful and fertile country, which might have been made the home of many thousands of industrious European families, was given away to any Bantu who chose to enter it. given away to any Bantu who chose to enter it. There are, of course, different ways of looking at this matter. The Bantu themselves, who regard their mode of life as vastly preferable to ours, inasmuch as it is comparatively free of care and toil, certainly think their possession of Natal proper and desirable. The missionary looking for raw material to work with is naturally of the same opinion. But the man who believes that the strengthening of the European element would be a blessing to Africa itself, who is convinced that the unprogressive people of the continent can never become civilised except under European govern- ment and under the guidance and control of a strong body of European settlers, must look upon INFLUX OF BANT 331 the alienation of the soil of Natal to the Bantu as a very great mistake. a very great mistake. As soon as Great Britain was dominant there, all who were in fear of Panda made their way into the country, where they were sure of being pro- tected and of being allowed to live as they chose. Their birthplace and that of their parents might be far away, but they were all termed natives by the government, and as soon as arrangements could be made tracts of land were assigned to them to live upon. Missionaries settled among them, and in course of time a few became converts to Christianity and made some advance towards civilisation. But the great majority re- mained what their forefathers had been, for it cannot be said that their use of a few articles of European manufacture was an indication of any real change. There are no people in the world more prolific than the Bantu of South Africa, and though their death-rate in places partly occupied by Europeans, where they contract chest diseases, is high, in their own kraals, where they live after the manner of their ancestors, it is low, and many of them attain a great age. THE COLONY OF NATAL FROM 1 842 TO 1 873. Their practice of polygamy has the result that among them there are no old maids. The consequence is an amazingly rapid increase of people, wherever the old checks of war, famine, and punish- ment by death on charges of dealing in witchcraft are removed. So very shortly there was a large Bantu population within the borders of Natal, where a few years before there had been hardly any in- THE COLONY OF NATAL FROM 1 842 TO 1 873. 332 habitants. They were permitted to live according to their own laws and customs, but they paid a small hut-tax, and the government exercised general control over them. This it was able to do, as there were no hereditary chiefs of high rank among them, and it was possible to use one of the little groups to keep another inclined to be unruly in check. to keep another inclined to be unruly in check. Owing to their presence in such numbers the country failed to attract European settlers, and in the early days after the British conquest only one large body of white immigrants entered it. Between 1848 and 185 1 some four or five thousand English people arrived, to whom small plots of ground were given, but man)- of them afterwards removed to Australia, and few remained as culti- vators of the soil. White people from abroad settled in the country every year, but never in large numbers, and most of them took up their residence in Durban or Maritzburg, or in one of the villages along the trading routes to the interior. In 1872, thirty years after the British conquest, including the Dutch-speaking families who remained on the uplands, there were not nineteen thousand white people living in Natal, and the Bantu had increased to over a quarter of a million. Although the disparity in number was so great, the Bantu did not often disturb the peace of the country, and only on two or three occasions was it necessary to use military force against defiant chieftains. They had ample space for living com- fortably in their own way, and taxation was so light that they did not feel it as a burden. But the THE HLUPI TRI 333 Europeans realised that this condition of things could not be permanent, for they were multiplying so rapidly that there must some day be a struggle for more room. THE COLONY OF NATAL FROM 1 842 TO 1 873. Europeans realised that this condition of things could not be permanent, for they were multiplying so rapidly that there must some day be a struggle for more room. So matters went on until 1873, when the first serious disturbance took place. In 1848 a section of the Hlubi tribe fled from Zululand, and had a location assigned to it at the sources of the Bush- man's river, at the foot of the Drakensberg. The Hlubi had once been the largest tribe in South- Eastern Africa, but in Tshaka's wars most of its members were killed, and those who survived were dispersed far and wide. " There was a white mark from the Tugela to Thaba Ntshu, and that mark was our bones," said once an old Hlubi to the writer of this volume, in recounting his personal adventures. He might have added that there was a similar line from the Tugela to the Kei. Along both these routes a few fugitives were scattered, and these have multiplied so greatly that if their descendants could all be collected together to-day the Hlubi would again stand out as the largest tribe of the country, except the Basuto. except the Basuto. The great chiefs had perished in Tshaka's wars, and the one of highest rank that was left was Lansralibalele — in English " The Sun is burning " — the head of the clan that sought refuge in Xatal. A stranger visiting his location in 1873 ^rould have regarded him as a man of little importance, with a following of not more than ten thousand souls, all told ; but those acquainted with his history knew that he was held in strong attachment by clans as 4 THE COLONY OF NATAL FROM 1 842 TO 1 873. 334 far away as the Caledon in one direction and the Fish river in another. There was a law in Natal, required for public safety, that no Bantu should have guns in their possession without being registered. In other parts of South Africa guns were obtainable, and Langa- libalele, setting the law at defiance, sent his young men away to earn money and purchase these weapons, which were brought by hundreds into his location without the necessary formalities being observed. When this became known, the chief was called upon to account for his guns, but he declined to do so. THE COLONY OF NATAL FROM 1 842 TO 1 873. Message after message was sent, requiring him to appear at Maritzburg, but he made excuses, and never went. It was subsequently proved that he was in treasonable correspondence with other chiefs beyond the Drakensberg, from whom he expected to obtain assistance, and he must have felt himself strong enough to maintain his independence against the white people. the white people. Peaceable means having failed to secure his obedience, an armed party was sent to enforce the demands of the government. Upon its approach Langalibalele abandoned his women and children, and with his cattle and most of his warriors fell back upon the mountains. In the Bushman's pass Major Durnford and a small party of volunteers overtook the rearguard of the rebels. The chief was in advance, and as the volunteers had orders not to fire first, they attempted to communicate with him. The induna in command pretended to send for the chief, and while waiting for him to arrive; the REBELLION OF LANGALIBALEL 335 volunteers were being surrounded. At the same time threatening gestures and language, coupled with taunts, were used towards them. They fell back in a panic, when too late, and as they did so five of them were shot down. The colonists at once awoke to a sense of their danger. They did not know how far the inclination to rebel extended, but of one thing they were certain : that nothing but the prompt punishment of the Hlubis would prevent all who were disaffected from rising in arms. Volunteers at once came forward. Everywhere in South Africa the Europeans were ready to help. The government of the Cape Colony took immediate measures to render effectual assist- ance, and the two republics expressed a willingness to give aid if needed. It was recognised that not only the peace of Natal, but of the entire country, was imperilled, for if time was given for all the sections of the Hlubi tribe to unite with the clan in rebellion a general war of races might ensue. in rebellion a general war of races might ensue. THE COLONY OF NATAL FROM 1 842 TO 1 873. Langalibalele and his warriors crossed the Dra- kensberg to Basutoland, in expectation of being joined there by one of Moshesh's sons ; but such prompt measures were taken by the governments of the Cape Colony and Natal that the rebels were surrounded before they reached their destination, and the chief, with some of his principal men, who were in advance, were obliged to surrender to a division of the Cape frontier armed and mounted police. The main body made an attempt to resist, but was dispersed after a sharp action, and all the cattle were captured. 336 THE COLONY OF NATAL FROM 1 842 TO 1 873. During this time the excitement of the Natal colonists was naturally very high, and what, under ordinary circumstances, would be regarded as undue severity was exercised towards the people Langali- balele had left behind, as well as to another clan living close by that sympathised with him. But as soon as the danger was over, violent measures of every kind ceased. every kind ceased. Langalibalele was tried by a special court, which sentenced him to banishment for life ; and as Natal had no outlying dependency to send him to, an act was passed by the Cape parliament authorising his detention on Robben Island. His clan was broken up, and the ground it had occupied was resumed by the government. the government. This event attracted a great deal of attention in England, chiefly through the action of the Aborigines Protection Society and of Bishop Colenso, who repre- sented the conduct of the white people and of the government as in the highest degree cruel and unjust towards the Hlubis. The Natal clergy, seventy-four ministers and missionaries of different denomina- tions, did their utmost to show that it was not so ; but their opinions were in general unheeded, as were also the statements of the South African press. also the statements of the South African press. The imperial ministry reflected the sentiments of the people. Sir Benjamin Pine, the lieutenant- governor of Natal, was recalled. THE COLONY OF NATAL FROM 1 842 TO 1 873. Compensation was ordered to be paid from the colonial treasury to the clan that had suffered loss owing to its sympathy with the rebels ; various Hlubis who had been condemned to terms of imprisonment had their TROPICAL PRODUCTS IN NATAL 337 sentences commuted ; and it was required that Langalibalele should be removed from Robben Island to a farm on the mainland, where he could have the society of his wives and be treated as a prisoner of state. These orders were of course promptly carried out. Langalibalele remained an exile for twelve years, during which time he was provided with every possible comfort. He lived in a house on a small farm in the Cape peninsula, and had part of his family with him. He was then permitted to return to Natal, and died there shortly afterwards. His rebellion was followed by an improvement in the mode of dealing with the black people. Bantu law was codified, so that thereafter it could not be interpreted differently by different magistrates. A Native High Court was created, which was presided over by a qualified judge, and to which there was an appeal from the decisions of the location magis- trates. A tax on marriages, recently imposed, was withdrawn, and the hut-tax was increased to four- teen shillings a year. The amount of marriage settlements was fixed and made payable only before the marriage, thus doing away with the most fruitful cause of litigation, and girls were amply protected from being forced to marry old men against their will. Owing to the hot Mozambique current flowing down close to it, the belt of land along the coast north of the Umzimkulu has a tropical vegetation, though it is perfectly healthy for Europeans. It seemed there- fore to present a favourable field for the production 23 THE COLONY OF NATAL FROM 1842 TO 1873. 33§ of coffee, sugar, ginger, arrowroot, cotton, and tea, and no long time elapsed before experiments began to be made. Not a plant among them all but throve wonderfully well, so that it was hoped and expected that Natal would shortly become one of the most valuable dependencies of Great Britain. THE COLONY OF NATAL FROM 1 842 TO 1 873. Here was a favourable soil and a favourable climate, and here, thought people at a distance, in the teeming Bantu locations was a great reservoir of labour that could be utilised for the good of both employers and employed. But the Bantu declined to be utilised in this way. Some of them were willing to work for a while when the whim seized them and they had a particular object in view, but they could never be depended upon, and were prone to leave service just when they were most needed. when they were most needed. The planters then turned to India for a supply of labour. Coolies were engaged there, and were brought over under contract for a term of years. By their assistance the soil was made to bring forth tropical products in considerable quantities, but eventually some were destroyed by diseases and others were found not to pay. Among these were cotton and coffee, both of which at one time gave promise of good returns. Sugar succeeded best, and its production remains to the present day an im- portant industry. It was supposed that the coolies would return to India when their contracts expired, as they were entitled to free passages back ; but they had found a goodly land, and many of them had no mind to leave it. Some of their countrymen of the trading class MODE OF LIFE OF EUROPEAN 339 were next attracted by the accounts spread by those who returned, and soon quite a little stream of Indian immigrants set in. As they can live upon the merest trifle, European competitors were rapidly driven out, and retail dealing, with all kinds of light labour, fell into their hands. They contributed nothing towards the military strength of the country and very little towards the revenue. They continued to arrive until at the present day they surpass the white people in number, but further immigration is now prohibitedc now prohibitedc The circumstances under which they lived deter- mined the mode of life of the Europeans in Natal. They became for the most part traders and for- warders of goods to the interior republics. THE COLONY OF NATAL FROM 1 842 TO 1 873. There were planters and farmers among them, and the Dutch-speaking colonists continued to be agricul- turists and cattle breeders, but more than half of the whole number of the English-speaking settlers resided in the two towns Maritzburg and Durban, and a large proportion of the remainder occupied villages along the trade routes. Maritzburg, and especially Durban, grew and prospered. In each of these towns improvements were seen every year. Newspapers, as well conducted as those of any other British colony, were published in both of these places, there were banks, insurance offices, churches of various denominations, and literary and benevolent institutions of different kinds. The education of children was carefully attended to. The religious controversy connected with the name of the right reverend Dr. Colenso, bishop of Natal, CONSTITUTION OF NATA 34 1 attracted the attention of English-speaking people throughout the civilised world and helped to bring the colony into notice. the colony into notice. The position of Durban made it a place of com- mercial importance. From early days it was realised that if the bar between the Point and the Bluff could be removed, so that ships could enter the inner sheet of water, it would become the gateway to a large area of the interior, and to effect this the energetic colonists turned their attention. A plan known ever since as [Milne's was adopted, and a considerable amount of money was expended upon it, when it was abandoned for another designed by Captain Vetch, of the admiralty. This was proceeded with for some time, but it proved a failure. Meantime ships lay at anchor in the outer roadstead, and communication with the inner harbour was maintained by means of lighters. From the Point a railway conveyed goods to Durban. For a few years after the British occupation Xatal was regarded as a dependency of the Cape Colony, and laws were made for it by the Cape parliament. Then a legislative council, consisting entirely of officials, was created, and the connection with the Cape Colony was severed, except that the high commissioner retained a good deal of power, and the head of the local administration ranked only as a lieutenant-governor. In 1856 a charter was granted by the queen, and a legislative council, with most of the members elected by the people, was constituted. THE COLONY OF NATAL FROM 1 842 TO 1 873. This with a few slight changes continued until 1873, when the imperial authorities considered it necessary THE COLONY OF NATAL FROM I 842 TO 1 873. 342 to increase the power of the crown, and the council was induced to pass an act admitting a number of nominee members. This act, however, was to be in force for only five years, and at the end of that period the more liberal constitution was restored. to increase the power of the crown, and the council was induced to pass an act admitting a number of nominee members. This act, however, was to be in force for only five years, and at the end of that period the more liberal constitution was restored. In 1866 Natal was enlarged by the addition to it of the tract of land between the Umzimkulu and Umtamvuna rivers, thereafter called the county of Alfred. It was part of the Pondo treaty state, but Faku found it impossible to bring the clans living in it under his authority, and was glad to get rid of them by ceding the territory to Natal. ZULULAND TO ITS ANNEXATION TO NATAL. Panda, who became independent of foreign control in 1843, was mucn ^ess intelligent than either of his predecessors, Tshaka or Dingana. Soon after his accession to power he grew so stout as to be un- wieldy, and never afterwards displayed activity of any kind, bodily or mental. Two of his sons, how- ever, Umbulazi and Ketshwayo by name, grew up to be men of superior ability. Though the discipline of the army was greatly relaxed, the military system introduced by Tshaka was still kept up, and the regiments were divided in their attachment to the young chiefs. " Two young bulls cannot live together in the same kraal," said Panda ; " one must drive the other out or be gored." The brothers were of the same opinion. In December 1856 a battle was fought between their adherents on the northern bank of the Tugela, which resulted in complete victory for Ketshwayo. His brother must have been killed, though the body was not found, for he was never seen again. Then a dreadful massacre of the defeated chiefs adherents took place, when not only the Panda, who became independent of foreign control in 1843, was mucn ^ess intelligent than either of his predecessors, Tshaka or Dingana. Soon after his accession to power he grew so stout as to be un- wieldy, and never afterwards displayed activity of any kind, bodily or mental. Two of his sons, how- ever, Umbulazi and Ketshwayo by name, grew up to be men of superior ability. Though the discipline of the army was greatly relaxed, the military system introduced by Tshaka was still kept up, and the regiments were divided in their attachment to the young chiefs. " Two young bulls cannot live together in the same kraal," said Panda ; " one must drive the other out or be gored." The brothers were of the same opinion. In December 1856 a battle was fought between their adherents on the northern bank of the Tugela, which resulted in complete victory for Ketshwayo. His brother must have been killed, though the body was not found, for he was never seen again. Then a dreadful massacre of the defeated chiefs adherents took place, when not only the 345 ZULU LAND TO ITS ANNEXATION TO NATAL. 344 men, but the women and children related to them, were put to death. About one-fourth of the Zulus perished. perished. ZULULAND TO ITS ANNEXATION TO NATAL. From that day Ketshwayo was the real ruler of the tribe, though his father lived until 1872. The young chief was a man of prepossessing appearance, dignified in manner, and gifted with mental power in a high degree. But he was as pitiless as a piece of steel, and human life under his government was sacrificed with as little compunction as the life of oxen and cows. Much as one could wish it other- wise, observation shows that this is the kind of rule which brings out what is best in the Bantu character as well as what is worst, and under Ketshwayo the Zulus were recognised by every one as the most intelligent, the most active, and the most fearless of all the blacks in South Africa. They were the most handsome too, for constant exercise in arms and in military drill greatly improved their appear- ance. Discipline had become relaxed during the fifteen years following Panda's accession, but by Ketshwayo it was restored to the same condition as under Tshaka. As time went on the Zulus became more and more a menace to their neighbours. Hemmed in between the South African Republic, Natal, and the sea, if they used their arms at all, it could only be against a civilised power. In 1877 Sir Bartle Frere became governor of the Cape Colony and high commissioner for South Africa. No man had a kinder heart or a more earnest desire to promote the welfare of the people of the country, KETSHW 345 white and black, the Zulus as well as British subjects. But war with Ketshwayo had become a necessity, and he could not avoid it without betraying his trust. It was his duty to protect the queen's subjects, and there was no question that many of them were in imminent peril, and must so remain until the Zulu military system came to an end. Unfortunately he did not know how strong the Zulu army really was, and none of those upon whom he depended for infor- mation were able to tell him. Ketshwayo gave him more than one provocation. A powerful Zulu force paraded along the British border, and the chief spoke of it as a mere hunting party. English officials who were sent into Zululand as envoys were treated by the indunas in a contemptuous manner. ZULULAND TO ITS ANNEXATION TO NATAL. Zulu subjects crossed the boundary, seized two women on Natal soil and carried them away to death, and Ketshwayo, when called upon for redress, treated the matter as of trifling importance. In several serious disturb- ances by Bantu tribes in distant parts of South Africa the agency of the Zulu chief was clearly traced, and in many other respects he showed him- self an enemy to the civilised governments of the country. In December 1878 Sir Bartle Frere, having col- lected a military force in Natal which every one believed to be strong enough for the purpose, sent an ultimatum to Ketshwayo, in which he demanded redress for the injuries sustained, and called upon the chief to disband his army. As no notice was taken of the message, on the 10th of January 1879 an English army entered Zululand in three divisions, ZULULAND TO ITS ANNEXATION TO NATAL. 346 consisting partly of British soldiers, partly of colonists, and partly of blacks. and partly of blacks. Ten days after crossing the Buffalo the central column formed a camp at the foot of the hill Isandlwana — that is The Little Hand — within sight of the Natal border. The country was so rough that it needed all that time to construct a road along which provisions could be conveyed. On the following morning part of the column, with Lord Chelmsford, the commander-in-chief, left the camp and moved away to attack a kraal several miles distant. Some Dutch farmers had advised the English officers to take precautions against surprise, and had told them of the encounters with Dingana, but their warnings were disregarded. Nothing was done for protection at Isandlwana, though there were waggons enough to form a lager. Not a trench was dug nor a spadeful of earth thrown up in a bank. No one there even dreamed of danger until a little before noon on the 22nd of January 1879, when the horns of a Zulu army about twenty thousand strong were closing around the camp. The fight for life was stubborn, but the odds on the enemy's side were too great, and all was soon over. ZULULAND TO ITS ANNEXATION TO NATAL. A few, principally mounted irregulars, managed to make their way out of the circle of Zulu spears before it was quite closed, but the ground was full of boulders and dry beds of occasional torrents, so that many of these even were overtaken and killed. With them were Lieutenants Melvill and Coghill, who were trying to save the colours of the first battalion of the 24th regiment, and who reached the Natal bank 1SANDLW 347 of the Buffalo before they were struck down. The colours were found in the river some days afterwards. At Isandlwana nearly seven hundred British soldiers and over one hundred and thirty colonists perished, for the Zulus gave no quarter. The victors lost about three thousand men. Information of the terrible disaster reached Lord Chelmsford in the afternoon. An officer had ridden towards the camp, and had seen it in possession of the Zulus. The party with the general, though weary from marching in the hot sun, at once com- menced to retreat, for all its stores of every kind were lost. Isandlwana was reached shortly after nightfall, and there, among the corpses of their slain comrades, officers and men, alike worn out with anxiety and fatigue, lay down and tried to rest. The Zulus, after plundering the camp, had retired. At early dawn the retreating band resumed its march, and reached Natal without being molested. without being molested. At Rorke's Drift, where the column had crossed the Buffalo, there was a small depot of provisions and a hospital, and there a hundred and thirty soldiers, under Lieutenants Bromhead and Chard, had been left to keep open communication with Natal. About five o'clock in the afternoon of the day of Isandlwana this post was attacked by between three and four thousand of the very best of the Zulu soldiers, com- manded by Dabulamanzi, a brother of Ketshwayo. Fortunately the garrison had received warning in time to enable them to make a lager of sacks of maize and boxes of biscuits, behind which they maintained such a gallant defence until four o'clock in the morn- ZULULAND TO ITS ANNEXATION TO NATAL. 348 ing of the 23rd that Dabulamanzi then thought it prudent to retire. Over three hundred of his men were lying dead around the lager. Of the garrison seventeen were dead and ten were wounded. ZULULAND TO ITS ANNEXATION TO NATAL. This splendid defence saved Natal from invasion, for if the post had fallen the colony would have been open to the Zulus. The other columns fared better than the one whose fate has been told. Colonel Pearson, with about two thousand European combatants and the same number of blacks, crossed the Tugela near the sea, and marched towards Ulundi, the Zulu capital, where the whole of the invading force intended to unite. At Inyesane he was attacked by a Zulu army between four and five thousand strong, but beat it back with heavy loss, and on the 23rd of January reached the Norwegian mission station Etshowe. Here he learned of the disaster at Isandlwana, so he sent his cavalry and blacks back to Natal, and fortified the station, where he remained until reinforcements arrived from England. England. The third column consisted of about seventeen hundred British soldiers, fifty farmers under Com- mandant Pieter Uys, and three or four hundred blacks. It was commanded by Colonel Evelyn Wood. This column was not attacked on its march, and after Isandlwana fortified a post at Kambula, where it remained. Colonel Wood managed to inflict much damage upon the Zulus in his neigh- bourhood by frequent sallies, but on one occasion, at a mountain named Hlobane, his patrol was nearly surrounded, and ninety-six of the party were killed. INVASION OF ZULU LAN 349 Among them were Commandant Uys, Colonel Weatherley, and the son of the latter, a mere youth, who died at his father's side just as Uys's brother had died by his father's side forty-one years before. On the day after this event the lager at Kambula was attacked by a great Zulu arm}', which suffered tremendous loss before it retired discomfited. In the beginning of April Lord Chelmsford, with a strong force of soldiers and sailors, marched from Natal to the relief of Colonel Pearson at Etshowe. On the way he was attacked at Ginginhlovu, but beat back his assailants, and succeeded in reaching the station. As soon as intelligence of the disaster at Isandlwana reached England strong reinforcements were sent out, and before June some nine thousand soldiers, cavalry and infantry, with a vast quantity of munitions of war and provisions, reached Natal. With them came the young prince imperial of France, who was fated to lose his life a few weeks later in a lonely dell in Zululand. ZULULAND TO ITS ANNEXATION TO NATAL. He went out from a camp with a small reconnoitring party, which was surprised by a band of Zulus while it was resting in a garden, and the prince, being unable to mount his horse, was stabbed to death, his companions having abandoned him and ridden away. Despatches now reached South Africa announcing that Sir Garnet Wolseley had been appointed com- mander-in-chief of the forces, high commissioner for South-Eastern Africa, and administrator of the territories bordering on the seat of war. Lord Chelmsford was at the time just completing his ZULULAND TO ITS ANNEXATION TO NATAI. 350 arrangements for an advance upon Ulundi. It seemed as if he was to be deprived of the satisfaction of bringing the war to an end, and, as actually happened, Sir Garnet Wolseley arrived before the 4th of July, when Ulundi was reached and the final battle was fought ; but Lord Chelmsford was still in command of the column. It is estimated that about ten thousand Zulu soldiers had been killed before the end of June. Some twenty thousand more had lost heart, as the)' had not succeeded in taking a single lager during the war, and they had consequently deserted and dispersed. With from fifteen to twenty thousand who were true to him still, Ketshwayo awaited the British army at Ulundi. Lord Chelmsford formed his troops in a hollow square, upon which the Zulus dashed them- selves in vain. Beaten back by a terrible storm of bullets, and having no hope of breaking the British square by even the heaviest sacrifice, they turned to retire, when the cavalry was let loose upon them. They dispersed, never again to rally, and Ketshwayo was a fugitive seeking only concealment. After Ulundi and the military kraals near it were burned, the army fell back upon its base of supplies, and Lord Chelmsford resigned his command. The war was over, the colonial volunteers were allowed to return home, and part of the large regular force in the field was sent to England, though until Ketshwayo's person could be secured it was not con- sidered advisable to remove the whole of the troops from the country. The people — all honour to them for it — were so loyal to their chief that for many CAPTURE AND RETURN OF KETSHWAYO. ZULULAND TO ITS ANNEXATION TO NATAL. 35 1 weeks not one could be found to betray him, though thousands must have been acquainted with his hiding-place. At length, however, a man, who was threatened with death if he did not divulge the secret, pointed out a secluded kraal on the border of a forest, and Ketshwayo became a prisoner. of a forest, and Ketshwayo became a prisoner. Xo captive ever conducted himself more decorously than the fallen chief of the Zulus. He was sent a prisoner to Capetown, and, after a short confinement in the castle, had a small farm close to the one occu- pied by Langalibalele assigned to him as a residence. There he was attended by servants of his own choice, and was well cared for in every respect. and was well cared for in every respect. Zululand was divided by Sir Garnet Wolseley into thirteen districts, each of which was placed under the government of a chief independent of all the others, and nominally guided by the advice of a single British resident. But this plan of settlement did not answer at all, and in 1883 Ketshwayo was allowed to return. In the meantime he had visited England, where he was very well received, and by his sensible observa- tions and dignified deportment had acquired the favourable opinion of even' one with whom he came in contact. It was thought that after the experience he had gone through he might without imprudence be allowed to return to his own country, upon making a promise to observe conditions that would prevent his power from becoming dangerous again. his power from becoming dangerous again. Some of the people welcomed him back, but others adhered to a rival chief named Sibepu, who had found means to secure a large following. War at once broke out between them, and when Ketshwayo died ZULULAND TO ITS ANNEXATION TO NATAL. 35^ in the following year, it continued between his son Dinizulu and Sibepu. Dinizulu secured the aid of a body of farmers, in return for which he ceded to them a large tract of land, which was formed into an in- dependent state, but was soon afterwards incorporated in the Transvaal Republic as the district of Vryheid. ZULULAND TO ITS ANNEXATION TO NATAL. With the assistance of the farmers he subdued his rival, but confusion and strife still continued, so that in ] 887 what remained of Zululand was of necessity annexed to the British dominions. On the 19th of May of that year a commission was issued appointing the governor of Natal governor of Zululand, but other- wise the two countries remained separate. Zululand was divided into six districts, and a European magis- trate was placed in charge of each, with a strong body of police to support his authority. of police to support his authority. This arrangement was acceptable to most of the people, but not unnaturally Dinizulu objected to it, and he caused such disturbances and opposed British authority so strongly that his arrest became unavoid- able. In 1888 he was tried by a specially appointed court of three judges, and was sentenced to banish- ment for life. Early in 1889 with two other chiefs he was sent to the island of St. Helena as a state prisoner, and perfect order was thereafter established in Zululand. As things were now going on smoothly, and imperial control was no longer needed, on the 30th of December 1897 the six districts were annexed to Natal and the system of government made uni- form throughout. At the same time Dinizulu accepted an offer to become government induna with a salary of £500 a •j- ?4 ZULU LAND TO ITS ANNEXATION TO NATAL. 354 year and a residence at Etshovve, and on the 5th of January 1898 he reached his native country again. A good house was built for him at Etshowe, but he soon got tired of it, and went to live at a kraal bear- ing the significant name Usutu, the battle cry of his partisans. In the disturbances of 1906, which will be related in the next chapter, he was implicated, and for which he was tried by a court similar to that which condemned him twenty years before. His case excited widespread interest, and he was defended gratuitously by Advocate W. P. Schreiner, now high commissioner in London. The trial took place at Greytown, and lasted from the 19th of November 1908 to the 3rd of March 1909. He was pronounced guilty of high treason, and sentenced to four years' imprisonment from the 9th of December 1907 and to pay a fine of £100 or undergo confinement for another year. ZULULAND TO ITS ANNEXATION TO NATAL. year. In 1910 the Union government took pity on him and released him from prison on condition of his residing on the farm Rietfontein, near Middelburg in the Transvaal province, which had been acquired for the purpose. His forfeited salary of £500 a year was also restored to him as a pension. He gladly embraced the proposal, and went to live at Rietfon- tein, where in October 191 3 he died. So ended the line of the most powerful military chiefs that ever ruled in South Africa, NATAL FROM 1 8/3 TO I9IO. AFTER 1873 the progress of Natal was much more rapid than before that date. The improvement, or rather the construction, of a sheltered harbour was a matter of the greatest importance, for if this could be effected prosperity would be certain. The eminent marine engineer Sir John Coode was requested to visit the place and ascertain what could be done, and he prepared a plan that differed very little from that of Mr. Milne, needing only more blasting and dredg- ing. This was taken in hand by the government, and was carried out with the greatest success. An excellent harbour was formed, accessible to any ships frequenting the Indian ocean, where they can lie in perfect safety beside wharves and discharge or take in cargo. There is even a dry dock for the repair of damaged vessels. The construction of railways came next in import- ance, and this was carried on until there were lines open from Durban to the heart of the Transvaal Republic and of the Orange Free State, with branches northward and southward opening up the whole pro- op 355 op 355 NATAL FROM 1 87 3 TO I9IO. 35 vince to commerce. The lines were constructed by the government, and had a gauge of forty-two inches. The main branch of this system of railways has the great advantage of passing through an extensive field of coal of good quality, from which fuel can be obtained at a cheap rate. It is on the plateau at the foot of the Drakensberg, so that it is centrally situated, and the coal, which is easily worked, is conveyed to Durban mainly along a descending gradient. It is not the least important of the natural riches of Natal. Besides furnishing fuel for the railways and the towns, an ever-increasing quantity is yearly disposed of to steamers, some of which call purposely to take it in, and an export trade is being opened up. The appliances for discharging it into ships are of the most perfect kind known anywhere. The coaling-station is on the southern side of the harbour, at a sufficient distance from the quays to prevent it from being unpleasant. unpleasant. NATAL FROM 1 8/3 TO I9IO. Durban grew to be a thriving city, with great warehouses, noble public buildings, beautiful private residences at the Berea in gardens adorned with almost every variety of trees and flowering shrubs and vines, and with all the conveniences of a modern European town. The large number of Indian resi- dents strikes the eye of a stranger at once, for they have a monopoly of nearly all light occupations. In the winter months it is regarded as one of the pleasantest places in South Africa for tourists to visit, as the climate is then most agreeable and the surroundings are in every way charming. EXTENSIONS OF THE COLON 357 The area of Natal during these years was greatly enlarged. On the 22nd of April 1895 the strip of land west of the Pongolo river was annexed to the empire, and on the 30th of May of the same year a British protectorate was proclaimed over Tongaland. On the 31st of December 1897 these territories together with Zululand were annexed to Natal, thus bringing the northern border up to the Portuguese territory surrounding Delagoa Bay. Then in January 1903 the districts of Utrecht and Vryheid and that portion of the district of Wakkerstroom lying east of the Drakensberg were cut off from the late Transvaal Republic and added to Natal. This was a very important addition, on account of the Europeans residing there. Natal in 19 10 thus embraced the whole territory between the Drakensberg and the sea, from Portuguese South-Eastern Africa to the Cape Colony. It covered an area of 36,173 square miles, or nearly double its extent (18,630 square miles) in 1873. Its European population was more than five times as great as in 1873, being now nearly one hundred thousand. In 1893 responsible government, that had long been desired, was introduced. Thereafter there was a legislative council of eleven members, who were nominated by the governor and selected by him to guard the rights and interests of the Bantu, holding their seats for ten years, and a legislative assembly of thirty-seven elected members, holding their seats for four years. The franchise differed in principle from that of the Cape Colony, or representative government of NAtAL FROM 1873 TO I916. 35 any kind would have been an impossibility. The Indians, as coming from a country where such institutions were unknown, were not given the right to take part in elections. NATAL FROM 1 8/3 TO I9IO. Male British subjects, not being Indians or Bantu, who owned land worth £$Gy or who paid ^10 a year for rent, or who had lived three years in the country and were in receipt of salaries of £96 a year, were entitled to vote. Bantu who possessed any of the above qualifications, and in addition had been by their own desire exempted from tribal and subject to colonial law for seven years, might have the franchise specially conferred upon them by the governor, but this was very rarely done. Individuals of mixed European and coloured blood who lived after the manner of white people and possessed the necessary qualifi- cations were entitled to vote, but they were few in number. The principle kept in view was that a privilege of which they were incapable of making proper use should be withheld from barbarians and ignorant people. In this respect Natal followed the system of the two republics rather than that of the Cape Colony. The effect of the war between Great Britain and the republics from 1899 to 1902 was the same upon Natal as upon the Cape Colony : there was a period when the influx of gold caused extravagance in building and in style of living, that was followed by great depression, from which the colony only recovered after several years. In 1906 there were more serious disturbances with some of the Bantu than any the colony had pre- SERIOUS DISTURBANCES WITH TtiE BANTU. 3§g viously to grapple with, and being under responsible government it had to meet them without any assistance from imperial forces. assistance from imperial forces. In 1905 an act was passed imposing a poll tax of £1 yearly upon all adult male Europeans, Asiatics, and Bantu except those who paid hut- tax, and this was regarded by some of the latter as a grievance. On the 7th of February 1906 two Europeans who were sent to collect the tax from a kraal in the south of Natal were attacked and murdered. The circumstance was attended with peculiar barbarity, but condign punishment was inflicted upon the perpetrators some months later, for no fewer than twelve men suffered the death penalty for it. penalty for it. NATAL FROM 1 8/3 TO I9IO. The next to take up arms was a petty chief named Bambata, who resided about two hours' ride from Greytown, in the county of Umvoti, and had a following of not more than five thousand souls all told. In March 1906 this man paid a visit to Dinizulu at the Usutu kraal, and upon his return home set his magistrate at defiance. On the 4th of April he attacked a party of police, killed four of them, and wounded four others, after which he fled to the Nkandla forest in Zululand. There he was joined by the powerful chief Siginanda, who was over ninety years of age, Methlokazulu, who was also a man of importance, and several others. The government felt the necessity of stamping out the rebellion without any delay, and at once put a very strong force in the field. Colonel Mackenzie, NATAL FROM 1 873 TO 1 9 10. 36 who was in chief command, had nearly ten thousand Europeans and over six thousand obedient Bantu under him, with whom he not only inflicted very heavy losses upon the rebels, but overawed others that were suspected of a disposition to join them. On the 8th of June the most severe action of the campaign was fought, in which both Bambata and Methlokazulu, with fully five hundred of their followers, were killed. Shortly afterwards Sigi- nanda surrendered, and some others of note were made prisoners. Before the end of July all resist- ance had ceased, and the disbandment of the colonial forces was proceeding rapidly. It was estimated that two thousand three hundred of the rebels had lost their lives, but thirty Europeans had also been killed and thirty-seven others wounded. The cost of suppressing the rebellion was nearly £900,000. A great number of prisoners were tried, and were sentenced to varying terms of confinement with hard labour. Twenty-five of them were banished to the island of St. Helena. But in 1910 the Union government, then just established, in a spirit of clemency set them all at liberty again. Natal had shown herself capable of quelling an insurrection on a large scale, but could not venture to be as lenient with rebels as the vastly stronger govern- ment of united South Africa. New and profitable industries were at this time being developed in Natal. NATAL FROM 1 8/3 TO I9IO. First among these was the cultivation of the black wattle for the sake of its bark, which is valuable for tanning purposes. NEW INDUSTR 36 1 It was found to pay for exportation to England, and the quantity produced increased yearly. and the quantity produced increased yearly. It was ascertained that there was a good market for maize in Europe, but its growth was not so much a European as a Bantu industry. If, how- ever, the Bantu .'grow it on a large scale and sell it for exportation, the advantage to the country is great, for whatever tends to make them more industrious and to enrich them tends to the general prosperity. They have been compelled to depend much more upon agriculture than formerly, as the cattle plague, which made its first appearance in Natal in 1897, swept off nearly all their herds, and deprived them not only of the means of purchasing any articles they needed by the sale of oxen, but of the milk which formed a very large part of their food. This loss of cattle has seriously affected even their increase, as the death-rate among little children has risen considerably owing to the want of milk. Another industry that has its base at Durban is that of whale fishing, which, though it is carried on by foreigners, adds to the purchasing power of the country. During recent years the number of Europeans in Natal engaged in agriculture has been increasing, and the tendency in that direction must become greater as the peril arising from the large number of Bantu on the soil diminishes. That this will be the result of union with the other provinces of South Africa and the establishment of a govern- ment with great resources is evident. THE ORANGE FREE STATE FROM 1 854 TO 1 899. There was a president, who was the principal executive officer, and who was elected by the burghers for a term of five years. He carried out the resolutions of the volksraad, and had no veto upon its proceedings. He was assisted by an executive council. The volksraad was the supreme authority, and consisted of a member for each field- cornetcy and one for each seat of magistracy. They were elected for four years. The president and the state secretary had the right of debate, but not of voting, in the volksraad. All European males of full age born in the State were electors, and also all European immigrant males of full age who had become burghers and were in possession of unmort- gaged land to the value of ,£150, or were lessees of landed property at a yearly rental of £36, or were in receipt of a fixed yearly income of £200, or had been resident in the State for three years and pos- sessed movable property worth ,£300. Men of coloured blood who lived in all respects as Euro- peans could have the privilege to vote accorded to them by special resolution of the volksraad, and some of very dark tint living in the same way went to the polls unquestioned. But the principle was kept clearly in view that the government was to represent the civilised inhabitants of the country, and that those who were uncivilised could have no voice in its for- mation, though they were to be protected equally with electors and had the same right in courts of justice. they were justly proud of. The form of government that they adopted was republican. There was a president, who was the principal executive officer, and who was elected by the burghers for a term of five years. He carried out the resolutions of the volksraad, and had no veto upon its proceedings. He was assisted by an executive council. The volksraad was the supreme authority, and consisted of a member for each field- cornetcy and one for each seat of magistracy. They were elected for four years. The president and the state secretary had the right of debate, but not of voting, in the volksraad. THE ORANGE FREE STATE FROM 1 854 TO 1 899. The history of the world may be searched in vain for an instance of a community in a more un- enviable position than the burghers of the territory between the Orange and Vaal rivers when they were abandoned by Great Britain. They were scattered thinly over a great plain, and beside them in a mountain land like a strong fortress was a hostile tribe armed to the teeth, under the ablest chief in South Africa, exulting in its recent victory over a British army, and vastly exceeding them in number of combatants. To the policy of forming a powerful Basuto state they had been resolutely opposed, yet now they were made to bear the con- sequences of its creation. Their country was with- out roads or bridges, almost without churches and schools, so that these were to be provided as well as the ordinary machinery of government, while they received no share of the customs duties on their trade levied in the ports of Natal and the Cape Colony. That they surmounted these diffi- culties and made their state one of the most The history of the world may be searched in vain for an instance of a community in a more un- enviable position than the burghers of the territory between the Orange and Vaal rivers when they were abandoned by Great Britain. They were scattered thinly over a great plain, and beside them in a mountain land like a strong fortress was a hostile tribe armed to the teeth, under the ablest chief in South Africa, exulting in its recent victory over a British army, and vastly exceeding them in number of combatants. To the policy of forming a powerful Basuto state they had been resolutely opposed, yet now they were made to bear the con- sequences of its creation. Their country was with- out roads or bridges, almost without churches and schools, so that these were to be provided as well as the ordinary machinery of government, while they received no share of the customs duties on their trade levied in the ports of Natal and the Cape Colony. That they surmounted these diffi- culties and made their state one of the most EORM OF GOVERNME 36^ flourishing in South Africa was something that they were justly proud of. they were justly proud of. The form of government that they adopted was republican. THE ORANGE FREE STATE FROM 1 854 TO 1 899. All European males of full age born in the State were electors, and also all European immigrant males of full age who had become burghers and were in possession of unmort- gaged land to the value of ,£150, or were lessees of landed property at a yearly rental of £36, or were in receipt of a fixed yearly income of £200, or had been resident in the State for three years and pos- sessed movable property worth ,£300. Men of coloured blood who lived in all respects as Euro- peans could have the privilege to vote accorded to them by special resolution of the volksraad, and some of very dark tint living in the same way went to the polls unquestioned. But the principle was kept clearly in view that the government was to represent the civilised inhabitants of the country, and that those who were uncivilised could have no voice in its for- mation, though they were to be protected equally with electors and had the same right in courts of justice. THE ORANGE FREE STATE. 364 The fundamental law of the state, like that of all other European communities in South Africa, was the Roman as modified by the legislature of Holland before 1652. The official language was Dutch, and the courts of law were after the Dutch pattern, though considerably modified. In each district there was a landdrost, with a body of heemraden to assist in settling important cases, and over all was a supreme court of judges, who must previously have been qualified barristers. When the British flag was withdrawn the com- munity was split into factions, but the common danger forced them to unite in choosing a president acceptable to Moshesh. Mr. Josias Hoffman, a farmer who was intimately acquainted with the great chief and on excellent terms with him, was therefore elected. During his short tenure of office, however, the burghers felt that concession to the Basuto power was carried further than was con- sistent with the dignity of an independent state, and early in 1855 the volksraad took advantage of his having made a present of a keg of gunpowder to Moshesh without reporting the circumstance and clamoured so loudly against him that he was compelled to resign. compelled to resign. Mr. Jacobus Nicoiaas Boshof, a man of education, of high moral character, and of considerable ability, was then elected president. THE ORANGE FREE STATE FROM 1 854 TO 1 899. Having had a training in official work in the civil service of the Cape Colony, he was able to put the different departments of the government in good order. But from the day of his assuming duty he was so incessantly harassed by CAUSES OF THE BASUTO WAR 365 the all-important question with Moshesh that he could do little or nothing else for the general welfare of the country. Moshesh's object, from a Bantu point of view, was so praiseworthy that his followers were ready to do everything in their power to promote it. He wished to recover for his tribe the whole of the territory south of the Vaal and west of the Drakensberg that had been in occupation of black people before the wars of Tshaka. The remnants of the tribes that in olden times had occupied the ground were now his followers, and he wanted the soil that once was theirs. Sir George Napier had given him by treaty a large portion of it, and that much, at any rate, he was determined to have. The object of the Free State government and burghers was to retain the boundary fixed by Sir Harry Smith, which, in their opinion, was a fair one. When white people moved into the territory it was a vast waste, and if they had not come Moshesh could never have had a quarter of the land that was now in his possession. Certainly Bantu had at some former period occupied ground nearly as far west as Bloem- fontein, but they had irrecoverably lost it during the Zulu wars. To admit Moshesh's claim would be to consent to the annihilation of the state, and the burghers had no mind for that. Here, in brief, was the cause of the long and desperate struggle between the Orange Free State and the Basuto tribe. the Orange Free State and the Basuto tribe. For several years Moshesh directed his efforts towards the south, leaving the farmers on his other borders undisturbed. In that direction the herds of THE ORANGE FREE STATE. 36 the white people were plundered mercilessly by his followers, and as a rule he would give no redress. Sir George Grey once tried to arrange matters amicably between the two parties, and through his agency an agreement of peace and friendship was signed ; but Moshesh did not long observe it. THE ORANGE FREE STATE FROM 1 854 TO 1 899. At length actual possession of occupied farms was taken by parties of armed Basuto, and hostilities could be staved off no longer. In March 1858 the burghers of all parts of the state were called out, and entered Basutoland in two divisions, the object being to try to confine the war to the enemy's country. But Moshesh was much too skilful a strategist for their commanders to cope with. He kept them closely occupied until after some severe fighting they arrived in front of Thaba Bosigo, where they learned that swarms of his light horsemen were ravaging their farms. Before them was a mountain stronghold which could not be taken by storm, and they had not the means of laying siege to it. Under these circumstances the burghers dis- persed, and made the best of their way to the localities where they had left their families. President Boshof had already applied to the South African Republic for aid, but as the burghers of the northern state were holding out for special terms of union, and there was no time to be lost, he requested the mediation of Sir George Grey. Moshesh con- sented to an armistice, and promised to abide by the decision of the governor. He was wise enough to recognise that if he pushed his advantage too far he would have to deal with the northern republic as END OF THE BASUTO WAR ify well as the southern, and he felt certain that the governor would give him, as the conqueror, a good strip of territory. strip of territory. Sir George Grey accepted the office of mediator. He took from Moshesh a far outlying mission station which was a kind of semi-independent fief of the Basuto chieftainship, but gave him a large extent of territory south of Sir Harry Smith's line. Thus the first struggle with the Basuto ended in very serious loss to the Orange Free State. Shortly after this Mr. Boshof resigned, and was succeeded as president by Mr. Marthinus Wessel Pretorius, son of the famous commandant-general. He represented a strong party that desired the union of the two republics, but as there were many con- flicting interests in the way, besides the declaration of the high commissioner that union would dissolve the conventions with Great Britain, every attempt at amalgamation failed. amalgamation failed. During these years the Free State was continually growing stronger. THE ORANGE FREE STATE FROM 1 854 TO 1 899. The Griqua captain Adam Kok sold his territorial rights to the republic, his people sold their ground to farmers from the Cape Colony, and then the whole clan moved away to a new country below the Drakensberg and south of Natal, given to them by Sir George Grey. With the ex- ception of a little tract of almost unoccupied land belonging to the Griqua captain Nicholas Waterboer between the Modder and Orange rivers and a loca- tion belonging to the Barolong chief Moroko, the whole territory between the Vaal and the Orange up to the Basuto border was now in possession of white THE ORANGE FREE STATE. 36 people, and sheep farming and cattle rearing could be carried on in a large portion of it with greater profit than in any other part of South Africa. profit than in any other part of South Africa. Along the Basuto border, however, there was a continual state of unrest. Moshesh had conquered the clans on the northern bank of the Caledon, and had annexed their ground. The white people main- tained that the boundary should remain as before, but the chief said contemptuously that he had never agreed to it, and he showed clearly that he would not respect it. President Pretorius did all that was possible to induce him to act fairly in the matter, but in vain. His people pressed across the line, settled on farms, and plundered the country for miles in front of them. In 1865 Advocate Jan Hendrik Brand was elected president. Of him it may be said that South Africa knows no worthier name, for no one has ever con- tributed more to the welfare of the people than he. Sir Philip Wodehouse had succeeded Sir George Grey as governor of the Cape Colony and her Majesty's high commissioner for the regions beyond. President Brand requested him to mark off the northern boundary between the Free State and Basutoland, and the volksraad empowered him to alter Sir Harry Smith's line if he saw fit to do so, as they were willing to lose some ground if only they could secure peace. Sir Philip was no lover of republics, and he never concealed his dislike of the South African farmers ; but in a matter of this kind he could be depended upon to act with the strictest justice. Moshesh very reluctantly agreed to abide PRESIDENT BRAND. THE ORANGE FREE STATE FROM 1 854 TO 1 899. 369 by his decision. He visited the country, carefully inspected it, heard the arguments on both sides, and after much consideration confirmed Sir Harry Smith's line. The president then called upon Moshesh to withdraw his followers from the farms they had taken possession of, and upon the chief's failure to PORTRAIT OF PRESIDENT BRAND. PORTRAIT OF PRESIDENT BRAND. do so, the burghers were called out to drive them back by force, when open war began. 25 back by force, when open war began. The laying waste of a large extent of territory in the Free State by Basuto horsemen could not be prevented, and a number of people were massacred in a shocking manner ; but, on the other hand, the 25 THE ORANGE FREE STATE. 37 burgher forces were victorious in several engagements and took some mountain strongholds by storm. Thaba Bosigo, however, resisted every attack upon it. After ten months' fighting Moshesh asked for peace, and offered to cede a considerable part of his country, but a year later he declared that he had only done so in order to secure a harvest. The president and the burghers were deceived, and in April 1866 terms were agreed to and a treaty of peace was signed. signed. His gardens were planted, and when his harvests were gathered Moshesh threw off the mask. He declined to fulfil the terms of the treaty, and in July 1 867 President Brand was obliged to call the burghers again to arms to compel him to observe his engage- ments. Both sides recognised that the struggle must now be a final one, and each put forth its utmost strength. But Moshesh no longer possessed the mental vigour of his earlier years, and in his tribe there was no one who could take his place as a strategist. His sons were mere ordinary barbarians. Soon fragments of the tribe began to drop off and move over the Drakensberg. Then one after another all the Basuto strongholds fell, except Thaba Bosigo, Moshesh's own mountain, which defied attack. The granaries were destroyed, and the bulk of the tribe, driven into the mountains, was suffering severely from hunger and disease. The Free State was in a fair way of being able very shortly to dictate its own terms when Sir Philip Wodehouse interfered. THE ORANGE FREE STATE FROM 1 854 TO 1 899. At Moshesh's request he proclaimed the Basuto tribe British subjects, and DISCOVERY OF DIAMOND 37 1 sent an armed force to protect them. British interests, he declared, would not permit of their being dispersed over the country as fugitives. Naturally the people of the Free State regarded this action as unfriendly and unfair, and the volksraad sent deputies to England to protest against it ; but the imperial authorities left the matter entirely in the governor's hands. The president then tried to secure as good terms as possible, and in February 1869 an agreement was concluded at Aliwal North by which the Free State obtained all the land north and west of the Caledon and south of a line almost identical with that of Sir Harry Smith between the Caledon and the Orange. and the Orange. The republic sorely needed rest when the struggle with the Basuto was over, though it was less exhausted than its opponent. Paper money had been issued to a large amount, and the notes were greatly depreciated in value. Farming operations had been neglected, and individuals as well as the state had been compelled to incur heavy debts. There was hardly a household in the land which did not bear evidence that a crisis of no ordinary nature had been experienced. The loss of life too had been heavy in proportion to the population. But the hearts of the people beat high, and burghers and government alike set to work resolutely to repair their losses. They had not time to get on their feet again when a new difficulty arose in the discovery of rich diamond mines in the western part of the state. An account of this has been given in another chapter, as well THE ORANGE FREE STATE. 3? as of the occupation of the mines by Great Britain and their ultimate annexation to the Cape Colony. The difficulty was at length settled by the payment of ^"90,000 as a solatium to the Free State, with which the public debt as represented by bluebacks was reduced. The sore feeling entertained by the burghers passed away, and they began to reflect that perhaps after all it was better for them to be relieved of the responsibility of maintaining order among the diggers. THE ORANGE FREE STATE FROM 1 854 TO 1 899. A diamond mine at Jagers- fontein had been left to them, and it was turning out much richer than had once been anticipated. Then they had all the advantages which the other mines offered as markets for farm produce, so that they might have a good deal of gain with no risk. After this settlement the Free State enjoyed constant peace. Roads, bridges, and good public buildings were constructed, and an excellent system of public schools was maintained by the government. In course of time railways were constructed, con- necting the state with Capetown, Port Elizabeth, East London, Durban, and Lourenco Marques, and a customs union with the Cape Colony was entered into. The boundaries of the republic on all sides were undisputed, and it had no longer independent tribes within its borders. The Barolong clan at Thaba Ntshu was the last to occupy that position. In 1880 the chief Moroko died at a very advanced age, and the succession was disputed by two men named Tsepinare and Samuel. Most of the people favoured the former, and when he was killed by THE ORANGE FREE STATE. 37 Samuel they appealed to President Brand to take them over as Free State subjects. This was the only way to restore order, so the president complied with their wish, and the ground they lived on was kept as a reserve for their use. It was named the district of Moroka. President Brand was elected again and again until 1888, when he died in office. Mr. F. W. Reitz, previously chief justice, was then chosen to fill the vacant place, which he did to the entire satisfaction of the people. Owing to ill health he was obliged to retire in 1895, and early in 1896 was succeeded by Mr. M. T. Steyn. The principal event of this time was a close alliance, defensive and under certain conditions offensive, with the South African Republic, which was then in a position of great difficulty, and with whose burghers those of the Free State naturally had strong sympathy. The industries of the state still remained almost entirely pastoral and agricultural, but valuable coal fields were beginning to be worked, and there were diamond mines at Jagersfontein and Koffyfontein, the last named, however, not being of great im- portance. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. THE vast tract of land north of the Vaal, that became the property of the emigrant farmers after the expulsion of Moselekatse, contained ground suit- able for almost every variety of agricultural and pastoral industry, and, though the circumstance was then unknown, in mineral wealth it was not surpassed by any country in the world. Its eastern and northern valleys, well watered and of great fertility, had for a time a strong attraction for settlers, but experience proved them to be less healthy than the open highlands, and they were therefore partly aban- doned. In some places the tsetse fly abounded, and this scourge of domestic cattle prevented settlement in its neighbourhood until the large game was ex- terminated, when it disappeared. The fever too, that was once so prevalent on the borders of forests and streams in the lowlands, in course of time became almost unknown in the same localities if the ground was cultivated and the rank grass burnt off before it began to decay. 375 375 375 375 THE SOUTH AFRICAN REPUBLIC. 37 The farmers were only fifteen or sixteen thousand in number, all told, so they naturally selected what appeared to them the choicest spots, and no one considered it worth his while to settle on the great plains of the west. There was no such thing as union among them. An attempt was made to form a kind of common government, by the election of a single volksraad for legislative purposes, but with four executive heads, one for each of the principal factions. This system, as may be imagined, was accompanied by much disorder, and was soon suc- ceeded by four republics, independent of each other : Potchefstroom, Zoutpansberg, Lydenburg, and Utrecht. Matters were not mended by this arrangement, and it may almost be said that the white people beyond the Vaal were without govern- ment at all. A notable evil that resulted from this condition of things was that the outskirts of the occupied area offered a refuge to vagabonds of every stamp, who resorted to them from other parts of South Africa. Men capable of the most abominable cruelty and meanness, but possessing the quality of brute courage, roamed along the frontier nominally as hunters and traders, and their lawless deeds were attributed by people at a distance to the whole community. people at a distance to the whole community. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. When Europeans first entered the country, it was in a similar condition to Mashonaland in 1890. North and west, as far as it was known, the Bantu tribes had been destroyed by the Matabele, and only a few wretched remnants were living either along the margin of the Kalahari desert or among almost LA WLESSN m inaccessible mountains. The greater portion of the territory south of the twenty-second parallel of lati- tude was literally without inhabitants, for Mosele- katse's bands were in the habit of traversing it yearly in one direction or other, and no clan could live in their way. The arrival of the white people and the flight of the Matabele gave new life to the dwellers in the mountains and deserts. They could come out into the open country once more, and make gardens and sleep in safety. The Europeans were masters and owners of the land, but in accordance with the ancient Dutch custom, they permitted each little Bantu community to be governed by its own chief in all matters that did not affect the ruling race. The kraals were made subject to a labour tax, and under a strong government no better tax could be imposed upon a barbarous people. But under the weak rule of the emigrant farmers the system was liable to great abuses, though the Bantu thought lightly of it until the dread of the Matabele was forgotten. Life was now safe, and the occupants of the kraals were multiplying at a prodigious rate, besides which fugitives were coming in from the regions beyond the Limpopo, where Moselekatse was lord. Ten years passed away, and the clans had become so strong that they began to chafe under the re- straints imposed upon them by the white men and to aspire to independence. The anarchy and strife among the Europeans appeared to give them the opportunity they wanted. But among themselves THE SOUTH AFRICAN REPUBLIC. 37 also there was the remembrance of ancient feuds, which caused so much jealousy that combination was impossible, and instead of rising all together, it was in succession that the most disaffected among them took up arms. Then, too, as will be seen on a much more memorable occasion at a later date, in presence of an opponent the farmers stood shoulder to shoulder, and were therefore able to suppress the various risings against their authority. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. various risings against their authority. These disturbances were brought prominently to the notice of the English people by the reverend Dr. Livingstone, the greatest explorer of modern times, who was then a missionary with the Bakwena under the chief Setsheli, and whose house and furni- ture were destroyed during the war. Dr. Livingstone was a strong partisan of the Bantu, and did his utmost to oppose the claim of the emigrant farmers to dominion over the clan with which he was living, so that his statements are those of an advocate rather than those of a judge. He represented Setsheli as wholly in the right, and the farmers as wholly in the wrong ; but any impartial writer who examines Setsheli's own account of the matter, as given by himself personally to the governor in Capetown, must come to a different conclusion. War cannot be carried on without cruelty, but in these contests acts were sometimes performed by the Europeans which exceeded the limit regarded as permissible by civilised nations. It should be re- membered, however, that the provocation on such occasions was very great, as, for instance, when white women and children were murdered in cold civil wa 379 blood, or when corpses were mutilated, or captives put to death by torture. Men belonging to the most refined circles in Europe would probably retaliate under such circumstances as cruelly as the emigrant farmers did. Early in 1 857 the Potchefstroom faction adopted a new constitution, under which Mr. Marthinus Wessel Pretorius became president, though with no other power than to carry out the resolutions of the volksraad. In the following year Zoutpansberg gave in its adhesion to this constitution, and, in i860, Lydenburg and Utrecht, previously united, were also incorporated, so that the whole country north of the Vaal became a single republic. The different factions now began to strive for the supreme power in the state, and a civil war broke out, in which some blood was shed. Peace was restored in May 1864, when Mr. Pretorius was accepted by all parties as the legally elected president, and Mr. S. J. Paul Kruger as commandant-general or military head. Kruger as commandant-general or military head. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. Meantime the Baramapulana tribe, which was living in a mountainous tract of land in the north of the republic, had become very strong in number, owing to an influx of broken clans from beyond the Limpopo. It was in possession of a good many guns, procured from the vagabond whites in the neighbourhood, and was disposed to resent any inter- ference with its actions. In a feud a brother of the chief was obliged to flee, and was protected by the government, a circumstance which greatly annoyed his opponents. In April 1865, when searching for a fugitive offender, some of the lawless Europeans and THE SOUTH AFRICAN REPUBLIC. 38 a party of blacks who were assisting them committed acts of great violence upon the outposts of the tribe, and a general war was brought on. and a general war was brought on. For more than three years the republic strove in vain to subdue the Baramapulana. There was no money in the treasury, and the government was actually at one time unable to raise funds sufficient to pay for the carriage of ammunition from Durban. The burghers of the southern part of the state re- fused to take part in the war. Commandant-General Kruger did all that man could do with the slender means at his disposal, but he was at length obliged to withdraw discomfited. The village of Schoe- mansdal, the centre of the ivory trade and the residence of a landdrost and a clergyman, was abandoned by its inhabitants when the feeble com- mando retired, and was afterwards burnt by the enemy. The Europeans were obliged for their safety to withdraw from a large part of the district of Zoutpansberg, to which they were never able to return. The Baramapulana, however, felt the want of commercial intercourse, and in July 1868 ex- pressed a desire for a renewal of friendship, at the same time offering to pay tribute, when peace was gladly made on conditions which by no means secured the absolute supremacy of the republican government. government. The white people had thus lost ground, and the fact of their having done so made it more difficult than before to preserve order among the Bantu farther south. In one respect only the country showed signs of progress : in the number of churches DIFFICULTIES WITH THE BAROLOXG 38 1 built and clergymen engaged. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. Yet even in religious matters there was constant strife among the sections of what outsiders can only regard as one church, so trifling are the differences that break it into distinct communions. A generation had grown up without a knowledge of books or of events beyond their own little circle. The rivers were unbridged, there were no public offices worthy of the name, the treasury was always empty, and the salaries of the officials, trifling as they were, could seldom or never be paid when they fell due. Commerce was carried on chiefly by means of barter, as gold and silver were exceeding scarce. Still on the farms anything like want was unknown, for the flocks and herds throve and in- creased in the rich pastures, and the fertile soil produced grain and vegetables and fruit in abun- dance. The war with the Baramapulana was hardly con- cluded when fresh difficulties arose through the Barolong of Montsiwa and other clans on the west setting up a claim to independence and to the possession of a territory of immense extent. The republic was not in a position to assert its authority by force of arms, and indeed the matter was hardly considered worth much notice until the discovery of diamonds along the lower Vaal gave importance to the claim. Then President Pretorius and her Majesty's high commissioner for South Africa arranged that it should be settled by arbitration, and each party appointed a representative to form a court, with Governor Keate. of Natal, as final umpire. The manner in which the case for the THE SOUTH AFRICAN REPUBLIC. 38 republic was conducted has been related in another chapter. Governor Keate's award gave to the tribes the independence and the territory that they claimed, and even took from the government at Pretoria a large district that had been occupied by white people ever since the great emigration. As soon as the award was known President Pretorius was obliged to resign, for the volksraad maintained that he had exceeded his authority in making the agreement with the high commissioner, and declared that they were not bound by his action. The high commissioner, however, announced that he would enforce the award, though he did not take possession of the territory cut off from the republic by it. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. And now there was a general cry that a clever man, capable of conducting business on equal terms with the queen's representative in Capetown, must be found to fill the office of president. The reverend Thomas Francois Burgers, a clergyman who had abandoned the orthodox church and whose name was then prominently before the public on account of the skilful manner in which he had conducted some difficult cases in the law courts of the Cape Colony, seemed to possess the requisite ability, and he was elected by a nearly unanimous vote. Mr. Burgers was an able and an active man, with large persuasive powers, but he was a dreamer. He dreamed of a powerful and prosperous republic, with colleges and telegraphs and railways, with a high name among the nations of the earth ; and he imagined that it could be formed off-hand out of a few thousand uneducated men with seventeenth REBELLION OF THE BAP EDI 383 century ideas and such immigrants as he could induce to join him from Holland. Two years after his election he induced the volksraad to send him to Europe to negotiate a loan for the purpose of constructing a railway from Pretoria to Delagoa Bay and to engage teachers for a number of state schools. In Holland ^"90,000 was subscribed towards the loan, and with the money railway material was purchased and sent out to rust and rot away at Lourenco Marques, for no more could be borrowed. A superintendent-general of education and a few other officials were engaged, with whom the president returned to Pretoria, to find that during his absence the Bapedi tribe, under the chief Sekukuni, that occupied a wild and rugged tract of land in the valley of the Olifants river, had acted in a manner that no government could tolerate, A large commando was called out to punish the insurgents, but the burghers assembled in fear and trembling. The president was to lead it in person, and as he was in religion an agnostic, they — with their thorough orthodox creed — feared much that the blessing of God could not rest upon the enterprise. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. So strong had this feeling become throughout the country that a large number of families, rather than remain under his government, were moving away to seek a new home beyond the Kalahari desert, and were even then marking the road to Mossamedes, in the Portuguese province of Benguela, where they ultimately settled, with a line of graves showing the terrible sufferings of their march. The passionate THE SOUTH AFRICAN REPUBLIC. 384 feeling at the time of his election had passed away, and hardly anything was now remembered except the failure of many of his plans. PORTRAIT OF SEKUKUNI. PORTRAIT OF SEKUKUNI. One strong place was taken, which the president in overdrawn language wrote of as the Gibraltar of the south, but this success did not give heart to the farmers. An attempt to take another stronghold failed chiefly owing to the conduct of the burghers REBELLION OF THE BAPED 385 themselves, and then there was a perfect stampede homeward, which all the efforts of Mr. Burgers could not prevent. Some days later the fugitives reached Pretoria, and no hope of suppressing the rebellion speedily was left. speedily was left. The volksraad was hastily convened, when it was resolved to engage men wherever they could be obtained, at £$ a month, rations, and a farm of four thousand acres when the disturbance was quelled. To meet the expense heavy war taxes were imposed. But the country was quite unable to bear this strain. The ordinary charges of government and the interest on the public debt could not be met, much less an additional burden. And so the whole administrative machinery broke down. The republic was really in a pitiable state, without money or an army, with rebellion triumphant, and a general election approaching that was feared might be attended with civil war. While things were in this condition Sir Theophilus Shepstone, previously secretary for native affairs in Natal, was sent by the British government as a com- missioner to Pretoria with very large powers. It is admitted by every one that a country is entitled to interfere with a neighbour whose weakness is a cause of common peril ; but whether Great Britain was justified in this instance in taking possession of the South African Republic is a question upon which opinions differ. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. One of the reasons assigned by Sir Theophilus Shepstone for the action which he took was that the territory was in danger of being overrun by the Zulus, and if that was really so, the 26 26 THE SOUTH AFRICAN REPUBLIC. 38 circumstance would go a long way to support his proceedings. But the farmers never expressed a fear of such a danger, and always alleged that they could repel Ketshwayo's armies much more easily than besiege a fortified mountain stronghold. The Zulu chief at the time was trying to play off the republic against Natal, and his declarations to one party concerning the other cannot be regarded as evidence, though the British commissioner seems to have attached much value to them. Further than this, the residents of the villages, who were principally English and Germans, requested the commissioner to declare the country a British dependency, as the only remedy against anarchy, and the farmers did nothing to oppose him and his slender escort. The government managed to patch up a kind of peace with Sekukuni, but otherwise matters remained in the condition described until the 12th of April 1877, when Sir Theophilus Shepstone issued a proclamation declaring the country a British possession, and there- upon assumed supreme control, the president retiring under protest. A considerable military force now entered the Transvaal territory, as the country was renamed, and apparently the new government was firmly established. Trade revived, money flowed in, and property of every kind increased in value. But the farmers were dis- satisfied with the loss of their independence, and sent Mr. Paul Kruger and Dr. Jorissen to England to endeavour to get the annexation withdrawn. The deputation failed in its purpose, and at that time the British ministry appear to have believed that a large BRITISH RU 38? proportion of the people of the territory — if not the majority — were in favour of English rule. As a proof that this was not the case, memorials were sent round against the annexation, and received the signatures of over six thousand five hundred individuals, represent- ing practically the whole rural population. Another deputation, consisting of Messrs. Paul Kruger and Pieter Joubert, with Mr. Eduard Bok as secretary, was now sent to England, in hope that with so strong an argument in its favour it would meet with success. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. But it returned disappointed, and there- after repeated declarations were made by the highest officials in South Africa that under no circumstances would the British flag be withdrawn from the Transvaal. Sir T. Shepstone was personally not greatly dis- liked, and if any one could have made the farmers contented under English rule he would have done it. But in March 1879 he was succeeded as adminis- trator by Sir Owen Lanyon, a man of haughty dis- position, who was incapable of even attempting to conciliate the people of the country. The feeling now rapidly gained ground that if peaceable means to obtain the restoration of independence did not soon succeed, an appeal to arms ought to be made. The women of South Africa have always had great influence in public affairs, and on this occasion their voice was decidedly in favour of war. Mothers en- couraged their sons, wives their husbands, to act as men, and if they were beaten they could die the death of patriots or move away to the unknown north as their fathers had done before them. 38 THE SOUTH AFRICAN REPUBLIC. At this time Sekukuni again gave trouble, but Sir Garnet Wolseley with a strong body of troops and a band of Svvazis marched against him, in- flicted great damage upon his tribe, and brought the chief himself a prisoner to Pretoria. Shortly after this event intelligence reached the country that Mr. Gladstone had succeeded the earl of Beacons- field as prime minister of England, and as the new premier when in opposition had denounced the annexation as unjust, the farmers not unnaturally thought that he would give them back their inde- pendence. For a while therefore the agitation almost ceased. Some of the troops were withdrawn from the territory, and Sir Garnet Wolseley, having been relieved as commander-in-chief by Sir George Colley, returned to Europe. returned to Europe. As soon as it was known, however, that Mr. Gladstone declined to withdraw the British flag, the general discontent came to a head. An attempt to seize the waggon of a farmer who refused to pay a tax brought a number of his friends to the rescue, and the officials at Potchefstroom, though supported by a strong military force, were openly set at defiance. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. A great meeting took place at Paardekraal, where Krugersdorp now stands, and after several days' dis- cussion it was resolved to commit their cause to the Almighty God and live or die together in a struggle for independence. Messrs. S. J. Paul Kruger, M. W. Pretorius, and Pieter J. Joubert were elected a triumvirate to conduct the government, and the volksraad resumed its functions as the supreme legislative power. It was decided that Heidelberg STRUGGLE FOR INDEPENDENC 389 should be the capital until Pretoria could be re- covered, and there, on Dingana's day, the 16th of December 1880, the flag of the republic was hoisted again. again. The act certainly proved that the European blood has not degenerated in courage by removal to South Africa, as many persons had previously assumed. And here it may be asked how it was that the same men who dared not face danger in the commando under President Burgers went through this war for independence with the bravery and devotion of ancient Spartans, yet afterwards claimed no glory for what they had done. The reply is short : religion caused the change. In one instance they believed that the Almighty was against them because their leader was not of the true faith, in the other they believed most thoroughly that the Almighty was with them, guiding and strengthening them in the unequal fight. It was this, and this alone, that turned the fugitives from Steelpoort into the men of Majuba hill. of Majuba hill. On the same day that the flag was hoisted the first blood was shed. A party of burghers, under Com- mandant Cronje, went to Potchefstroom to have a proclamation printed, and was fired upon by the soldiers there, when one of them was badly wounded. Colonel Winsloe, who was in command of the soldiers, had a camp outside the village, and had also fortified the landdrost's office and some adjoining buildings, in which a garrison was stationed under Major Clarke. Commandant Cronje returned the fire, and then laid siege to the buildings occupied by Major Clarke, who THE SOUTH AFRICAN REPUBLIC. 39 after holding out two days was obliged to surrender. Colonel Winsloe held the camp throughout the war, and only surrendered it after an armistice was entered into. Disaster after disaster now attended the British arms. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. Colonel Anstruther was directed to march from Lydenburg with two hundred and sixty-four men to reinforce the garrison of Pretoria, and was warned that he might meet with resistance on the way, but having a very poor opinion of the fighting powers of the farmers, he took no precautions whatever. On the 20th of December he was marching carelessly with a long waggon train, when at Bronkhorst Spruit, thirty-eight miles from Pretoria, he suddenly found himself in front of a force of about the same number of farmers under Commandant Frans Joubert. The commandant demanded that he should proceed no farther, and upon his replying that he would go on, a volley was poured in by the farmers. The soldiers made a very feeble resistance, and in a few minutes so many were disabled that the colonel — who was mortally wounded — was obliged to surrender. mortally wounded — was obliged to surrender. From the garrisons in Natal Sir George Colley now collected a body of rather over a thousand men, and set out to assist the troops in the Transvaal, who with the loyalists were beleaguered in the various villages. On learning of this movement, Commandant-General Pieter Joubert, who was one of the triumvirate, entered Natal with a force superior in number, and occupied a strong position at Lang's Nek, on the road along which the British general must march. On the 28th BRITISH DISAST 39 1 of January 1881 Sir George Colley attempted to force the passage of the Nek, but was beaten back with heavy loss. He then fortified a camp at Mount Pros- pect, four miles distant, and awaited reinforcements which were on the way from England. On the 8th of February with nearly three hundred men General Colley left his camp to patrol the road towards Newcastle, and near the Ingogo river was drawn into an engagement with a body of farmers under Commandant Nicolaas Smit. Up to dusk neither side could claim victory, but when night fell the remnant of the English patrol returned to camp in a heavy fall of rain, leaving two-thirds of those who went out in the morning dead and wounded on the field. In the three engagements here mentioned the British loss was about six hundred men, nearly half of whom were killed. The farmers had seventeen men killed and twenty-eight wounded. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. Military critics attribute the difference largely to steady aim and skill in shooting on one side, the farmers attribute it entirely to the working of Providence in their favour. The crowning disaster was yet to come. During the night of the 26th of February General Colley left his camp with six hundred men, and climbed to the top of Majuba hill, posting two pickets on the way. From this position he hoped to command the farmers' camp at the Nek two thousand feet below, and when at dawn on the next morning Commandant-General Joubert observed the soldiers on the crest above him, he at once realised the danger. Some hundred and THE SOUTH AFRICAN REPUBLIC. 39 fifty volunteers now offered to try to take the hill, and actually made their way up in face of the superior force above, the bullets passing over them as if they were charmed. A little before noon seventy or eighty men reached the crest at different points, and then the soldiers were seized with a panic and fled. Ninety-two killed, one hundred and thirty-four wounded, and fifty-nine prisoners repre- sented the British loss that day, against one man killed and five wounded on the farmers' side. General Colley himself was among the slain. General Colley himself was among the slain. In the meantime the soldiers and British adherents in the Transvaal villages were closely besieged, and a good many lives were lost in sorties and skirmishes, but none were reduced to surrender. After Sir George Colley's death, Sir Evelyn Wood took command of the English forces, and as troops were fast arriving in Natal, he soon found himself at the head of twelve thousand men. But now came instructions from the imperial government not to advance, and on the 5th of March an armistice was concluded between the general and the triumvirate. This led to the arrangement of terms of peace and the restoration of semi-independence to a portion of the former republic, Swaziland being cut out and the boundary on the western side being drawn in so as to exclude nearly the whole of the Betshuana and Korana clans living there. It was to be under the suzerainty of her Majesty the queen, who was to have control of all its ex- ternal relations and of all legislation affecting the Bantu residents. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. On the 3rd of August a document THE CONVENTION OF PRETORI 393 termed the convention of Pretoria was signed to this effect, and five days later the British forces were withdrawn. A volksraad was elected, but for some time it was doubtful whether the convention would be ratified. The condition of things which the Sand river convention laid down was what the members wanted and what they believed they had a right to, not such shackled privileges as were described in the convention of Pretoria. But it was clearly explained to them by the triumvirate that their only choice in the matter was that or renewal of the war with Great Britain. The volksraad then yielded, and on the 25th of October 1881 the convention was formally ratified, but to say that it was approved of would be incorrect. Mr. Kruger was shortly afterwards elected presi- dent of the vassal state, which entered upon a career beset with difficulties. Things could not work smoothly under such an arrangement, and the British government soon came to consider that a really friendly independent Trans- vaal republic would be better than such a discon- tented inimical vassal state as then existed. So in October 1883 a deputation, consisting of President Kruger, the reverend S. J. Dutoit, superintendent- general of education, and Commandant Nicolaas Smit, proceeded to England and concluded an agreement by which the obnoxious suzerainty was withdrawn and all the other restraints upon inde- pendence were removed, though the full extent of ground claimed by the old republic was not given back, because England regarded it as imperative THE SOUTH AFRICAN REPUBLIC. 39 on her to retain the highway to the centre of the continent. THE SOUTH AFRICAN REPUBLIC FROM 1 852 TO 1884. On the 27th of February 1884 a docu- ment known as the convention of London was signed, which restored independence to the South African Republic, with limited boundaries, and in the words of the earl of Derby, who was then secretary of state for the colonies, its administra- tion " was left free to govern the country without interference, and to conduct its diplomatic corre- spondence and shape its foreign policy, subject only to the requirement that any treaty with a foreign state should not have effect without the approval of the queen." approval of the queen." Even this convention did not fully satisfy the Transvaal people, because of the restriction on their freedom to make treaties with foreign states, and more particularly because it did not restore to them all the land on their south-western border that they believed themselves entitled to. For these reasons many of the members of the volksraad refused to agree to it, and it was only owing to President Kruger's influence that it was at last ratified. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. An event of primary importance, not only to the Transvaal Republic, but to all South Africa, was the discovery of gold on the Witvvatersrand, the water- shed from which the streams flow northward on one side and southward on the other. Ever since 1872 two or three hundred gold-diggers had been working at Pilgrim's Rest and other localities in the district of Lydenburg, but for many years the quantity of the precious metal found was too small to attract the attention of people in other parts of the world. In 1885 richer and more extensive fields were opened up, and a town named Barberton, which for some months was the busiest place in the country, sprang into existence. the country, sprang into existence. In 1886 some gentlemen named Struben, who had been for over a year prospecting on the high belt of land between Pretoria and the Vaal river, proved the existence of gold there in payable quantities. It was soon found that the extent of the gold-bearing reefs was very great, though some parts were much 395 6 THE SOUTH AFRICAN REPUBLIC FROM 1884-99. 396 richer than others. It was not a field for individual diggers, because the precious metal was embedded in rock or conglomerate, which had first to be removed, then to be crushed by powerful machinery, and finally to be treated by different processes, all of which required large capital and special skill. But companies were rapidly formed, and for a distance of thirty miles along the reef mining rights were secured and men were set to work. In the centre of this line, 5,600 feet above the level of the sea, rose almost as by magic the largest city in South Africa, the world-famed Johannesburg, a city of massive edifices, many of architectural beauty that would not be out of place in any capital of Europe, a city of parks, and luxurious clubs, and magnificent hotels, and churches of all denomina- tions, with trams and telephones and all other appliances of modern times. To it people migrated from Europe and America, with ideas and habits different from those of the people of the country. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. Every year saw the production of gold increasing, until it has now, in 19 16, with that from Lydenburg and Rhodesia, reached nearly forty million pounds sterling, and South Africa furnishes more of that metal than any other country in the world. metal than any other country in the world. The republic possessed also iron in the greatest abundance, coal of good quality and practically limitless in quantity, silver, copper, lead, and several other minerals. The people brought into the country by the attrac- tion of gold were mostly English-speakers, and between their interests and those of the farmers DIVERGENT VIE 397 there was a great difference. The farmers regarded the country as theirs, bought with the blood of their kindred, and converted by them from a waste, such as Moselekatse had made it, into a civilised state covered with peaceful homesteads and places for worshipping God. They were determined not to surrender their right of governing it to strangers, who, if admitted into the legislature, would certainly use their power to subvert the constitution, if not the independence of the republic. Before 1877 the fran- chise was probably the lowest in the world, but now it was raised so that foreigners, without renouncing their allegiance to the governments under which they were born and without long residence, could not become voters. The burghers regarded the mineral wealth of the country also as something in which the state was entitled to share largely, and which should not there- fore be removed without leaving in its stead public works and buildings and improvements of other kinds. With this view, taxation was so arranged as to bear much more heavily upon the mining than upon the farming industry. The great mass of the miners and speculators they regarded not as men who had come there to make the republic their permanent home, but as men who had come there to make as much money as possible in as short a time as possible, and then to return to their own countries to spend it. The gold, though plentiful, was not inexhaustible, and when the mines were worked out, these strangers would certainly not remain. 39§ THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. The newcomers, on the other hand, maintained that as they were the wealth producers, the persons who brought the revenue to a sum that would have been deemed fabulous before, they were entitled to representation in the legislature. They had many other grievances, among which was the discourage- ment of the use of the English language in schools, but all centred in want of representation. but all centred in want of representation. A second volksraad was introduced by the govern- ment to deal principally with mining matters, and in it the newcomers were represented, but as its powers were subordinate to those of the first volksraad, it did not give much satisfaction. not give much satisfaction. At this time goldmining overshadowed all other matters in the South African Republic, still there were many changes going on besides that. A market in which prices unknown before for all kinds of farm produce could be obtained stimulated agriculture and made money plentiful. Railroads were constructed in different directions, and public works of various kinds were undertaken by the government. The various Bantu tribes and clans that had given so much trouble in earlier days were reduced to subjection and made to observe quietness' and order. Among others the Baramapulana in Zoutpansberg were compelled to submit once again to the authority of the republic, so that the supremacy of the Europeans was undisputed throughout the whole country. At this time the government was unfriendly to the Cape Colony, and by heavy taxation of colonial produce to encourage home industry and by an attempt to close the drifts of the Vaal river to THE JAMESON RAI 399 colonial trade in order to force importers to make use of Delagoa Bay, so many well-wishers in other parts of South Africa were turned into ill-wishers that the disaffected party at Johannesburg reckoned largely upon outside support. They then entered into a conspiracy to overturn the government at Pretoria, which they believed could be easily effected. Their plan was to intro- duce rifles and ammunition secretly, with which to arm the workmen, and to seize by night the state arsenal at Pretoria, which was almost without means of defence, thus adding to their own supply of materials of war while depriving the government of its reserve stock. It is to be deplored that the right honourable Cecil J. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. Rhodes, then prime minister of the Cape Colony, made the single great mistake in his brilliant career by taking part in this conspiracy and promising to aid it with a strong contingent of Rhodesian police, that was at his disposal, as he was managing director of the chartered company. The police were accordingly massed near Mafeking under pretence that trouble was feared from the Bantu there, and Dr. L. S. Jameson, then administrator of Rhodesia, took immediate command of them. But nothing was in readiness at Johannesburg when in the evening of Sunday, the 29th of December 1 895, with almost inconceivable rashness Dr. Jameson crossed the border with five hundred mounted men and eleven pieces of artillery, to make his way to the goldfields, one hundred and seventy miles distant. He feared that if he delayed any longer the repub- lican government would suspect his purpose and 00 THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. 400 prepare to oppose him, and further he believed that the leaders of the conspiracy were wrangling and wasting time, so that his presence was necessary to force them to take immediate action. Intelligence of the invasion spread rapidly, and the farmers hastily saddled and rode, each one instinctively, to the hilly country about twelve miles west of Krugersdorp, through which Dr. Jameson would have to pass. On Tuesday evening he reached that part of the road, and skirmishing commenced. It continued until the morning of Thursday, the 2nd of January 1896, when his position was utterly hopeless, and he was obliged to surrender to Commandant Cronje. He had lost eighteen men killed and about forty wounded. On Monday morning, the 30th of December 1896, intelligence was received at Johannesburg that Dr. Jameson had set out, when the leaders of the plot at once recognised that all prospect of its success was destroyed. There was now no possibility of seizing the arsenal at Pretoria, and they could not arm more than three thousand men. They took possession of the hills commanding the city, however, and threw up entrenchments, enrolled a strong body of police, and assumed the local government, in hope that by putting on a bold face they might secure good terms from the authorities at Pretoria. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. At the urgent request of the leaders of the revolt and with the concurrence of the president and the executive council, Sir Hercules Robinson, her Majesty's high commissioner, hastened to Pretoria, where he was informed that Johannesburg must lay EFFECTS OF THE RAI 4OI down its arms and surrender unconditionally before anything else could be determined, and this was accordingly done. Dr. Jameson and his band were then handed over to the high commissioner, who released the rank and file and sent the officers to England for trial, where they were sentenced to short terms of imprisonment. Sixty-four of the leaders of the plot were arrested and put upon their trial at Pretoria, where they received various sen- tences, which were afterwards commuted into payment of heavy fines. of heavy fines. The attempt to overturn the republican govern- ment had completely failed, and its evil consequences remained. Not the least of these was the retirement of Mr. Rhodes from public life, for he immediately resigned both the important positions he had pre- viously held. That it increased President Kruger's distrust of Englishmen goes without saying, and that their antipathy to him was henceforward more bitter is equally obvious. bitter is equally obvious. It has been stated that the policy of the republican government was unfriendly towards the British possessions in South Africa. But the unfriend- liness was not confined to one side, though neither the colonial governments nor the colonial people were to blame in the matter. The long delay in connection with the transfer of Swaziland and the annexation to the British dominions in April and May 1895 of the territory between the Portuguese dominions and Zululand, whereby the republic was shut in from extending to the sea, cannot be ignored as weights in the other scale. 27 cale. cale. THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. 402 After the discovery of gold at Witvvatersrand the revenue of the republic rose by leaps and bounds, and public works could be undertaken that were undreamed of before. A system of state-aided schools was established. Many new towns were laid out, and some of the buildings in them are among the best in South Africa. Good roads were made in various places, rivers were bridged, and lines of telegraph were constructed. A railway was laid down from Lourenco Marques to Pretoria, with a branch to Barberton. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. It was constructed from the Portuguese border by a company called the Nether- lands South African, which was aided by the republican government. A line from Pretoria to Vereeniging on the Vaal river, passing through Johannesburg, was also constructed by the same company, with assistance from the Cape govern- ment. At Vereeniging it joined the trunk line through the Orange Free State, which branched off to the three chief ports of the Cape Colony. From Krugersdorp a railway was constructed through Johannesburg to the Springs, passing over a great coal mine which supplied fuel to the city and to the quartz-crushing machinery along a route of fifty-four miles. Another line connected Johannesburg with Natal, so that a large portion of the present network of railways was completed before the close of the nineteenth century. At that time the country was governed by a presi- dent, elected for five years. After the restoration of independence Mr. S. J. Paul Kruger filled that office uninterruptedly. He was aided by an execu- INCREASE OF MILITARY STRENGTH. 403 tive council, consisting of three heads of departments and two non-official members appointed by the first volksraad. For military purposes a commandant- general was elected by the burghers every ten years. years. The legislative power was vested in two chambers, each consisting of twenty-four members, elected for four years. The first volksraad was by far the more important of the two. Its members were elected by burghers of European blood, either born in the republic or naturalised after a difficult probation. The second volksraad had very little power, being intended chiefly as an advisory body in matters connected with the mining industry, and its enact- ments being subject to review by the first volksraad. Its constituency was larger than that of the first volksraad, as foreigners could make themselves eligible as voters after a residence of two years. Dr. Jameson's raid at the close of 1895 caused the government of the republic to devote a very large part of its resources to the increase of its military strength. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. It had not been neglectful in that respect before, but now large sums of money were devoted to the importation of munitions of war and to the construction of forts, until the whole of the burghers were armed with the most perfect modern weapons, Johannesburg, with its people utterly defenceless, lay commanded by the great guns of a battery, and Pretoria was fortified as if it had been in the condition of Metz or Strassburg. This, which seemed to the republican government to be merely a prudent measure to maintain its inde- THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. 404 pendence if again attacked, was regarded by Great Britain in another light. Here was a state making itself the first military power in South Africa, a position that could not be conceded to it if English prestige was to be preserved. Hence the British garrisons were strengthened, and a word that had not been pronounced for thirteen years was used to denote the relationship of the republic to the British government, the word "suzerainty." government, the word "suzerainty." By far the greatest benefit that the burghers of the republic regarded as gained by them in the substitu- tion of the convention of 1884 for that of 1881 was the elimination of that word, with whatever it meant or might be made to mean, for nobody could define its exact purport. From the moment it was used again — in 1897 — the difficulty of negotiation was increased a hundredfold, for to the mind of the South African burghers of Dutch descent it seemed as if Great Britain had resolved to withdraw from the agreement she had entered into in 1884, and by some means or other to destroy the independence of the northern republic. In Johannesburg the English residents were seeth- ing with discontent. Apart from the humiliation caused by their want of success in 1895-6, and their being deprived of arms, they had many grievances. They had not even a real municipal government, while the want of proper sanitary arrangements caused not only much discomfort, but also much sickness, and a death-rate considerably in excess of what under other circumstances it would have been. This was due to the resolution of the government to GRIEVANCES OF ENGLISH RESIDENTS. 405 withhold even limited power from all except burghers of the state. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. Then there was the subject of language in public schools frequented by English children. The govern- ment, forgetting what resentment this question had caused among the Dutch colonists when an attempt was made to force the use of English upon them, made Dutch the principal medium of instruction, and although this rule had been relaxed, the bitter feeling it engendered could not easily pass away. A system of granting monopolies — especially one for the manufacture of dynamite, which raised the price of that necessary article in mining to an exorbi- tant rate — was another grievance, though perhaps not much felt except by the proprietary classes. much felt except by the proprietary classes. Taxation was so adjusted as to fall chiefly upon those inhabitants who were not burghers, and by it the cost of living was greatly increased. The means of obtaining the right of voting for the head of the state and members of the first volksraad were so restricted that practically Englishmen could not obtain that privilege. To those accustomed to the highly trained and splendidly conducted body of men who preserve order in English cities, the police appeared to be a mere rabble, and many of its members were believed to be corrupt to the last degree. This vice was also said to extend to not a few men in higher positions. Petitions had been sent to the volksraad asking for more liberal treatment, and had been rejected — even worse, had been received with taunts by indi- vidual members. 406 THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. Without going further, a position existed here which enabled men of influence to bend the masses easily to their will. An Englishman not allowed to carry arms, with the great guns of a fortress frowning upon him, paying taxes without being represented in the government, unable to send his children to a public school, liable to abuse from an ignorant and corrupt policeman, taunted by an equally ignorant member of the volksraad when petitioning for re- dress : such a man needed very little persuasion to induce him to follow any one in opposition to the rulers of the land. There is in brief one side of the question ; but it must be looked at also from the opposite direction. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. To the foregoing observations the government at Pretoria would reply as follows : — Pretoria would reply as follows : — In no other country in the world is a foreign language the medium of instruction in state-aided schools. Dutch is the language of the South African Republic, and yet English children are taught in their own tongue up to a certain standard. own tongue up to a certain standard. The monopolies complained of and the high import duties on all articles that can be produced in the republic are to encourage home industries. Other countries — notably the United States — have protec- tive tariffs for the same purpose. tive tariffs for the same purpose. The system of taxation is not unfair. The burghers have military duties to perform without payment, which fully compensate for the smaller amount of money they contribute. The right of voting carries with it duties which the great majority of Englishmen would not perform. THE OPPOSITE VIE 407 The privilege cannot be given without adoption of the burghers' burdens as well as their rights. the burghers' burdens as well as their rights. The police are the best that can be obtained, and it is unreasonable to expect from them the demeanour of those of London. If corruption existed to the extent that is stated there would be no reason for further opposition, because all that is asked for could be bought. The existence of corruption in high places implies the existence of corrupters as well. existence of corrupters as well. The taunting language of a few individuals should not be regarded as an offence committed by a community, neither has it been confined to one side only. Nearly all other Europeans are satisfied, and sub- mit without demur to the government of the country; it is only Englishmen who complain. it is only Englishmen who complain. That was the other side of the question, and every reader can decide for himself as to the merits of the respective cases. But at the back of each stood a principle which was really the governing factor of the whole position. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. According to the authorities at Pretoria the South African Republic was a sovereign state subordinate to Great Britain only to the extent that treaties with foreign powers were subject to the queen's approval before they could be put in force, and they rested this claim upon the wording of the convention of 1884. They therefore maintained that they had the same right with regard to Englishmen in the republic as, for instance, Germany exercised with regard to Englishmen in Metz or Berlin, and whether it was prudent or not for them to exercise THE SOUTH AFRICAN REPUBLIC FROM 1 884-99. 408 such power was for them to decide, the fact that the number o( Englishmen in the German cities was exceedingly small in comparison with the whole population, whereas the number on the goldfields approached that of the burghers themselves, not affecting the question of right. According to this view Great Britain could interfere only under inter- national law on behalf of any of her subjects who believed themselves to be suffering wrongs, in pre- cisely the same way that France or Germany could. On the other hand, Great Britain maintained that the republic was really a subordinate state, moving in the political sphere not as a primary but as a secondary body, and not justified in attempting to carry out a policy opposed to that of the power whose interests and responsibilities in South Africa were immeasurably greater. That was the real question at issue, and neither side would recede from its position by a hair's- breadth. The claim to suzerainty made on behalf of Great Britain in 1897 exemplified this in a striking manner. The matter was brought to a point by a petition to her Majesty the queen from the great body of Englishmen on the goldfields, asking for interference on their behalf. President Steyn, of the Orange Free State, invited Sir Alfred Milner, governor of the Cape Colony, and President Kruger, the head of the South African Republic, to meet in conference at Bloem- fontein, in hope that an amicable arrangement could be made. The governor proposed that all foreigners THE CONFERENCE AT BLOEMFONTEIN. 4OC) should be admitted to full burgher rights after a residence of five years in the republic, and that they should be allowed a certain number of representatives in the volksraad. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. The president declined, as he believed the concession would jeopardise the inde- pendence of the republic, and the conference came to an end. By independence President Kruger meant not only the retention of a separate flag and adminis- tration, but the retention of power by the Dutch- speaking burghers of the country. To him and to them the conversion of the state into an English republic, in which they would form a mere party — possibly in time a mere minority — signified a loss of independence quite as complete as if the four-coloured flag should be replaced by the British ensign. The volksraad then passed an act to admit foreigners to the franchise after a residence of seven years and to increase the number of representatives for the districts in which they resided ; but this did not satisfy the British government. Negotiations, however, were continued, and at one stage an offer was made in practical agreement with the governor's proposal, provided the suzerainty claim was withdrawn. This was declined, but no exact and formal demand embodying everything that Great Britain required was presented in its place, and the government at Pretoria was left to believe that no matter what it conceded short of independence some fresh claim upon it would be made. 4-10 THE SOOTH AFRICAN REPUBLIC FROM 1 884-99. Meantime troops were being massed on the borders of the state, chiefly in Natal, and an enormous force was in preparation to be sent to South Africa. A great proportion of the British press was advocating war as the only means by which the supremacy of England could be maintained. On the 7th of October 1899 the British reserves were called out, which was a practical intimation that military force was to be resorted to. As such it was received by the governments of the two republics, then in the closest alliance, and on the 9th an ultimatum was handed to the British agent at Pretoria, demanding the removal of the forces from the border and an engagement that the troops then on the way out should not be landed in South Africa, failure of compliance before the evening of the nth to be regarded as a declaration of war. THE SOUTH AFRICAN REPUBLIC FROM 1 884 TO 1899. The object of the ultimatum was to bring on hostilities, which were then inevitable, before the arrival of the main body of the army, and thus open a way for the mastery of the troops in Natal. The reply of the British government was the recall of its agent, and at five o'clock in the evening of the nth of October 1899 a state of war existed between Great Britain and the two republics. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. It is not necessary to enter into the details of the war, which would occupy too much space in a book like this, and of which there are scores of volumes for any one desiring complete information to refer to. At its commencement the two republics could put sixty thousand combatants into the field with the foreigners that joined them, and during its course they were aided by a few thousand of their kinsmen in the Cape Colony, whose sympathies with them were so strong as to overcome the fear of danger that such action involved. But beyond these they had no reserves, for that number included every boy that could carry a rifle as well as every greybeard capable of enduring the fatigue of a campaign. When any were lost therefore by death or capture they could not be replaced, so that attrition became the determining factor in the strife. Before the con- clusion of peace thirty-one thousand four hundred combatants had been captured and sent abroad, three thousand eisrht hundred had been killed in THE TRANSVAAL AND ORANGE FREE STATE. 412 action or had died of wounds, some ten thousand had either abandoned the cause in utter despair of success or had tendered their submission and pro- mised fidelity to the British government, and only fifteen thousand remained in the field at last. Of those who had submitted, some, who were called by friends the national scouts, by opponents the handsoppers, actually fought on the British side and from their knowledge of the country and of the habits of their countrymen were of great ser- vice to the cause they had adopted. vice to the cause they had adopted. On the other side Great Britain employed con- siderably over three hundred thousand fighting men under her ablest generals, Lord Roberts and Lord Kitchener, and without difficulty replaced her losses : nine thousand killed, thirteen thousand deaths from disease, and seventy-five thousand wounded and invalided. She was supported by her great over- sea dependencies too, for as soon as her supremacy in South Africa was endangered, regiments from Canada and Australia hastened to her aid. They were Canadian troops that forced the first large commando in the Free State to surrender. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. The great distance of the scene of operations from the coast made it necessary to employ a very large number of men to guard the lines of communication, and also made the expense of supplying the forces with food and war material enormous, the cost of the war to the imperial treasury being at least £250,000,000. But this sum represents only a fraction of what the war really cost Great Britain, for the creation of a powerful German navy received COST OF THE WA 413 from it a tremendous stimulus. The want of a navy alone prevented Germany's interference in a quarrel that might have given her the dominion of a very large part of the continent, and at once associations to promote the building of a fleet of enormous strength sprang up throughout that empire. Very largely to the South African war of 1 899-1902 Germany owes her possession of a navy second only in fighting strength among all those of the world to that of England. But if the cost of the war to Great Britain was enormous, to the two republics it was even more in comparison, so great was the destruction of pro- perty, so widespread the impoverishment of the people. There were whole districts in which hardly a farmhouse was left standing and not a single ox or sheep remained. Nearly all the women and children were in concentration camps, where over twenty-six thousand of them perished from sickness and diet they were unaccustomed to, and more than half of the men still alive were prisoners in distant lands. In modern times there had been no such instance of prolonged endurance of suffering and of resistance to overwhelming force, but at last even the coarsest food was almost exhausted, and the horses were all worn out. Most of the men of the Transvaal and the Orange Free State felt that they could do no more, though some of them were still averse to submission and the women encouraged them to hold on till death. They had hoped that some European power would have interfered on their behalf, and had sent THE TRANSVAAL AND ORANGE FREE STATE. 414 delegates to try to obtain assistance, but without success. President Kruger himself had gone on a like mission, leaving Mr. Schalk W. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. Burger to act during his absence ; but though he had been received — especially in Holland — with warm sympathy, no power would give more than that. The plain fact had to be faced that to prolong the strife could only entail more suffering, possibly the entire destruction of the people of the two republics, without the slightest hope of ultimate success. slightest hope of ultimate success. In January 1902 the government of Holland requested the secretary of state for foreign affairs in London to permit the republican delegates to return to South Africa to consult the leaders of their people with a view of bringing the war to an end, and then return to Europe with the in- formation they should gather. As this implied mediation, Lord Lansdowne declined to do so, and stated that the South African leaders could enter into direct communication with the commander-in- chief of the British forces, who had already been instructed to forward immediately any offers he might receive for the consideration of his Majesty's government. government. This led to a correspondence between Acting President Burger and Lord Kitchener, which resulted in safe conducts being granted to the prin- cipal men on the republican side to meet at Klerks- dorp and confer together as to terms of peace. Accordingly, on the 9th of April 1902 there assem- bled Messrs. S. W. Burger, acting president, F. W. Reitz, state secretary, Louis Botha, commandant- THE MEETING AT VEREENIGIN 415 general, J. H. de la Rey, commandant, and six others, forming the government of the South African Republic, and M. T. Steyn, president, C. R. de Wet, chief commandant, J. B. M. Hertzog, commandant, and four others, forming the government of the Orange Free State. Orange Free State. On the nth of April the two governments requested Lord Kitchener to grant them a personal interview, and on his consenting they went to Pre- toria, and proposed to enter into a customs, postal, telegraph, and railway union with the two British colonies, to grant the English language equal rights with the Dutch in schools, and that there should be an amnesty on both sides. This proposal was com- municated by telegraph to England, and a reply was sent that his Majesty's government could not enter- tain any proposals that were based on the continued independence of the former republics. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. The imperial authorities regarded them as extinct, because Lord Roberts, on the 24th of May 1900, had proclaimed the Orange Free State, and on the 1st of September of the same year the South African Republic, British possessions. The republican governments having no power to agree to the surrender of independence, it was arranged with Lord Kitchener that thirty represen- tatives should be elected by the people of each state purposely to instruct them how to act, and that they should meet at Vereeniging on the 15th of May, where the members of the two governments would await them. When they assembled the greatest diversity of opinion was expressed, but after a time THE TRANSVAAL AND ORANGE FREE STATE. 4l6 it was resolved by a majority to send a commission to Pretoria to offer the Witwatersrand and Swaziland fully to Great Britain, to admit a British protectorate, with the entire control of foreign relations, over the remainder of the country, and if this offer should not be accepted, to ascertain what terms would be granted. granted. As members of this commission Commandant- General Botha, Chief Commandant De Wet, and Commandants De la Rey, Smuts, and Hertzog were appointed. They proceeded to Pretoria, where Lords Kitchener and Milner rejected their pro- posal, and the following terms, which the British representatives said could not be altered, were submitted to them : — The burgher forces in the field immediately to lay down their arms, to surrender all material of war, to acknowledge King Edward VII as their sovereign, and to desist from further opposition to his Majesty's authority. The burghers sent abroad as prisoners of war to be brought back to their former residences. The burghers not to be deprived of personal liberty or property, and no civil or criminal proceedings to be taken against them for anything clone in accord- ance with the rules of war. The Dutch language to be taught in schools where the parents desired it, and to be used where necessary in courts of law. The possession of rifles for defence to be permitted on taking out licenses. Military administration on the earliest possible date to be followed by civil govern- ment, and as soon as circumstances would permit representative institutions to be introduced, ending THE PEACE OF VEREEN1GIN 417 in responsible parliamentary government. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. The ques- tion of conferring the franchise on natives not to be determined before the introduction of self-govern- ment. No special taxation to be laid on landed property to assist in defraying the cost of the war. His Majesty's government to place ^"3,000,000 at the disposal of a repatriation commission, and to permit all notes and requisition receipts given by burgher officers in the field to be paid out of it. Where urgently needed to re-establish individuals on the land, money to be lent without interest for two years, to be repaid thereafter on easy terms. The commission returned to Vereeniging, and on the 29th of May delivered a report to the above effect. Earnest discussion followed, a (g\v of the representatives declaring themselves in favour of continuing the war rather than consent to the loss of independence ; but the good sense of the majority prevailed, and on the 31st it was resolved by fifty- four votes against six to empower the members of the two governments to accept these terms. Those gentlemen immediately proceeded to Pretoria, and at eleven o'clock that evening a document embodying them was signed in Lord Kitchener's residence by them and by Lords Kitchener and Milner. Mr. Steyn's health had completely broken down, and two days before he had formally resigned his office, when Chief Commandant Christian de Wet was appointed acting president, and in that capacity he signed the document. In this manner the two republics came to an end, and thereafter they took the status of British colonies, THE TRANSVAAL AND ORANGE FREE STATE. 41 8 The position of those individuals in the Cape Colony and Natal who had assisted the republics was a matter that affected the early proceedings, as they could not be abandoned without dishonour. Lords Kitchener and Milner declined to make any written stipulations concerning them, but gave a verbal assurance that those of them — three in number — who were accused of committing crimes not permissible by the laws of war, though they would be tried, should not suffer the death penalty, and all the others would merely be deprived of the right to vote at parliamentary or municipal elections. In point of fact, even this punishment was only temporary, for in 1906 the Cape parliament passed an act conferring the franchise on them again. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. One good effect of the war was that it created in England a feeling of greater respect for the Dutch- speaking farmers than had existed before. They had proved themselves valiant, if stubborn, oppo- nents, in many instances they had acted chivalrously, and, in sporting language, they had played the game fairly and kept it up well. For such a people the feeling could only be one of admiration. Co-opera- tion between them and English-speaking people now became possible, for such prejudice as was caused by the misrepresentations of former times could no longer exist: Immediately after the terms of peace were signed the fullest confidence in each other was shown by those who had just before been enemies, and all combined in a strenuous effort to repair as much as possible the damage that war had wrought. It was CHINESE LABO 419 a difficult task to undertake. A very large propor- tion of the adult males of the late republics were prisoners in Ceylon, St. Helena, and the West Indies, and these had to be brought back to their former homes in South Africa. Their families were in concentration camps, their homesteads had been destroyed, and their cattle of all kinds had disap- peared. If they had been left to themselves very many of the country people of the Transvaal and the Orange Free State must have perished ; but with the assistance of the British government they were able to commence agriculture and cattle breeding again. again. Of course they had a very hard struggle before them, but they faced the position so bravely that ten years later the traces of the tremendous conflict they had gone through could hardly be observed. Great Britain did not limit her assistance to the terms of the agreement of peace, but expended very much larger sums than those mentioned in that document in endeavouring to restore prosperity to the country. This was necessary, as unfortunately 1903 was a year of drought, when no crops could be raised, besides which disease destroyed many of the cattle imported. The kindly feeling then exhibited by the new autho- rities had an excellent effect upon the bulk of the people, and hope for better days was felt. people, and hope for better days was felt. To promote gold-mining, from which alone a revenue could be expected for some time to come, Chinese labourers were introduced. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. Bantu were not then to be obtained in sufficient numbers, so Lord Milner hit upon this expedient. They were engaged THE TRANSVAAL AND ORANGE FREE STATE. 420 for a fixed time under contracts, and were to be sent back to China when the terms of service expired. At one time there were over fifty thousand of them working at the mines. But a more undesirable class of labourers could not have been found, and as for- tunately a majority of the English people chose to regard them as slaves, while by every one in South Africa except by the mine-owners they were objected to, their further introduction was quickly stopped, and those already here were sent back as soon as their contracts would admit of it. In 1910 the last of them had gone, and Bantu labourers were again solely depended upon to do the rough work of mining. After a brief interval, so brief that complete order was hardly restored, came an act of unexampled generosity : the grant by Great Britain of full parlia- mentary government with a responsible ministry to both the new crown colonies. This was indeed provided for in the conditions of peace, but no time was fixed, and only the most liberal interpretation of the clause could have brought it into effect at so early a date as 1906. It was certainly the wisest thing that could be done. It placed the leaders of the men recently in arms against Great Britain in political power, as had been foreseen, for General Botha became prime minister of the Transvaal, but it turned them into firm adherents of connection with the widespread British realm. It was now the general opinion that even more liberty was enjoyed than would have been the case under the proposal made at Vereeniging to come PARLIAMENTARY GOVERNMEN 42 1 under British protection, and the revenue derived from the goldfields was not lost, as it would have been under that scheme of settlement. There were indeed some fractious individuals — in what country in the world are there none? — who remained discon- tented, but they formed a small minority, and were not so much opposed to British sovereignty as to the policy of those of their own people who were willing to work cordially with the English colonists for the common good of all. common good of all. THE TRANSVAAL AND ORANGE FREE STATE FROM 1899 TO I9IO. So matters went on in the Transvaal and in the Orange Free State, without the occurrence of any- thing requiring special notice, until 1910, when an event of supreme importance to South Africa that will be related in the next chapter took place. THE UNION OF SOUTH AFRICA, 1910 TO I916. The period of depression that followed the war had not entirely passed away when the minds of men throughout South Africa turned towards the desir- ability of closer union between the colonies over which the British flag was flying. The interests of these colonies were the same, and they were inhabited by closely related people, so that there was no reason why they should not become a single state now that the grant of responsible government to the Transvaal and the Orange Free State made voluntary union possible. Instead of pursuing four different policies with regard to the Bantu, as had previously been the case, a single system, dealing with those people kindly and justly, but firmly, could be carried out, and as this was, and would long remain, the most important question to be dealt with everywhere in South Africa, the advantage of union of the European governments would be great. European governments would be great. The labour of devoted missionaries during nearly a century, coupled with the exertions of the ruling authorities and of the white people generally, had THE NEW CONSTITUTI 423 effected great improvement in the condition of a large number of the Bantu, but had not by any means eradicated their desire to be free of European control. The Bambata rebellion in Natal and the Ethiopian movement — or attempt to form inde- pendent church communities — elsewhere were plain manifestations of this feeling. The Bantu were multiplying rapidly, and in all parts were pressing upon the ground occupied by their white neighbours. No other oversea possession of Great Britain had so difficult a question as this to deal with, and a stronger government than any of those then existing was needed for the purpose. But this was only one, though far the most pressing one, of the many reasons that caused the people of the four colonies to desire to be united as a single community. It was evident also that one strong government would command greater respect abroad than several weak administrations jarring with, and sometimes opposing, each other. sometimes opposing, each other. After some preliminary arrangements, in 1908 the different parliaments appointed delegates, who met first at Durban and then at Capetown, and after much discussion agreed upon the form of the new constitution. THE UNION OF SOUTH AFRICA, 1910 TO I916. It differed from that of either Canada or Australia, inasmuch as it was not a federation of provinces, leaving to each a local government, but actual union, like that between England and Scotland, that it was intended to bring about. There was indeed to be a council in each of the existing colonies, thenceforth to be termed provinces, provided for, but it was wholly subservient to the general parliament, THE UNION OF SOUTH AFRICA, I9I0 TO I916. 4^4 was in fact little more than a magnified divisional council such as then existed in the Cape Colony. council such as then existed in the Cape Colony. The constitution thus formed was submitted to each of the four parliaments early in 1909, and was approved of by those of the Cape Colony, Transvaal, and Orange Free State. In Natal, where a section of the people, fearing the loss of English pre- dominance, would have preferred federation to unification, it was submitted to a plebiscite, and was adopted by a majority of votes. It was then brought before the British parliament, and being approved of, it received the signature of the king, and on the 31st of May 1 9 10 — the eighth anniversary of the con- clusion of peace between Great Britain and the former republics — it came into effect. former republics — it came into effect. Under this constitution there is a governor- general, who is appointed by the king, and who is also high commissioner for those parts of South Africa not included in the Union. Lord Gladstone was the first to fill this office and Lord Buxton the second. There is a house of assembly, consisting of one hundred and twenty-one members — 51 from the Cape province, 36 from the Transvaal, 17 from Natal, and 17 from the Orange Free State. They are elected for five years, each province retaining its old franchise, so that in the Cape province coloured people can still vote, but only Europeans can be elected. There is provision for a periodical re- arrangement of the electoral districts, as the popu- lation may increase in some localities and diminish in others. ADMINISTRATIVE AND LEGISLATIVE CAPITALS. 42$ As a check upon hasty legislation, more than for any other purpose, there is an upper house, termed the senate, consisting of forty members. THE UNION OF SOUTH AFRICA, 1910 TO I916. How it is to be chosen in future has not yet been decided. In 1910 eight members were elected by each of the four parliaments then existing, and eight were nominated by the governor-general in council, to hold their seats for ten years. Four of the nominated members were men specially qualified to look after the interests of the Bantu, by their intimate acquaintance with the habits and wishes of those people. The senate has the power of delaying, but not of preventing legislation, for in cases of disagreement between it and the assembly, after it has twice rejected any measure the question is decided by a majority of votes in a joint sitting of both houses. Each member of parliament receives ,£400 per annum and is entitled to travel free on the railways. The English and Dutch languages have equal rights in parliament, as well as in the public offices and courts of law. It was a difficult matter to decide where the capital of the Union was to be, as each of three of the provinces laid claim to it. It became necessary to make a compromise, and so Pretoria is the administrative capital, and there magnificent public offices have been built. Capetown is the legislative capita], where only a new wing was needed to make the parliament house suitable for the accom- modation of the larger number of members, and at Bloemfontein the supreme court of appeal holds THE UNION OF SOUTH AFRICA, I9IO TO I916. 426 its sessions. This is generally admitted not to be a satisfactory state of things, but it was the best arrangement that could be made in 1909. arrangement that could be made in 1909. The first session of the Union parliament was opened in Capetown by his royal highness the Duke of Connaught, who came to South Africa for the special purpose, on the 4th of December 1910. The ministry contained men whose names were of world-wide reputation. General Louis Botha was, and is still, prime minister, General (now Lieutenant-General in the British army) J. C. Smuts was minister for the interior, and Mr. J. W. Sauer (since deceased) was minister for railways and telegraphs. The cabinet consisted of ten ministers. Several changes caused by death, resig- nation, or disagreement in policy have since taken place, but practically the ministry is still the same. Mr. F. W. THE UNION OF SOUTH AFRICA, 1910 TO I916. Reitz became president of the senate. A large portion of the time since 1910 has been devoted by the Union parliament to bringing into uniformity various laws of the former colonies on several subjects in which they differed, and, of course, to financial matters. Railway construction and other internal improvements have also occupied much attention, and a great deal has been done to promote agriculture. At least three other measures deserve to be specially mentioned. One of these is the creation of an army by the Defence Force Act of 191 2, which makes it compulsory on all healthy European males, with few exceptions, between sixteen and sixty-one years of age, to render military service in any part of South Africa Photo] {Topical, GENERAL BOTHA. GENERAL BOTHA. THE UNION OF SOUTH AFRICA, I91O TO I916. 428 when required. It provides for a permanent stand- ing force of five regiments of mounted riflemen, with artillery attached, and requires men twenty- one years of age to commence a course of training, to last four years, unless they volunteer to do so when younger. They are formed into classes of those to be first called out and of first and second reserves, and are distributed in certain proportions among different branches of the service. In 1914 over forty thousand young men were placed on active service in the field, without any difficulty or delay, enabling Great Britain to withdraw every imperial soldier from the country, and providing an ample -force to garrison the towns on the coast and to undertake the conquest of German South- West Africa. For the first time in our history on a large scale English and Dutch speaking men lived together in the same camp, drilled together, formed close friendships, lay side by side in the hospitals, and, as the casualty lists showed, shed their blood together for the same cause. This is hopeful indeed for the future of South Africa. Another measure is an attempt to solve the Bantu difficulty, or at least one element in it, by defin- ing certain areas in which it shall not be lawful for Europeans to purchase or own land, and other areas in which the same law shall apply to blacks. But whether segregation is possible or not has yet to be proved, and under any circumstances there will be great difficulty in carrying it out. THE UNION OF SOUTH AFRICA, 1910 TO I916. will be great difficulty in carrying it out. The creation in 19 16 of three universities — two of them teaching — to take the place of the old THE POPULATION AND AREA OF THE UNION. 429 purely examining university of the Cape of Good Hope, is also a measure of importance. They may not all be urgently needed, but there was no other way of satisfying rival interests. The South African college has now acquired the dignity of being termed the University of Capetown, the Victoria college has become the University of Stellenbosch, and the South African University will be domiciled at Pretoria. The population of the Union in 191 3 consisted of: Europeans. Bantu. Other. Cape province ... ... 583,227 1.551,854 482,763 Transvaal province ... 470,175 1,327,731 49,694 Natal province ... ... 98,439 969,830 156,009 Orange Free State ... 187,138 366,950 30,196 1,338,979 4,216.365 718,662 The last column covers the Indians in Natal, the Malays in the Cape province, and the mixed breeds, Hottentots, and progeny of slaves wherever living. The area of the Union is 479,114 square miles, equal to a square 692 miles each way. Of this great extent of surface, the Cape province covers 276,565 square miles, Transvaal 111,196 square miles, Natal 36,173 square miles, and the Orange Free State 55,180 square 'miles. 55,180 square 'miles. The Union government took over the public debts of the four provinces, which amounted altogether in round numbers to £110,000,000. Chiefly owing to war expenses, that amount has since greatly increased, and now, in June 1916, it stands at £142,851,000, which is still being added to. THE UNION OF SOUTH AFRICA, IQIO TO I916. 430 The greater portion of this sum, however, is invested in railways, telegraphs, harbours, and other public works, which bring in a fair return for the capital expended in their construction, and which are necessary for the proper development of the country. country. On the 1st of January 191 6 there were in the Union 8,874 miles of railway belonging to govern- ment open for traffic, besides a line for military purposes, ^>Z miles in length, from Upington to Kalk- fontein, and one, 20 miles in length, from Walvis Bay to the mouth of the Swakop river. These lines, with the stations and rolling stock, had cost ^88,494,328. THE UNION OF SOUTH AFRICA, 1910 TO I916. In the Cape province there were 3,677 miles with a gauge of 42 inches and 287 miles with a gauge of 24 inches ; in Transvaal 2,465 miles of the former and 279 of the latter ; in the Orange Free State 1,265 miles of the former ; and in Natal 997 miles of the former and 156 of the latter. In addition to the above the government controlled 50 miles leased from the Natal-Zululand Railway Company, and there were 496 miles owned by companies, making altogether 9,528 miles open in the Union. On harbours ^"12,326,915 had been expended. On harbours ^"12,326,915 had been expended. There were about 15,000 miles of telegraph, with nearly 50,000 miles of wire, in the Union. The war which broke out in 19 14 had such a disturbing effect upon South Africa that any statistics of trade since 191 3 would be misleading. The figures given here are those for the year ending 31st of December 19 1 3, and represent the normal EXPORTS. condition of the Union were The exports of that year Gold Diamonds Coal Copper ... Tin Silver . . . Wool ... Ostrich feathers Hides and skins Mohair Wattle bark ... Maize Fruit Buchu Whale oil Animals... Wine Other articles £37.374,553 11,389,807 1,390,908 507,856 436,55° 102,471 5,719,288 2,953,5^7 2,008,857 876,255 309=329 65,169 51,858 32,071 122,692 38,106 n,5i5 74,678 £63,465,550 Of the exports £59,492,629 went to Great Britain and £3,972,921 to foreign countries. and £3,972,921 to foreign countries. In the same year the imports from the British dominions were valued at £25,566,314 and from foreign countries at £12,842,284. The importance attained by Durban is shown by no less than £12,252,845 worth of goods being received from abroad at that port. Through Delagoa Bay the imports for the Union were valued at £5,329,1 14. The balance of exports over imports amounted to £25,056,952, which covered the interest on the public debt, £4,911,948, and paid the dividends on mining shares owned abroad. The revenue of the Union in 191 3 was £15,805,076. RHODESIA SOUTH OF THE ZAMBESI. The vast territory between the Zambesi river on the north, the Union of South Africa on the south, the Portuguese possessions on the east, and the Betshuanaland Protectorate on the west, is called Rhodesia, after the man through whose ability and energy it exists as a British possession. It came to be very thinly inhabited in exactly the same manner as the country south of the Limpopo river before 1836. When Moselekatse was driven to the north by the emigrant farmers he commenced to destroy the tribes there just as he had destroyed the Betshuana, and Lobengula, his son and successor, continued the murderous practice. Large areas were utterly wasted, and the remnants of the tribes retreated to the hills, where they made their kraals among rocks in places difficult of access. These situations, if they could not be defended, offered facilities for escape when the Matabele bands approached in their yearly raids. By those terrible invaders the lives of the Makalaka and Mashona were held in no more esteem than the lives of so many antelopes. The ravagers built The vast territory between the Zambesi river on the north, the Union of South Africa on the south, the Portuguese possessions on the east, and the Betshuanaland Protectorate on the west, is called Rhodesia, after the man through whose ability and energy it exists as a British possession. It came to be very thinly inhabited in exactly the same manner as the country south of the Limpopo river before 1836. When Moselekatse was driven to the north by the emigrant farmers he commenced to destroy the tribes there just as he had destroyed the Betshuana, and Lobengula, his son and successor, continued the murderous practice. Large areas were utterly wasted, and the remnants of the tribes retreated to the hills, where they made their kraals among rocks in places difficult of access. These situations, if they could not be defended, offered facilities for escape when the Matabele bands approached in their yearly raids. By those terrible invaders the lives of the Makalaka and Mashona were held in no more esteem than the lives of so many antelopes. The ravagers built GRANT OF A CHART 433 military kraals in the best situations, and at Bulawayo, the principal of these, the chief himself resided. This was the condition of the country down to the year 1890. 1890. RHODESIA SOUTH OF THE ZAMBESI. According to modern ideas it is outrageous to enter a country occupied by an inferior race and take pos- session of anything in it by the right of the strong hand, but it is perfectly legitimate and moral to induce a chief to attach a mark to a document, the full pur- port of which he is incapable of comprehending, and thus securing under the name of a concession what- ever is desired. On the 30th of November 1888 Messrs. Rudd, Maguire, and Thompson, after con- siderable negotiation, obtained the mark of Lobengula to a document giving them the right to search for and extract minerals in the territory under his con- trol. Other concessions were obtained by different individuals, and ultimately all were united, when their proprietors became the British South Africa Company. To enable this great association to carry out its projects a charter was applied for, which was granted by the imperial government on the 29th of October 1889. By it a Company was created with a capital of one million pounds sterling, and with a board of eight directors, three appointed by the crown and five elected by the shareholders. Power was given to it to appoint and remove officers, except judges and magistrates, within the sphere of its operations, and to issue ordinances for common purposes. Judges and magistrates were to be appointed by the high commissioner, who was also to proclaim important 29 RHODESIA SOUTH OF THE ZAMBESI. 434 laws, in both instances on the recommendation of the Company. Mr. Cecil John Rhodes, the leading spirit in the enterprise, was appointed the managing director. The Company's first object was to take possession of some commanding positions in the country, and for this purpose to send in a strong armed force. Dr. Leander Starr Jameson was directed to proceed to Bulawayo to arrange matters. He had been there before, in 1888, when he had cured Lobengula of an attack of gout, and he had thus acquired much influence. The chief gave his consent to a body of Europeans entering Mashonaland, but it was evident that the Matabele army — especially the young regi- ments— could not be trusted to let the expedition pass onward unmolested. RHODESIA SOUTH OF THE ZAMBESI. The leaders of the pioneer force therefore resolved to take a circuitous route, so as to avoid the neighbourhood of the principal military kraals and to do away as much as possible with the danger of a collision. The pioneer party left the Cape Colony in March 1890, and on the 12th of September reached the spot where now the town of Salisbury stands. Dr. Jameson then explored the country to the eastward, and ascertained that with little difficulty a railroad could be constructed from Salisbury to Fontesville on the Pungwe river, a stream that is navigable for large lighters from that place to Port Beira, an excellent harbour at the mouth. Forts Tuli, Victoria, Charter, and Salisbury — form- ing a line of posts from south to north — were built and occupied by the Company's police, the pioneers, THE LIP PERT CONCESSI 435 consisting of one hundred and ninety-two young men, dispersed in search of gold-bearing reefs, and numerous small parties joined them from the Cape Colony and the two republics. But as these parties did not take with them a sufficient quantity of food or medical requisites, and as the summer of 1890-91 was exceptionally wet, they suffered great discomfort, and many died of fever brought on by exposure and want. The occupation of Mashonaland by white people in this manner was not at all to Lobengula's liking. He had anticipated that a few men would have gone there to look for gold, and here they were in hundreds, occupying strong posts and acting as if they were the real masters of the country. Still he did not wish to quarrel with them, for he appreciated the value of his subsidies. He chose rather to create a rival interest to theirs, and without any conception of the consequences of what he was doing, he granted to Mr. Edward Lippert a concession to dispose of farms, townships, and pasture lands within the sphere of operations of the British South Africa Company, the agreement to hold good for one hundred years. the agreement to hold good for one hundred years. When this important concession was purchased by the Chartered Company, whose capital was now doubled, the sovereign power in Mashonaland in name as well as in reality was claimed. RHODESIA SOUTH OF THE ZAMBESI. That terri- tory was thenceforth regarded as embracing the whole plateau from the Portuguese border on the east to the Shashi streamlet, which flows in a southerly direction to the Sabi river, and the Umnyati, a tribu- tary of the Zambesi, on the west. These streams RHODESIA SOUTH OF THE ZAMBESI. 436 were thereafter commonly termed the boundary, west of which Europeans were not permitted to search for gold and Lobengula's right to do as he pleased was neither disputed nor interfered with. East of it his rights were regarded by the Company as altogether extinguished, though neither he nor his army acquiesced in this view of the matter. acquiesced in this view of the matter. The Company now considered its hold upon Mashonaland so secure that it could disband the greater number of its police and trust for defence to volunteers and burghers. The European population was increasing, and as there were ample supplies of every kind in the country there was hardly any sick- ness in the summer of 1891-92. On the 20th of February 1892 the telegraph was opened from Cape- town to Salisbury. Farming was tested with good results, and the limits of the part that is healthy for Europeans were soon ascertained. Many appliances of modern times, including even printing presses, were introduced. In July 1892 the first building lots were sold by auction in the village* of Salis- bury, Victoria, and Umtali, when high prices were realised. Prospecting for gold was extensively carried on, and mines worked in ages long gone by were reopened and found to contain quartz that would pay for crushing, though until the heavy machinery required for that process could be brought in by rail it was not possible to do more than make experiments. experiments. Meantime some farmers in the South African Republic obtained a concession of land from a petty chief in the country, and attempted to move in and UNSUCCESSFUL RIVAL 437 form a settlement on their own account, but were met at the Limpopo by a strong force of police, who required them either to turn back or to proceed on their journey as subjects of the Chartered Com- pany. Some of them accepted the Company's terms and moved on, the others drew back, as President Kruger disowned their action and they were without support. support. RHODESIA SOUTH OF THE ZAMBESI. Nothing further of much interest occurred until April 1893, when the telegraph wire was cut by the people of a Mashona kraal near Fort Victoria, and a piece of considerable length wTas carried away. Dr. Jameson, the administrator for the Chartered Com- pany, imposed a fine upon the offenders, which was paid by them with cattle stolen from the Matabele. As soon as this became known Lobengula was informed that his cattle would be restored to him, but his indignation towards the Mashona was too great to be appeased, and he directed a division of his army to punish them. Messengers were sent to tell the Europeans that they need not be alarmed, for no harm would be done to them. But when, on the 9th of July, Mashona fugitives came panting into Victoria with information that their relatives were being murdered, when the ferocious Matabele warriors pursued their victims into the township and stabbed to death servants before the eyes of their masters, English blood was unequal to the task of submission. Accordingly protection was given within the fort to as many Mashona as requested shelter. When the raid commenced Dr. Jameson was at When the raid commenced Dr. Jameson was at Salisbury, but as soon as the wire conveyed the RHODESIA SOUTH OF THE ZAMBESI. 43§ intelligence to him he left for Victoria. Upon his arrival he found that the European prospectors and farmers had abandoned the open country, and that the Matabele were still destroying Mashona kraals in the neighbourhood. He therefore sent for the indunas, with whom on the 18th he had a short conference. He informed them that within an hour they must retire towards Bulawayo, or he would drive them away. The indunas would make no promise, but said they could not control their young men, though in point of fact they withdrew most of their force, and when a couple of hours later Captain Lendy with a small party of mounted men was sent out to drive away any who were not retreating, he found on the Victoria commonage only about three hundred Matabele, who were besieging a hill upon which some Mashona were trying to defend them- selves. Between these and Captain Lendy's party a collision occurred. The Matabele were driven away, and were followed about nine miles, losing nine or ten men in the pursuit. RHODESIA SOUTH OF THE ZAMBESI. It was then ascertained that the European inhabi- tants of the country had not been altogether unmo- lested, as a farmhouse had been pillaged, and some hundreds of cattle of different kinds had been driven off. No European, however, had been harmed in person. person. A peaceful settlement of this matter was impos- sible. On the one hand Lobengula dared not punish his warriors for what they had done and could not give security against a repetition of the offence, and on the other the white men in the territory with a OVERTHROW OF THE MATABELE POWER. 439 single voice declared that they could not continue their ordinary pursuits if they were to remain exposed to Matabele raids. But neither party was prepared for immediate war. In June Lobengula had sent a strong division of his army across the Zambesi against the Barotse chief Lewanika, so that he was for the time being crippled. The British South Africa Company had no horses and no troops even for the protection of its magazine of arms at Tuli. But both parties recognised that a contest was inevitable, and without loss of time began to make preparations for it. to make preparations for it. The day that Lobengula's people returned from Fort Victoria and reported what had occurred the chief sent messengers to recall his army from the Barotse country, and he refused thereafter to receive his monthly subsidy of ^"ioo from the Company. When a demand was made upon him for compen- sation for the damage done to the farmhouse and for the cattle taken from the white people, he declined to give it until the Mashona who were protected by the Europeans were surrendered to him with their families and all their belongings. He and his indunas displayed the utmost insensibility to any argument that the Mashona and Makalaka had rights of person and property, and they could not be brought to acknowledge that those miserable creatures were in that respect in any way different from the chief's cattle. from the chief's cattle. The high commissioner was apprehensive that if open war was commenced between the Chartered Company and the Matabele it might not be possible RHODESIA SOUTH OF THE ZAMBESI. 440 to prevent it spreading to the British Protectorate, and he therefore exerted himself to the utmost to restore concord. RHODESIA SOUTH OF THE ZAMBESI. The Company was prevented from pressing the claim for compensation, and was limited to the defence of its occupied territory while he was endeavouring to negotiate with Lobengula. If the chief could have guaranteed that the Mashona beyond a recognised boundary would not again be molested, the imperial authorities would have been satisfied. But Lobengula could not do that, nor could he even long restrain his army from attacking the white people, as the young regiments were clamouring for war. as the young regiments were clamouring for war. In one instance only the high commissioner acted in a manner that must have irritated Lobengula. Some guns and ammunition were on the way inland to the chief, and by Sir Henry Loch's order they were detained at Palapye. A letter was sent to Lobengula to acquaint him that they would be allowed to go on as soon as all cause of trouble was removed, and expressing a desire for peace, but informing him distinctly that the Mashona protected at Fort Victoria would not be surrendered. Towards a missionary family and the white traders at and near Bulawayo Lobengula acted in a manner deserving the highest commendation. He informed them that he might not be able to protect them after the return of the regiments from beyond the Zambesi, and advised them therefore to leave the country. Not a European who could be regarded as having a claim to his protection received the slightest harm. Some he assisted to remove, and those who chose to remain he treated with all possible kindness, OVERTHROW OF THE MATABELE POWER. 44 1 Personally the chief was anxious for peace, but as head of the Matabele he was unable to modify his claim that the Mashona should be left to the will of his warriors. The Chartered Company was equally unable to permit his bands to ravage and murder within the limits of its operations. No issue could be clearer. Accordingly on both sides preparations for the inevitable struggle were hurried on. The forts Victoria and Salisbury were made impregnable against any native force, and Charter and Tuli were provided with garrisons. The Company pur- chased a large number of horses, and enrolled a body of the hardiest young men in South Africa. RHODESIA SOUTH OF THE ZAMBESI. These engaged to serve throughout the war without money payment, but were to be armed and main- tained, and upon the conclusion of peace each was to receive a farm three thousand morgen in extent, twenty gold reef claims, and an equal share in half the cattle captured, the other half to be retained by the Company. By the beginning of October this force was in readiness, but could not advance upon Bulawayo without the consent of the high com- missioner. On the 30th of September a patrol sent out from Victoria to examine the country around ascertained that a Matabele army of six or seven thousand men was encamped in a bushy spot only twenty-five miles north-west of the fort, and two of the European scouts were fired upon. The Matabele were in a position where it would be difficult to attack them, but as the white people had abandoned the open RHODESIA SOUTH OF THE ZAMBEST. 44 2 country and Fort Victoria was impregnable, they could not do any harm where they were. could not do any harm where they were. Five days later, on the 5th of October, a patrol of three men of the Betshuanaland border police was fired upon by a party of about thirty Matabele on the southern bank of the Shashi river, near Macloutsie, within the Protectorate and beyond the Chartered Company's territory. The policemen returned the fire, and upon some others riding up the Matabele retired. It was subsequently ascertained that they were scouts of an army seven or eight thousand strong, under Gambo, a son-in-law of Lobengula. This affray made it evident to the high commissioner that the Matabele were incapable of distinguishing between the Chartered Company's forces and those of the imperial government, and brought all hope of a peaceful settlement to an end. It also showed that Lobengula, reserving only a body-guard, had massed his army in two great divisions, to act on the east and on the south. That part of his force which had returned from the Barotse country had been attacked by small-pox, and many of the warriors had died. It had therefore been put in quarantine, but was now released and in the field, though the disease had not entirely disappeared. RHODESIA SOUTH OF THE ZAMBESI. Intelligence that the imperial police had been fired upon in territory under imperial protection reached Capetown by telegraph the same day, and the high commissioner at once gave Dr. Jameson permission to send his forces forward. There was no time to be lost, for in five or six weeks the rainy season might be expected to set in. OVERTHROW OF THE MATABELE POWER. 443 The plan of the campaign was that two columns should advance directly upon Bulawayo from dif- ferent directions. The first was under Major Patrick William Forbes as commander in chief, with Major Allan Wilson as next in rank. On the 17th of October this column marched from the Ironmine hill, at the source of the Tokwe river. It was com- posed of between six and seven hundred Europeans, nearly three-fifths of whom were mounted, and it had five maxim and five other field guns of great destruc- tive power. It was provided with a sufficient number of waggons to carry its stores and to form lagers, and it was accompanied by five or six hundred blacks, who were needed to tend the cattle, assist in scouting, cut bushes for strengthening lagers, and do other work of the kind. It kept as much as possible in open country, so that the Matabele army following it might not have an opportunity to attack it by surprise. surprise. The southern column consisted of Betshuanaland border police and British South Africa Company's men in equal numbers, only four hundred and fifty in all, but nearly every man was mounted. Lieu- tenant-Colonel Goold-Adams was in command. The column was splendidly equipped with maxims and other guns, and had a sufficient waggon train. It was accompanied by seventeen or eighteen hundred Bamangwato under the chief Khama. Bamangwato under the chief Khama. On the 1 8th of October an unfortunate incident occurred. Three Matabele indunas, with a European trader as their protector, who were sent by Lobengula to confer with the high commissioner, though they RHODESIA SOUTH OF THE ZAMBESI. 444 had no authority to make the concessions necessary for peace, arrived at Colonel Goold-Adams' camp at Tati. The European, without giving any information as to the character of his companions, went to get some refreshment, and during his absence the indunas were arrested on suspicion of being spies. RHODESIA SOUTH OF THE ZAMBESI. Two of them, without comprehending that a little patience would ensure their release, broke away from con- finement, and were shot down when attempting to escape. It was a deplorable affair, but it can only be regarded as an accident. The third induna, who was the highest of them all in rank, was afterwards found to be an envoy only in name, as he had no proposals of a practical nature to make. The column under Major Forbes was accompanied by Dr. Jameson, Sir John Willoughby, and Bishop Knight-Bruce. It destroyed the Matabele military kraals along its line of march, and had frequent skirmishes with parties of the enemy, but took such precautions that it could not be attacked by a large force except when in lager. On two occasions only it had encounters worthy of the name of battles, but on the last of these the power of Lobengula was completely shattered. completely shattered. A little before daylight on the morning of the 25th of October the column was in lager on the head- waters of the Shangani river when it was attacked by a Matabele army about five thousand strong. Three times within about four hours bands of the savage warriors advanced upon the lager, but on each occasion they were driven back with heavy loss by the machine guns. OVERTHROW OF THE MATABELE POWER. 445 After the fight at Shangani the column continued to advance, and was closely followed by the defeated Matabele. As soon as intelligence of the battle and its results reached Lobengula he sent nearly the whole of his body-guard, including the Imbezu and Ingubo regiments, the very choicest of his warriors, to reinforce his army. The Imbezu and Ingubo, pure Zulus, believed that they alone could destroy the advancing whites, and were eager to display their superiority to the mongrel regiments. superiority to the mongrel regiments. A little after noon on the 1st of November the lager, which was being formed near the source of the Imbembesi river, was suddenly attacked by the Matabele army, that had managed to approach under cover of a thick forest. The Imbezu and the Ingubo were in front, and excited the admiration of the Europeans by their splendid bravery and coolness. RHODESIA SOUTH OF THE ZAMBESI. But they could not get through the storm of bullets poured upon them from the maxim and other machine guns, and in less than an hour the engage- ment— which proved to be a decisive one — was over. The two gallant regiments had lost half their number in killed and wounded, and some others had also suffered severely. Fugitives from the battle of Imbembesi conveyed to Lobengula tidings that all was lost, when by his orders his principal kraal was set on fire, and with the whole of his people he fled northward towards the Zambesi. On the 3rd of November Major Forbes had reached a point about eight miles distant from Bulawayo when he heard a tremendous report and saw a column of smoke shoot up to the sky. It RHODESIA SOUTH OF THE ZAMBESI. 446 was Lobengula's magazine, which the fire had reached and caused to explode. On the following day — 4th of November 1893 — the Chartered Company's force arrived at the still smoking kraal, and found in its neighbourhood only two individuals alive, two white men, traders, whom the chief had protected to the last, and who were still unharmed in property and in person. in person. The column under Colonel Goold-Adams advanced more slowly, and met with no opposition except on one occasion. On the 2nd of November the rear of the waggon train was attacked by a few hundred men of Gambo's army, and an action followed which ended in the defeat of the Matabele. Three days later Khama and his people abandoned the expe- dition, and set out to return to Palapye. The Europeans continued to advance, and shortly after- wards learned that Gambo, having heard of the defeat at Imbembesi of an army superior to his own, had retreated northward with his regiments. Being apprehensive, however, that they might meet with resistance in a range of hills through which they were obliged to pass, they marched slowly and took pre- cautions against surprise, so that they only reached Bulawayo on the 15th of November. Bulawayo on the 15th of November. Various messages were sent to Lobengula assuring him of personal safety if he would surrender, but as these had no effect, Dr. Jameson resolved to send a party to capture him, and so bring hostilities to an end. RHODESIA SOUTH OF THE ZAMBESI. As soon therefore as the southern column reached Bulawayo, Major Forbes with two hundred and ten of the Chartered Company's men and ninety OVERTHROW OF THE MATABELE POWER. 447 of the Betshuanaland border police, well provided with maxims, set out in pursuit. The rainy season had now commenced, and the difficulty of traversing the country was greatly increased. The horses soon became unfit for much exertion, and it was necessary to send the greater number of the men back to Bula- wayo, causing considerable delay before reinforce- ments could arrive. A great many Matabele were dispersed over the country along the line of march, but they offered no resistance, and it was evident that if the chief could be captured they would at once lay down their arms and submit. On the 3rd of December Major Forbes' force was on the left bank of the Shangani river, and it was ascertained that Lobengula's waggons, in which he was travelling, had crossed the preceding day. The chief was known to be ill, and it was believed that there were not many soldiers with him prepared to fight. Major Wilson, with a picked body of men mounted on the best horses of the patrol, was there- fore sent on with directions to follow the spoor, but under any circumstances to return before dark. The main body of the patrol formed a lager on the bank of the stream. Instead of returning before dark Major Wilson remained beyond the Shangani that night, and at daylight on the morning of the 4th rode up to the chiefs waggons. The men who were with Lobengula showed fight, and the little band of Europeans was attacked and several of the horses were killed. Some might have escaped if they had chosen to ride away, but no one thought of abandoning his comrades, RHODESIA SOUTH OF THE ZAMBESI. 448 and so they stood side by side till their ammunition was all expended, and then, with heaps of Matabele dead around them, the last of the brave thirty-four met their fate as became gallant men. met their fate as became gallant men. While this tragedy was being enacted on the eastern side of the Shangani, Major Forbes was attacked on the western bank, but succeeded in beating off his assailants. The river meantime had come down in flood, and communication between the banks was cut off. RHODESIA SOUTH OF THE ZAMBESI. The firing in the action with Major Wilson's party was distinctly heard, but it was not possible to give assistance, and without being certain whether any had escaped or not, on the 5th of December Major Forbes was obliged to retire towards Bulawayo. The return march was attended with much suffering, but without further mishap. An occurrence must here be related which is the with much suffering, but without further mishap. An occurrence must here be related which is the only disgraceful event of the war. Lobengula had sent several messages to say he intended giving himself up, but had taken no steps to make his assertions good. That he ever intended to surrender to an armed force is in the highest degree improb- able, but that he would have been willing to meet Dr. Jameson and other white men for the purpose of arranging terms by which he might retain his position as chief in a modified form is tolerably certain. He had a thousand sovereigns in his waggon when he fled from Bulawayo, and when ill and hard pressed by his pursuers he sent mes- sengers with this money as a peace offering to the English commander, hoping to be granted terms. On the way his messengers fell in with two scouts COXCLUSIOX OF PEAC 449 of the Betshuanaland border police, to whom they delivered their message and the money, and these scoundrels concealed the matter and kept the gold. Whether Wilson's party would have been destroyed if the message had been conveyed to Major Forbes can be only conjecture, but the probabilities are that a satisfactory arrangement would have been made in time to prevent that lamentable occurrence. After all was over the matter was discovered, and the two policemen were tried and sentenced to fourteen years' imprisonment with hard labour, surely a light punishment for a crime of such magnitude. punishment for a crime of such magnitude. On the 22nd of November Babyane and four other indunas came in to inquire what terms of peace would be granted, and they were followed two days later by fourteen others, whose object was to obtain the like information. They were told that if they would surrender their arms they might return to their kraals and make gardens without being molested. The military huts were, however, destroyed. RHODESIA SOUTH OF THE ZAMBESI. On the 23rd of January 1894 Lobengula died of fever and gout about forty miles south of the Zambesi, and his death removed the only barrier to a pacification of the country. Before that time the greater number of his people had accepted British rule, and expressed themselves satisfied with the change ; though, of course, at heart they were far from being so. During the campaign nineteen Europeans, besides Wilson's party, lost their lives from all causes. Wilson's party, lost their lives from all causes. In this manner Matabeleland, the vast healthy plain in the centre of the continent, from four to six thousand feet above the level of the sea, was acquired RHODESIA SOUTH OF THE ZAMBESI. 450 by the British South Africa Company, and with Mashonaland took the name Rhodesia. Salisbury remained the seat of government, but on the 14th of November the high commissioner issued a pro- clamation constituting Matabeleland a subordinate district, and appointing a magistrate to reside at Bulawayo. Two townships were laid out : one at Bulawayo, and the other at Gwelo, the centre of what is believed to be a rich mining territory, some distance beyond. A few building sites were sold at Bulawayo privately before the 30th of July 1894, and on that and the two succeeding days three hundred and fifty-nine plots of ground were disposed of there by auction, and realised on an average rather over £\o\ each. On the 1st of August sixty-three building lots were sold by auction at Gwelo, and realised rather over £63 each. No town in South Africa, except Johannesburg, ever made such rapid progress in building as Bulawayo in the first two years of its existence. Courts of justice, churches, schools, and telegraph offices took the place of military kraals, and the lives and property of black men, as well as of white, became secure in a region where, just before, the midnight executioner and the ruthless despoiler were the savage agents of a despotic chief. The astonishing progress which the country was making was checked in 1895 by a severe drought and by the ravages of swarms of locusts, followed by an outbreak of rinderpest in a most destructive form among the cattle, The disbanded Matabele STATUE OF RHODES AT BULAWAYO. STATUE OF RHODES AT BULAWAYO. RHODESIA SOUTH OF THE ZAMBESI. RHODESIA SOUTH OF THE ZAMBESI. 45- warriors, weary of the monotony of life under a European government, and longing for excitement of some kind, were under any circumstances unre- liable subjects ; and when the priests or ministers of the mighty spirit that dwelt in a cave announced that the white men were sorcerers and the cause of the disasters, they accepted the statement as true. For various reasons they did not regard themselves as beaten in the war of 1893. They had not been punished in the way they had been accustomed to deal with defeated enemies, and could not understand why a conqueror should spare his opponents, except on the assumption that he was afraid of them. Much discontent also was caused by the manner in which the captured cattle had been disposed of. Towards the close of 1895 the country was denuded of the mounted police force for the purpose related in the chapter on the South African Republic, and when tidings were received that Dr. Jameson, the conqueror of Lobengula, was himself conquered and a prisoner, the greater part of the Matabele tribe rose in rebellion. Many of the miners* prospectors, and farmers were fortunate enough to be able to make their way to the towns, but some hundreds of men, women, and children were cruelly murdered. In every instance in which the Europeans met them in battle the insurgents were defeated, but a good many white men were killed while fighting. A large section of the Mashona, in the belief that the Matabele would be the victors, also rose in arms, proving how kAILRO 453 slender the tie of gratitude is with these people. Before December 1896 most of the chiefs had accepted the terms of peace which were offered, and which left considerable power in their hands, but parts of the territory remained for some months longer in a disturbed condition. Since that time the Bantu in Rhodesia have been peaceable, and some of them are showing an inclination to improve their condition. There has been a considerable influx from the territory north of the Zambesi, and about half of those working in the mines are foreigners by birth. foreigners by birth. To open up the country railroads were necessary, and nowhere else in South Africa were they con- structed more rapidly. RHODESIA SOUTH OF THE ZAMBESI. Very shortly two great trunk lines were completed, one joining the territory to the southern coast ports and, crossing the Zambesi at the Victoria Fails, penetrating Central Africa, the other connecting that line with Beira on the eastern coast. There are now, in 1916, 1,762 miles of railway open for traffic. Rhodesia suffered from the depression general in South Africa after the war of 1899- 1902, but it was the first of the different communities to show indica- tions of returning prosperity. A good class of settlers was attracted to it, who set themselves to develop both its agricultural and mineral resources. The war now taxing the energies of the British people everywhere is affecting Rhodesia seriously, for fully one-tenth of her adult male European resi- dents are serving in the field, and consequently industrial progress is retarded. Gold mining, how- RHObESlA SOUTH OF THE ZAMBESI. 454 ever, is being prosecuted vigorously, as the following figures will show: produced in 1913 to the value of £2,903,268, in 1914 £3,580,209, in 1915 £3,823,168. The mining industry differs from that of the Wit- watersrand, in being in the hands of small companies scattered over the whole country. scattered over the whole country. Other metals exported from Rhodesia in 191 5 were copper to the value of £224,314, chrome to the value of £175,792, asbestos £32,190, and silver £17,144. Of vegetable productions maize was the principal, of which 340,772 bags of 200 pounds each were ex- ported. Cattle breeding is extensively carried on by Europeans, who owned in 191 5 395,000 head. Europeans, who owned in 191 5 395,000 head. The area of Rhodesia south of the Zambesi is 143,830 square miles, or a square with a side 380 miles long, and the population consists of 24,000 Europeans, 750,000 Bantu, and 3,000 Asiatics and mixed breeds. BASUTOLAND, THE BETSHUANALAND PROTECTOR- ATE, AND SWAZILAND. BASUTOLAND, though lying in the very centre of the Union of South Africa, is a Bantu reserve quite independent of it except for a customs union. When it was taken over as a British possession, an agent was appointed by the high commissioner, who, with a few magistrates and some police, guided rather than governed the tribe. Moshesh died soon after- wards, and his principal heir, Letsie by name, had none of the old chief's ability. Molapo and Masupha, two other sons, were at the head of considerable sections of the tribe. All were unwilling to part with any real authority over the people, and gave just sufficient obedience to the British officials to secure protection, but carefully avoided conceding more. protection, but carefully avoided conceding more. In 1 87 1 the territory was attached to the Cape Colony, which thus became responsible for the preservation of order within it. The system of administration continued as before. Bantu law was recognised, except in a few of its worst features, but it was intended gradually to assimilate it to the law )3ASt)T0LAN£>. 4 of the colony. Sufficient hut-tax was easily collected to cover the cost of administration and to leave a small amount for public works, besides providing for liberal allowances to the chiefs and for the maintenance of schools. In a short time the tribe recovered from its losses in property, and increased in number as only Bantu can in a period of peace. Europeans believed that the British officials were gaining control over the people, and that the power of the chiefs was waning, but it was soon to be proved how little foundation there was for such a belief. In 1877 a wave of disturbance began to pass along the Bantu tribes connected with the Cape Colony, and when it subsided the govern- ment resolved upon a general disarmament. As soon as the measure was applied to Basutoland, the people rose in rebellion. Some clans, indeed, pro- fessed to be loyal, but only because others with whom they were at feud were on the opposite side. The colony spent a vast amount of money in trying to reduce the rebels to submission, but failed in the attempt, and the end was that in 1884 Basutoland was transferred back to the imperial government. BASUTOLAND, THE BETSHUANALAND PROTECTOR- ATE, AND SWAZILAND. The country since that date has been legally under the control of his Majesty's high commissioner for South Africa, and is under the direction of a British administrator, with magistrates to assist him. These officers have some moral influence, though the people obey only when it pleases them. Practically the government is carried on through the chiefs. Upon Letsie's death he was succeeded as paramount chief by his son Lerothodi, a man of considerable ability. AREA AND POPULATI 457 He again was succeeded by his son Letsie II, who died in 19 14, leaving no son, and was followed by his brother Griffith, the present paramount chief, who is a Christian of the Roman catholic faith. The people, who have profited greatly by the teaching of mission- aries of the Paris evangelical society, are the most industrious Bantu in South Africa, and are making good progress towards a civilised mode of life. good progress towards a civilised mode of life. Basutoland has an area of 10,293 square miles, equal to a square block of land with a side a little over one hundred and one miles in length. Its lower valleys are exceedingly fertile, but much of its surface is mountainous and unfit for agriculture. Its population has increased so rapidly that it now contains fully 350,000 Bantu, but only 900 Europeans. The white people are officials, missionaries, and traders, with their families. No others are permitted to settle in the country. The Betshuanaland Protectorate. The Betshuanaland Protectorate. The territory between the Molopo river on the south, the Botletle river (or now practically the Zambesi) on the north, the Union and Rhodesia on the east, and the late German protectorate on the west, is under the protection of Great Britain, which means that all white people living in it are under the jurisdiction of magistrates appointed by the high commissioner, and that the relationship of the Bantu tribes to each other is controlled by the same authority, though the government of the chiefs over their own people is not often interfered with. Much the greater portion of this territory is without surface THE DETSHUANALAND PROTECTORATE. 458 water, and is very thinly inhabited by Bushmen, Masarwa of mixed Betshuana and Bushman blood, and Betshuana who were formerly held by the tribes with fixed residences at the fountains in a condition of the most abject slavery. The principal compact tribes in the territory are the Bangwaketse, the Bakwena, the Bakatla, and the Bamangwato, the last under the far-famed Christian chief Khama, now a very old man. All of these tribes suffered greatly from Moselekatse, and indeed narrowly escaped complete destruction. The nature of the country to the westward of their principal kraals was such, however, that they were able to take refuge in it without fear of pursuit by the Matabele, who were unacquainted with the places where underground water could be found. Still, they lost all their property, and the greater number of them perished of hunger. The principal compact tribes in the territory are the Bangwaketse, the Bakwena, the Bakatla, and the Bamangwato, the last under the far-famed Christian chief Khama, now a very old man. All of these tribes suffered greatly from Moselekatse, and indeed narrowly escaped complete destruction. The principal kraal of the Bangwaketse is Kanye, and close by is the place where Makaba defeated the great Mantati horde that laid waste the southern portion of what is now the Transvaal province of the Union. The horde was at the time in a famishing condition, and was encamped in two sections some distance apart, so that Makaba was able to fall upon and defeat one division before the other could come to its aid. This Makaba, who is the legendary hero of the tribe, was afterwards killed in battle by the Makololo under Sebetoane, then moving northward to the Zambesi. The Betshuanaland Protectorate. The best of the Bangwaketse warriors were slain at the same time. It was only after Moselekatse was driven to the north by the TRIBES IN THE TERRITOR 459 emigrant farmers that the Bangwaketse were able to rally and settle down, but since that time they have lived unmolested, and have grown strong again. lived unmolested, and have grown strong again. Next to the north are the Bakwena, whose prin- cipal kraal is named Molopolole. This is the tribe with which the reverend Dr. Livingstone lived when Setsheli, who collected its fragments together and brought them out of the desert, was its chief. Setsheli acted generally with prudence, but on one occasion he forced war on the Transvaal farmers, and for many years there was strife between him and the Bakatla under the chiefs Khamanyane and his son Lintshwe. Still he guided his people so prudently that the Bakwena tribe grew and prospered. that the Bakwena tribe grew and prospered. The Bakatla are recent immigrants from the country to the eastward. Setsheli gave Khamanyane ground to live upon at Motshudi, and accepted him as a vassal, but as soon as he gathered strength he made war upon the Bakwena. A little later he died, but his son Lintshwe continued the strife until his independence was acknowledged. This chief, Lintshwe, who is now the head of the tribe, is a convert to Christianity, and keeps his people, who are prosperous, in good order. are prosperous, in good order. The Bamangwato are farther to the north. Moselekatse committed dreadful havoc with this tribe, but spared the heir to the chieftainship, whom he made a soldier in one of his regiments. As soon as it was safe to do so, the remnant of the tribe came out of the desert, and under the leadership of a man named Sekhomi, who was a member of the ruling house, went to reside at Shoshong, a place which THE BETSHUANALAND PROTECTORATE. 460 could be easily defended, though it was poorly sup- plied with water and otherwise an uninviting locality. Years passed away, and Matsheng, the legitimate chief of the Bamangwato, still remained a Matabele soldier. At last, through the influence of the reverend Dr. Moffat with Moselekatse, he was released, and was received with great joy as their ruler by the people at Shoshong. The Betshuanaland Protectorate. But he had acquired Matabele habits and tried to govern in the Matabele way, so his subjects became dissatisfied, and at length there was a revolution, when he was obliged to seek the protection of the Bakwena chief Setsheli, and Sekhomi became again the head of the Bamangwato. It was not possible for the tribe to grow powerful at a place like Shoshong, yet it dared not move away through fear of the Matabele. It managed to exist, and that was all. Sekhomi's eldest son, Khama by name, grew up to be a man of great ability, but between him and his father and his brother Khamane a feeling of estrangement arose. Khama, who had embraced the Christian religion, refused to comply with the ancient customs, and this was regarded by Sekhomi as rebellion. The young man, with a large party attached to him, then moved far away to the north- west, and settled on the banks of the Botletle river, but sickness carried off many of his followers there. With the remainder he returned, overthrew his father and his brother, and made himself chief at Shoshong. Khamane fled to the South African Republic, where a location was given to him. KHA 461 Khama proved an exceptionally able ruler, and by his skilful dealing with the British authorities secured the recognition by the imperial government of his claim to the enormous tract of country stretch- ing from below Shoshong to the Botletle river. As soon as a strong detachmeut of European police was stationed between him and the Matabele he removed from Shoshong to Palapye, and still later to Serowe, where there is no want of water and ground for gardens. He then permitted Kha- mane to occupy Shoshong. mane to occupy Shoshong. The Bakalahari, Masarwa, and Bushmen who roam over the waste parts of the protectorate are among the most miserable inhabitants of this earth. The former are Betshuana by blood, but long service to pitiless masters took all spirit out of them and made them intensely stupid. Since there have been European officials in the country, the lot of these poor creatures has been ameliorated, and their lives are now secure. In well-frequented places they are able to hold property, and even in the desert they are beginning to realise that their former masters have no longer any right to deal with them accord- ing to inclination. The Betshuanaland Protectorate. ing to inclination. At the principal kraals in the protectorate mis- sionaries of the London society have been stationed for many years, and large numbers of the people have embraced the Christian religion. The railroad from Capetown to the Zambesi passes near Kanye, Molopolole, and Serowe, thus bringing the most thickly settled part of the territory into connection with seaports, Three hundred and THE BETSHUANALAND PROTECTORATE. 462 ninety-four miles of its length, from Ramathlabama to Ramakvvabane, are within the protectorate. The territory is divided into two districts, over each of which an officer presides who receives his instructions from the high commissioner. All Europeans in it and all others who are not subjects of the chiefs, who commit offences, are tried by these magistrates. The chiefs govern their own people without any interference, except in very serious cases of crime, but their relationship to each other is subject to control, and they are required to collect money towards the maintenance of the white officials. In 1895, 6, and 7 there was a very severe drought, so that no crops could be raised. At the same time the rinderpest destroyed nearly all the horned cattle. The result was great distress, but the pro- tecting power came to the aid of the people by importing sufficient maize to prevent starvation and by employing many hundreds of young men in the construction of railroads. The protectorate is within the South African customs union, Its area is in round numbers 275,000 square miles, or equal to a square with a side of 525 miles, and its population consists of about one thousand Europeans, 175,000 Bantu, and 20,000 Masarwa and Bushmen. Swaziland. Swaziland. A tract of land enclosed on the north, west, and south by the Union, and on the east by the Portuguese territory, is a protectorate in which the European A tract of land enclosed on the north, west, and south by the Union, and on the east by the Portuguese territory, is a protectorate in which the European 463 THE SWAZ residents are ruled by a British official, and the Swazis are governed by their own chief under the supervision of an English commissioner. supervision of an English commissioner. The Swazis are closely akin to the Zulus, and were formed into a military tribe by an energetic chief named Sopuza, who was the only adversary that Tshaka was unable entirely to destroy. The tribe was greatly reduced in number, and was com- pelled to pay tribute to the Zulu chief, but was able to preserve a separate existence in its moun- tains. Sopuza was succeeded by his son Swazi, who managed to free himself from allegiance to Panda, and whose name the tribe then assumed. Swazi was succeeded by his son Umbandini, a good- natured but very weak and simple man, who was easily persuaded by Europeans to grant concessions of all kinds until there was nothing more left to part with. As long as his rebel and rival brother Umbelini lived he was, indeed, entirely subservient to white men. The South African Republic was always on good terms with the Swazis, and indeed regarded them as allies under its protection, but in 188 1 was obliged to acknowledge their independence in the convention of Pretoria. From that date until the close of 1906 various plans were adopted by Great Britain for maintaining order among the European residents in Swaziland, but the concessions granted by Umbandini — many of which had been acquired by the government of the South African Republic — prevented any of them from being successful. In 1906 the present system of dealing with the country SWAZILAND. 4 and people was introduced, and things have gone on smoothly since. His Majesty's high commis- sioner in South Africa has the supervision of all matters connected with the protectorate, and ap- points the local officials. The area of Swaziland is only 6,536 square miles, or a square block 81 miles each way, and the popu- lation consists of 200 Europeans and 90,000 Bantu. Swaziland. lation consists of 200 Europeans and 90,000 Bantu. Thus there are in the whole of British Africa south of the Zambesi about 1,365,079 Europeans, 5,581,365 Bantu, and 741,662 Asiatics, Hottentots, descendants of slaves, and mixed breeds. The natural rate of increase of the Bantu has been marvellous in the past, but is likely to be less rapid in the future, owing to the altered conditions of life of many of them, the greater cares and wants that are now felt, and the spread during recent years of chest diseases along the eastern coast and of syphilis among the Betshuana. PORTUGUESE SOUTH AFRICA. When the Portuguese at the close of the fifteenth century made their appearance in the Indian sea, they found stations on the African coast as far south as Sofala occupied by Arabs who carried on an extensive trade with Hindostan, Persia, and the adjacent countries. The people among whom the Arabs lived were not subject to them, except on the islands, but tolerated their presence and left them generally undisturbed on account of the benefits derived from trade. They formed many independent communities, generally intensely jealous of each other, and they had greatly deteriorated in blood owing to their ancestors having taken Bantu women as wives, so that they were easily reduced to subjection by the Europeans. Sofala was at that time a place of great reputation throughout the east, on account of the gold that was exported through it. This gold was collected by Bantu in places some distance from the coast, and was then obtained by the Arabs in barter. It is not possible to ascertain what quantity was sent 465 3 PORTUGUESE SOUTH AFRICA. 46 away yearly, but it was probably much greater in the fifteenth century than at a later date, because just when the Portuguese arrived the Makaranga tribe, that occupied the country in which the mines were situated, began to break into fragments bitterly hostile to each other. The paramount chief of this tribe had the title of Monomotapa, a word that by some means became misunderstood by Europeans, and was afterwards used as the name of the whole country from the lower Zambesi to Cape Agulhas. The ruins of buildings constructed in Mashonaland by some remote race of gold-seekers were known to the Arabs and Bantu, but no traditions extended back to their origin. back to their origin. Sofala has not a good harbour ; but, owing to its reputation, the king of Portugal resolved to take possession of it, and in September 1505 an expe- dition under Pedro da Nhaya arrived there with that object. The Arabs at first offered no oppo- sition, and a fort was built before hostilities broke out, when the sheikh was killed, and his people became vassals of the Europeans. The place proved exceedingly unhealthy, but it was maintained from that date onward as a trading station. PORTUGUESE SOUTH AFRICA. The mongrel Arabs were provided with goods, and went inland bartering gold and ivory, with which they returned to the Portuguese fort. Supplies were received from the magazines at Mozambique by means of small vessels manned by blacks under European officers, and the gold and ivory were sent to that station, which was the headquarters of the Portuguese on the East African coast. FORMATION OF TRADING STATIONS 46? For more than a quarter of a century Sofala remained the only post occupied by white people in the country. Then two outstations were formed on the southern bank of the Zambesi : one, named Sena, about a hundred and forty miles from the mouth of the river, the other, named Tete, a little over three hundred miles from the sea. Each was a trading station, to and from which merchandise was conveyed up and down the stream in boats. In 1 544 a man named Lourengo Marques ascertained that ivory was to be had from Bantu on the shores of Delagoa Bay, and thereafter a small vessel was sent yearly from Mozambique and remained for a few weeks trading for that article. But no fort or durable building of any kind was erected there, except by the Dutch, before 1787. except by the Dutch, before 1787. After the middle of the sixteenth century also Inhambane was visited occasionally by trading vessels from Mozambique, though permanent occu- pation commenced only in 1730. pation commenced only in 1730. Colonisation was not even attempted. The forts were guarded by a few soldiers, and the officers were almost the only traders. Whether soldiers or civilians, the Portuguese in East Africa degenerated rapidly. A European female was very rarely seen, and nearly every .white man consorted with Bantu women. Fever, when it did not kill them outright, deprived them of energy, and there was nothing to stimulate them to exertion. Cut off from all society but that of barbarians, often, until towards the close of the sixteenth century, without any religious minis- trations, sunk in sloth, and suffering from excessive PORTUGUESE SOUTH AFRICA. 46 heat and deadly malaria, no lives led by Europeans anywhere could be more miserable than theirs. The gold obtained in commerce was of trifling value, but it was believed that if the mines were in the hands of white men great wealth could be drawn from them. PORTUGUESE SOUTH AFRICA. Accordingly, in 1569 a splendidly equipped force of a thousand men, under an experienced officer named Francisco Barreto, was sent from Lisbon to effect the conquest. This expedition ascended the Zambesi to the junction of the Mazoe, and then attempted to proceed up the valley of the last-named stream. In several hotly contested actions with the natives it was victorious ; but fever, loss of all the transport cattle, the nature of the country, and the wiliness of the enemy, soon brought about its destruc- tion. Very (ew indeed escaped to return to Portugal. A little later another attempt was made, and an army went inland from Sofala, but effected nothing. went inland from Sofala, but effected nothing. In 1560 some missionaries of the Society of Jesus tried to establish themselves in South-Eastern Africa. One of them, by name Goncalvo da Silveira, was murdered by the monomotapa, and the others were obliged to withdraw, but successors resumed the work. About 1 580 the Dominicans entered the territory, and from that date until 1775 they had numerous stations scattered among the people. Their converts were told by thousands, yet within a single generation after the removal of the European teachers hardly a trace of Christianity was visible. In 1592 the garrisons of Tete and Sena were exter- minated by an invading horde, and when an attempt to recover those places was made by a body of troops 469 GENERAL DECLI from Mozambique, it failed disastrously. An arrange- ment, however, was made with the blacks by which the stations were reoccupied purely as trading posts. stations were reoccupied purely as trading posts. When at the highest point of their prosperity the Portuguese possessed, south of the Zambesi, only the forts that have been named and a few trading stations inland, where individuals were permitted to reside and carry on barter, upon payment to the chiefs of heavy taxes under the name of presents. In 1609, by taking the part of one of the rival claim- ants to the dignity of monomotapa, the Portuguese government appeared to be in a fair way of securing a firm footing, a prospect which was soon destroyed, however, by the incompetency of its officials. PORTUGUESE SOUTH AFRICA. After this many Portuguese took part in tribal wars, and there was even an instance of a powerful chief being deposed and replaced by another through the instru- mentality of a few missionaries and traders, but the government never obtained actual control over any large body of Bantu in the territory beyond gunshot of the forts. After the destruction of the Portuguese power in the eastern seas by the Dutch, the stations on the East African coast have no history worth recording until our own times. So low had they sunk that at the beginning of the nineteenth century there were only twelve hundred and seventy-seven professing Christians in the whole territory south of the Zambesi, and these included Asiatics, mixed breeds, and blacks, as well as the Europeans of all ages and both sexes. The only occupied places were Lourenco Marques in Delagoa Bay, Inhambane, Sofala, Sena, and Tete, PORTUGUESE SOUTH AFRICA. 47 but there were a few Europeans and Asiatics scattered about among the Bantu. The old trading and mission stations in the interior were so completely lost that even their sites were forgotten. even their sites were forgotten. The wars that followed the establishment of the Zulu power by Tshaka drove two bodies of fugitives northward along the coast, both of whom created fearful havoc. First came the Angoni, slaughtering all before them until they reached the border of the lake Nyassa, where they are living to-day. Follow- ing them came the followers of Manikusa, now called the Matshangana, who remained as lords in the belt of land between the central plateau and the sea, from the Zambesi to Delagoa Bay. In 1833 they captured the garrison of Lourenco Marques, and put every man to death. In 1834 they destroyed Inhambane, and killed all the inhabitants except ten. In 1836 they took Sofala, and left no Portuguese living there. Next they pillaged Sena, but some of the inhabitants took refusre on islands in the river, and were allowed to return upon payment of tribute. Towards the other Bantu they acted as wolves do towards sheep, for none were able to resist them. When the first fury of the conquest of the country by Manikusa was over, the Portuguese stations were reoccupied by feeble garrisons of convicts and blacks, who existed there on sufferance. PORTUGUESE SOUTH AFRICA. After 1838, when the emigrant farmers from the Cape Colony began to settle on the highlands of the interior between the Vaal and Limpopo rivers, Dela- goa Bay acquired a value it never had before. It was the nearest port to the newly occupied territory, MACMAHOrfS AWAR 47 1 and efforts were repeatedly made to open a road to it. These did not succeed for many years, owing to the prevalence of fever near the coast and to the inter- mediate belt of land being infested with the tsetse fly, but the position of the bay made it certain that in time all the difficulties of establishing communication through it between the South African Republic and the outer world would be overcome. In 1 86 1 an event took place which greatly bene- fited the Portuguese. This was the death of Manikusa, chief of the Matshangana, whose name was a watchword of terror in the country. Two of his sons fought with each other for the chieftainship, and the Portuguese, by aiding Umzila, who conquered his opponent, obtained a cession of territory, and what was of more importance, a declaration of vassalage and a friendly neighbour. vassalage and a friendly neighbour. In July 1869 a commercial treaty was concluded between the governments of Portugal and of the South African Republic, and in it a boundary line — the one now existing — was laid down. This treaty was viewed unfavourably by Great Britain, and that country thereupon put forward a claim to the southern and eastern shore of Delagoa Bay, based on certain documents obtained from Bantu chiefs by Captain Owen, of the royal navy, in 1823. The Portuguese government refused to acknowledge this claim, and it was referred to the arbitration of the president of the French republic, who in July 1875 decided against Great Britain. In March 1887 a Company was formed in London to construct a railway from Lourenco Marques to PORTUGUESE SOUTH AFRICA. 47 the border of the South African Republic, under a concession from the Portuguese government. The line when completed was seized by the Portuguese authorities, owing to a clause of the concession not having been carried out, and the amount of compensation to be paid to the shareholders was not settled until 1899. PORTUGUESE SOUTH AFRICA. From the border to Pretoria the line was continued by the Netherlands South Africa Company, and in July 1895 was opened for traffic. A large proportion of the commerce of the republic then found its way to Delagoa Bay, and with the development of the goldfields the traffic became almost as much as the line could carry. Lourenco Marques then became an important town, and has continued to grow and prosper ever since. Great steamers began to frequent the bay, and as no expensive harbour works were needed, as at Capetown, East London, and Durban, the shipping and landing charges were relatively small. At present ships of all sizes lie as safely as in a dock along a quay of great length, on which there are all modern appliances for discharging and receiving cargo. The town is now the capital of the whole province of Mozambique, and in it the governor-general resides and the legislative council meets. But it differs from all the towns of British South Africa, inasmuch as five men live in it for every woman, because very few people wish to make it their permanent home. Its population, according to a recent census, consisted in round numbers of 3,000 Portuguese, of whom 500 were females, 1,600 other BEI 473 Europeans, of whom 340 were females, 1,700 Asiatics, of whom only 70 were females, and 3,500 Bantu labourers, of whom 50 were females. Nearly 50,000 Bantu labourers, engaged in different parts of the Portuguese territory, are forwarded every year through Lourenco Marques to work in the gold- mines at the Witwatersrand. There is not much commerce with the surrounding territory, the existence of Lourenco Marques as an important town being due almost entirely to the transit through it of passengers and merchandise to and from the interior. Yet it is to-day much the most important place in the Portuguese possessions in South-Eastern Africa. Europeans, of whom 340 were females, 1,700 Asiatics, of whom only 70 were females, and 3,500 Bantu labourers, of whom 50 were females. Nearly 50,000 Bantu labourers, engaged in different parts of the Portuguese territory, are forwarded every year through Lourenco Marques to work in the gold- mines at the Witwatersrand. PORTUGUESE SOUTH AFRICA. There is not much commerce with the surrounding territory, the existence of Lourenco Marques as an important town being due almost entirely to the transit through it of passengers and merchandise to and from the interior. Yet it is to-day much the most important place in the Portuguese possessions in South-Eastern Africa. Next to it comes Beira, a town unknown before 1890, and which sprang into being as the ocean terminus of a railroad from Rhodesia. Beira is at the mouth of the Pungwe river, not far north of Sofala. It has a good harbour, capacious, with considerable depth of water, and is easy of access. The Arabs had once a small settlement there, but the Portuguese never occupied the place in olden times, and when the Asiatics retired it fell into such utter decay that for more than three centuries it was completely forgotten. completely forgotten. When the pioneer expedition of the British South Africa Company was on the way from the Cape Colony to Salisbury in September 1890, Messrs. Colquhoun and Selous, two of its officers, turned out of the road to visit the chief Umtasa at Manika, who was induced by them to place himself under British protection and to grant a concession of PORTUGUESE SOUTH AFRICA. 4/ mineral and other rights in his country. This chief was claimed by the Portuguese as a subject, and some little time afterwards several Portuguese officials visited his kraal. There, on the 14th of November, they were arrested by Major Forbes, of the British South Africa Company, and were sent to Salisbury as prisoners. to Salisbury as prisoners. There was great excitement in Portugal when intelligence of the events at Umtasa's kraal reached that country. Bands of students pressed forward as volunteers to defend the honour of their flag, and were sent with all haste to Beira. Upon the arrival of the first party at that port, they, with some negroes from Angola, were directed to occupy a recently erected trading station named Andrada, about twenty English miles from Umtasa's kraal. They arrived at that station on the 5th of May 1891. Not far distant was a camp of the British South Africa Company's police, fifty-three in number, com- manded by Captain Heyman. PORTUGUESE SOUTH AFRICA. On the nth of May a Portuguese force, consisting of about a hundred Europeans and three or four hundred Angola blacks, was sent out to make a reconnais- sance, and at two in the afternoon fell in with the English pickets, who retired upon the camp. The Portuguese followed, and an action was brought on, which resulted in their total defeat, with a heavy loss in killed and wounded. There were no casualties on the British side. The whole Portuguese force then fled precipitately to the sea coast, abandoning Andrada, which the British Chartered Company's men occupied on the THE TREATY OF LISB 475 following day. They found there some stores, of which they took possession as lawful spoil of war, but the most valuable part of the booty con- sisted of eleven machine guns that had been left behind. Negotiations between the two governments in Europe were shortly afterwards completed, and in June 1 89 1 a treaty was signed at Lisbon in which a boundary between the British and Portuguese possessions was defined, the same as exists at present. The treaty provided further that in the event of either of the powers proposing to part with any territory belonging to it south of the Zambesi the other should have a preferential right to purchase the territory in question or any portion of it. A very important clause provided for the immediate survey and speedy construction of a railroad from the British territory to Beira, and for encouraging commerce by that route. And now, for the first time, the Portuguese territory in South Africa was defined on all sides, and was secured from invasion by Bantu tribes beyond the border. In accordance with the terms of the treaty, a com- pany was formed and a railway was constructed con- necting Beira by the line through Rhodesia with the great northern trunk road, so that unbroken com- munication was secured between the mouth of the Pungwe river and all the ports on the southern coast. Beira, which is built on a tongue of sand projecting into the estuary of the Pungwe, is the healthiest site PORTUGUESE SOUTH AFRICA. 47 on that part of the coast. After the construction of the railway the town advanced with rapid strides, and soon became a place of considerable importance. PORTUGUESE SOUTH AFRICA. The population at the close of 191 3 consisted of 778 male and 309 female Europeans, 582 male and 53 female Asiatics, 5,207 male and 1,420 female Bantu labourers, and 185 male and 176 female mixed breeds. and 185 male and 176 female mixed breeds. The whole of Portuguese South Africa between the Zambesi and Sabi rivers, except the district of which Tete is the centre, is now ruled by the Mozambique Company. This Company was formed in 1888 as a mining corporation, the acquisition of the goldfields of Manika being the inducement to the shareholders to subscribe the capital. In February 1891, however, the Company obtained a charter, which conferred upon it extensive privileges. It has a monopoly of all mineral and commercial rights, which it may lease in detail to associations or individuals ; it is under an obligation to introduce a limited number of colonists, and it has taxing and govern- ing powers subject to the supreme authorities at Lisbon. The chief official of the Mozambique Company in the territory between the Zambesi and Sabi rivers has the title of governor, and resides at Beira. The country is divided into districts, over each of which a commissioner, subordinate to the governor, pre- sides. The officers who administer justice are ap- pointed by the supreme authorities in Lisbon, and are not subject to the authority or superintendence of the Company, but to the governor-general of Mozambique. THE MOZAMBIQUE COMPAN 477 The Company has carried out its agreement with regard to the introduction of European settlers, and has given liberal assistance to those who were desirous of becoming farmers. In consequence of this, re- markable progress has been made, though gold- mining has only produced about ^24,000 yearly. The sugar cane has been introduced, and has been found to thrive exceedingly well along the banks of the rivers. In 191 3 23,000 tons of sugar were exported, and there is a good prospect of a great development of this industry. Ten tons of cotton, 2,600 tons of maize, 1,100 tons of oil seeds, and 3,800 tons of mangrove bark were also exported in that year. year. PORTUGUESE SOUTH AFRICA. A census on the 31st of December 191 3 showed the population of the territory under the administra- tion of the Mozambique Company to consist of 1,401 male and 471 female Europeans, 1,033 male and 62 female Asiatics, 131,488 male and 140,073 female Bantu, and 779 male and 705 female mixed breeds. Sena and Sofala have not recovered their old im- portance, small as that was, and are now insignificant places compared with Beira. At Sofala the ocean has recently so encroached upon the land that the ancient fort has quite disappeared. The tract of land between the Limpopo and Manisa rivers from the border of the Union to the sea is held by another Company under a concession from the government in Lisbon granted in November 1893. government in Lisbon granted in November 1893. Inhambane, the port of the territory between the Limpopo and the Sabi, has made some progress of PORTUGUESE SOUTH AFRICA. 47 late years, though, as it is mainly dependent upon trade with the Bantu, it is far less important than Lourenco Marques or Beira. The village consists of a church and a few houses and shops. a church and a few houses and shops. There remains the territory of which Tete is the seat of government, between the Zambesi and the Anglo-Portuguese border west of the Mozambique Company's district. It is occupied entirely by Bantu, except the village, which contains a church and from twenty to thirty stone houses of European pattern. It is protected by a small garrison of black troops with white officers, who occupy a fort overlooking the river. The European residents, officials included, do not number more than twenty-five or thirty, for the commerce of the place is small. the commerce of the place is small. The government of Tete is military in form and subordinate to Mozambique. The Jesuits have established a mission here, and also at a station a few miles distant. There are extensive coalfields in the neighbourhood, and it is possible that, owing to them, the village may some day become a thriving place. The banks of the Zambesi are nowhere perfectly healthly for Europeans, but there is less fever at Tete than at any place farther down the stream, and the Portuguese residents, who are ac- climatised, do not trouble themselves about it. PORTUGUESE SOUTH AFRICA. Throughout the whole territory from the Zambesi to Lourenco Marques difficulties in controlling the Bantu were experienced until recently ; but Portugal has opened her eyes to the fact that it is necessary to employ other and better forces than convicts and un- civilised negroes, such as were formerly engaged, and PRESENT CONDIT 4/9 she has succeeded in establishing her authority firmly. Towards the close of 1895 the great chief Gungunhana, son and successor of Umzila, assumed an attitude which compelled the government to bring him to account by force of arms. He was captured and banished in January 1896, and his defeat secured the peace of the country. With his downfall the last of the great military tribes that had their origin in the time of Tshaka disappeared, to the great relief, not only of the Europeans, but of all the other Bantu in South Africa. Lines of splendid steamships now connect Lourenco Marques, Inhambane, and Beira with Europe by way of the Red sea, and with all the British ports along the southern coast. The commerce of the territory has made rapid progress, and the prospects of Portuguese South Africa are brighter to-day than at any previous time since Pedro da Xhaya built the first fort on the river bank of Sofala. (LATE) GERMAN SOUTH-WEST AFRICA. ALONG the western coast of South Africa, between the Kunene river on the north and the Orange river on the south, and extending eastward to the twentieth meridian from Greenwich, with an arm thrust out north of Betshuanaland, is a great tract of land, some 325,000 square miles in extent, that until the nine- teenth century was far advanced was regarded as little better than a desert. Instead of rising in a series of steps to an elevated plain, as on the eastern side of the continent, there is here a narrow belt along the Atlantic shore sloping upward to the escarpment of the interior plateau, which is almost on a level with the Kalahari desert and Betshuana- land. ALONG the western coast of South Africa, between the Kunene river on the north and the Orange river on the south, and extending eastward to the twentieth meridian from Greenwich, with an arm thrust out north of Betshuanaland, is a great tract of land, some 325,000 square miles in extent, that until the nine- teenth century was far advanced was regarded as little better than a desert. Instead of rising in a series of steps to an elevated plain, as on the eastern side of the continent, there is here a narrow belt along the Atlantic shore sloping upward to the escarpment of the interior plateau, which is almost on a level with the Kalahari desert and Betshuana- land. The coast belt is a waste of sand blown about by the wind, so that where a hill stands one week there may be a hollow the next, and where there is usually less than an inch of rainfall in a year. In a few limited localities, however, it is possible for human beings to live there. The only harbours on the coast are Walvis Bay, a sheet of deep water between the RAINF 48 1 mainland and a long sandspit curving round and then running parallel with it, and Angra Pequena fcalled by the Germans Luderitzbucht), a little inlet farther south, where Bartholomeu Dias landed in 1487, and which he named Angra dos Ilheos. No fresh water is found at either of these places, so that what is needed for drinking must be condensed from the ocean. (LATE) GERMAN SOUTH-WEST AFRICA. The habitable part of the country is the elevated land between the coast belt and the Kalahari, so that it has a desert on each side. The part between the tropic and the Orange river is called Great Nama- qualand. Before the German occupation it was thinly peopled by Hottentots, who were divided into a number of little clans independent of each other, and who maintained themselves chiefly by herding a few cows and sheep that needed exten- sive areas to pasture on. The surface is from three to six thousand feet above the level of the sea, and is broken up by mountain ranges and hills and sandy plains. The rainfall is only six to eight inches in a year, and even this is confined to a few heavy storms. The country is drained by a nullah — for there is seldom flowing water in it, — running from north to south into the Orange river. Proceeding northward, the country improves, and the rainfall increases. At Windhoek, a little north of the tropic, there is an average yearly fall of four- teen inches, and at Grootfontein, in 19J0 south latitude, as much as twenty-four inches falls. Cattle breeding can be carried on here with success, and even agriculture is possible in some limited localities, (LATE) GERMAN SOUTH-WEST AFRICA. 482 This part of the territory is termed Hereroland or Damaraland, and is occupied by the Herero tribe of Bantu and the people called by Europeans Berg Damaras, who are Bantu by blood, but speak a dialect of the Hottentot language and live after the manner of Bushmen. Still farther north, approaching the Kunene river, is Ovamboland, whose inhabitants are Bantu, forming several tribes independent of each other, but all called Ovambo by Europeans. other, but all called Ovambo by Europeans. Between the Herero tribe and the Hottentots war had been carried on ever since the former entered the country, sometimes one party, sometimes the other, getting the upper hand for a short time. In 1 79 1, when Europeans first became acquainted with them, the Swakop river was, roughly speaking, the dividing line between them, and so it is to-day. In 1 8 14 the London society commenced mission work in the country, and later the Wesleyans followed, but both were replaced by Rhenish missionaries, who made Great Namaqualand and Hereroland their special field of labour. special field of labour. (LATE) GERMAN SOUTH-WEST AFRICA. In 1840 the Hereros were thoroughly beaten by the Hottentot captain Yonker Afrikaner and his allies, and were reduced to a state of abject servi- tude. Their principal chief was compelled to tend Afrikaner's cattle, most of which had once been his own property and had been taken from him. For more than twenty years this was the condition of things, but then the Hereros managed to get pos- session of weapons, and in 1863 the chief and some of his people fled with the cattle they were tending, and took refuge at the Rhenish mission station HERERO WAR OF INDEPENDENCE 483 Otjimbingue. There they were attacked by the Hottentots, but they defended themselves bravely and beat back their assailants, of whom some two hundred were killed. This was the commencement of what was after- wards termed the Herero war of independence. Jan, son of Yonker Afrikaner, was the leader on the Hottentot side, but some of the clans of his people refused to assist him, and several able white men, such as Mr. Frederick Green and the Swedish naturalist and author Charles J. Andersson, led his opponents. Hostilities were carried on for seven years, during which time great atrocities were com- mitted by the Hottentots on several occasions. Otjimbingue was more than once attacked, without being taken, but most of the other mission stations were pillaged and destroyed. Many hundreds of Hottentots lost their lives, as did scores of Hereros, till at length the Europeans in the country managed to bring about peace. In September 1870 Jan Yonker Afrikaner agreed to the terms dictated by the reverend Dr. Hahn, which declared the Hereros to be perfectly independent, and assigned Windhoek as his (Jan Yonker's) residence, where a Rhenish missionary would also be stationed. So ended the Herero war of independence, entirely in favour of that party. A little later a number of farmers from the South African Republic, with their families, made their appearance in Damaraland. They had abandoned their homes because the president was not of the orthodox faith, and withal most incredible suffering (LATE) GJSXMAN SOUTH- WFST AFRICA. 484 had crossed the Kalahari in search of a country to settle in. This they found at last at Mossamedes, in Portuguese territory, but for several years they remained in Damaraland. (LATE) GERMAN SOUTH-WEST AFRICA. Their presence alarmed the Herero chiefs, who, acting by the advice of their European friends, in June 1874 sent to ask protec- tion from the governor of the Cape Colony. This led to the matter being brought before parliament, and the adoption in 1875 °f a resolution in favour of annexing the coast as far north as Walvis Bay and making inquiry into the condition of the country generally. generally. To obtain information Mr. W. C. Falgrave was appointed, and in 1876 he went inland from Walvis Bay and made a tour through the country. He found the Herero chiefs and Hottentot captains willing to come under British protection and be guided by the advice of an English official, but not to relinquish any authority over their own people. The Hereros offered to cede a large tract of good pasture land for the use of Europeans, to raise a revenue for the payment of the white official. revenue for the payment of the white official. In the following year Mr. Falgrave was sent back to make inquiries into details, but before anything further could be done in the matter, the ninth Kaffir war broke out, and the Sprigg ministry, that then came into power, was disinclined to burden the colony with fresh responsibilities in connection with the tribes beyond the northern border. Sir Bartle Frere, looking ahead to what might happen, desired to pro- claim a British protectorate over the whole country, but Lord Carnarvon refused to give his consent, and BEGINNING OF GERMAN OCCUPATION 485 would only permit the occupation of Walvis Bay and a small tract of land about it. In 1880 war between the Hereros and Hottentots was resumed, and so bitter was the feeling between them that the principal Herero chief gave orders that all Hottentots, irrespective of age or sex, who could be found were to be put to death, and from one hundred and fifty to two hundred were actually murdered in cold blood. Attacks and forays on both sides went on after this, without either party gaining a decided advantage, until June 1882, when the southern Hottentot captains agreed to peace, but Jan Yonker and another continued a guerrilla warfare. At this time a scramble by European powers for unappropriated spots on the earth's surface was com- mencing, and Germany was looking keenly for places on which to found colonies. (LATE) GERMAN SOUTH-WEST AFRICA. She had as yet no foreign possessions, so the hosts of people that left her shores yearly were all lost to her. Yet it was not supposed in British South Africa that she would plant her flag upon the least inviting part of the continent, the Sahara alone excepted, where the only attraction for Europeans was the healthiness of the climate. But a surprise was in store for those who were allowing things to go on in fancied security. In 18S3 a merchant of Bremen named Luderitz estab- lished a trading station at Angra Pequena, and on the 1st of May of that year purchased the inlet and a small tract of sand dunes about it from the Hottentot captain of Bethany for two hundred rifles and ^100 {LATE) GERMAN -SOUTH-WEST AFRICA. 4$6 in money, and in August he completed the pur- chase of another and much larger tract of sand from the same captain for sixty rifles and .£600. He then applied to his government for protection, and communications were opened between the German authorities in Berlin and the foreign office in London on the subject, though nothing was said or written that could lead to the supposition that Germany had any design of taking possession of the whole country. The secretary of state now signified his consent to the annexation of the coast line by the Cape Colony, provided Great Britain should not be called upon to bear any portion of the expense, and this being agreed to, in July 1884 both houses of the Cape parliament resolved that such annexation was expedient. Meantime Mr. Palgrave had been sent again to Great Namaqualand and Hereroland to ascertain whether the various chiefs and clans there were still desirous of coming under British sovereignty. He proceeded to various parts of the territory, and received the assurances of the leading chiefs that they were not only desirous, but anxious, to place themselves under British rule as a means of restor- ing and preserving peace, but he had not concluded his task when he was recalled, as the action of the German government had made his mission useless. At that time British subjects held concessions from Herero and Namaqua chiefs of greater value than that which Mr. Luderitz had obtained from GERMAN PROTECTORATE PROCLAIMED. 487 the captain of Bethany. (LATE) GERMAN SOUTH-WEST AFRICA. The establishments along the coast for catching and drying fish were owned in Capetown, and part of the trade of the country was carried on by Englishmen. On the other hand the missionaries were Germans, except in Ovamboland, where they were Finnish, and part of the little trade was in German hands. On the 7th of August 1884, by order of the emperor Wilhelm I, Captain Schering, of the corvette Elizabeth, hoisted the German flag at Angra Pequena and proclaimed a protectorate over the coast and a belt of land along it twenty geographical miles in width from the Orange river to the 26th degree of south latitude, and a day or two later Captain Von Raven, of the German gunboat Wolf, issued at Cape Frio a similar proclamation respecting the coast from the 26th degree of latitude northward to Cape Frio, excepting the British territory at Walvis Bay. On the 15th of August the German consul in Capetown informed the government of the first of these facts, and soon the full significance of the occurrence — that another and possibly disturbing factor had been introduced into South African affairs — came to be realised. The twelve guano islands off the coast and Walvis Bay with the little territory around it re- mained under the British flag and connected with the Cape Colony, but shortly all the remainder of the country from the Orange river in the south to the Portuguese possessions in the north, and from the Atlantic ocean on the west to the twentieth (LATE) GERMAN SOUTH-WEST AFRICA. 488 meridian from Greenwich on the east, with an additional tract in the shape of a rectangle on the north-east, which made the Zambesi its border there, became a dependency of the German empire. With characteristic energy the new African pro- prietor set to work to make something of the country that to all appearance at the time was hardly worth having. The first seat of the local government was at Otjimbingue, but Windhoek was soon acquired from Jan Yonker, and the authorities then removed to that place. Here money was lavishly expended in the erection of substantial public buildings, and in ornamenting a town that would be worthy of its position as the capital of the tflrst of Germany's oversea colonies. (LATE) GERMAN SOUTH-WEST AFRICA. Here, within a quarter of a century was to be seen one of the loftiest wireless telegraph stations in the world, where messages were received from and transmitted to Berlin. Here was a town with big hotels and stores, with churches and newspapers, with a museum and public library and railway station, all lit by electricity, where a few years before nothing more pretentious than Jan Yonker's huts was standing. It was strongly fortified, and was the headquarters of the troops in the country. Around it were located a number of farmers, dams were constructed and fountains were opened, so that patches of land could be irrigated, and all possible encouragement and assistance was given to the settlers. This was the case in every part of the country This was the case in every part of the country where water could be obtained by sinking artesian CATTLE REARING. 489 wells or opening fountains, and wherever it could be conserved dams or reservoirs of some kind were constructed. By a very large expenditure of money it was found possible to irrigate little plots of ground here and there, but never sufficient to raise the grain and vegetables needed for home consumption. With cattle rearing it was very different, for all kinds of stock throve well. In many parts the AFrlMm1-. WEI. I. AT UKAMAS, SHOWING METIIOH OF RAISING WATER. AFrlMm1-. WEI. I. AT UKAMAS, SHOWING METIIOH OF RAISING WATER. farms needed to be fifteen or twenty thousand acres in extent to support a family, but in others five thousand or even less would suffice. Foreign stock of the best breeds — horses, horned cattle, asses, camels, sheep, goats, and pigs — were imported, and for their propagation the country was found to be eminently suitable. There can be very little doubt that this will be the permanent industry of the future, (LATE) GERMAN SOUTH-WEST AFRICA. 49° Copper-mining from the first occupied the atten- tion of the German authorities. It had long been known that rich deposits of this metal were to be found at Otavi, in Hereroland, but it was supposed that its distance from the sea would make it too costly to remove it. (LATE) GERMAN SOUTH-WEST AFRICA. The Germans carefully in- spected the locality, found another mine at a place called Tsumeb, a little farther away, constructed a railroad to connect both these mines with Swakop- mund, where they could ship the ore, and then commenced working the deposits with a good profit profit They were certainly very far from being idle. But for years they were unable to devote their full energy to opening up the country, owing to wars with the Hottentots and the Hereros, who resented their methods of protection. In 1893 the war with the Hottentots, who were under the leadership of an able captain named Hendrik Witbooi, com- menced. It was carried on after the guerrilla manner by the Hottentots, who moved about in the moun- tains where no European force could follow them, and made their appearance where and when they were least expected. The war lasted nearly ten years, and was followed in 1904 by hostilities with the Ovaherero. Most fearful atrocities were committed by these barbarians, who murdered German men, women, and children indiscriminately, but the retaliations of the white soldiers were hardly less reprehensible. There are many honourable exceptions, but as a general rule the German, particularly if he is a DISCOVERY OF DIAMON 49 1 Prussian, does not permit feelings of humanity to stay his hand in striking a foe in any way or with any weapon, and in this instance full play was given to his passions. Herero men were shot, women and children were driven into the desert without food and left to die of starvation. Some fled across the Kalahari and took refuge in British territory, but more than half of the tribe perished. The war lasted nearly five years, and as many as 19,000 German soldiers were employed in it. It and the war with the Hottentots cost the lives of 1,487 men, besides 907 wounded who recovered, and an expenditure in money of £30,000,000. and an expenditure in money of £30,000,000. The discovery early in 1908 of diamonds beneath the sand in the neighbourhood of Angra Pequena gave great impetus to the settlement. The gems were mostly very small, but they were in immense numbers, and their collection proved exceedingly profitable. (LATE) GERMAN SOUTH-WEST AFRICA. Every atom of food consumed on the fields had to be imported, every drop of water used had to be condensed from the sea, so that only companies with considerable capital could engage in the undertaking, but very shortly the desert waste presented a busy scene. The revenue was so swollen by the taxes on diamonds that it soon came to equal the expenditure for ordinary — not military — purposes, a substantially built town sprang into existence at Angra Pequena, and the exports rose to large figures. rose to large figures. In 191 3 diamonds were exported to the value of nearly £3,000,000, copper to the value of nearly £400,000, and tin to the value of £31,600. The {LATE) GERMAN SOUTH-WEST AFRICA. 492 European civil population consisted then of 8,328 men, 4,683 women, and 1,625 children. There was also a regular military force of nearly 2,000 men in the country, and not only were the able-bodied civilians liable to be called out for purposes of war, but coloured men also. There were approximately 15,000 Hottentots, 18,000 Berg Damaras, and 20,000 Hereros left. With the Ovambo in the extreme north the Germans never had much to do, and their land had not been exploited. They were supposed to number about 80,000 souls. How many people there were in the strip of land along the upper Zambesi or how many Bushmen were wandering about in waste places cannot be stated even approximately. approximately. The government was far more bureaucratic than that of any British colony. The governor, ap- pointed by the kaiser in Berlin, was practically almost absolute. There was indeed a council, but half the members were nominated by the governor, and it had no power of initiating measures. Great Namaqualand and Hereroland were divided into fifteen districts, each under an official subject to the governor. According to our ideas, there was too much control and direction, too little left to individual initiative, more regard than necessary paid to military formality ; but to the German mind this did not seem objectionable. Owing to the Kalahari desert being on its eastern border, there was no commerce overland between the German and British possessions in South Africa, but MILITARY PRECAUTIOXS. 493 steamers were continually plying between Table Bay, Angra Pequena, and Swakopmund, and a good deal of agricultural produce found a ready sale there for money. (LATE) GERMAN SOUTH-WEST AFRICA. For military purposes forts had been built in various places and lines of observation posts esta- blished, which were connected with each other by telegraphs and telephones. There were 1,297 miles OBSERVATION POST OBSERVATION POST of railway in the country, a large portion of which was designed for strategic purposes in case of war with Great Britain. To the German mind this was not acting in a hostile manner, but merely using necessary precautions against possible clanger. On the 4th of August 1 9 14, when war actually did break out, this was the condition o( things in German South-West Africa. of railway in the country, a large portion of which was designed for strategic purposes in case of war with Great Britain. To the German mind this was not acting in a hostile manner, but merely using necessary precautions against possible clanger. On the 4th of August 1 9 14, when war actually did break out, this was the condition o( things in German South-West Africa. {LATE) GERMAN SOUTH-WEST A ERICA. 494 To enable the imperial troops to be withdrawn for service in Europe, the Union government immediately proposed to take upon itself the full burden of defence of the country, including the naval station in Simon's Bay, which offer was accepted by Great Britain. The authorities in England considered it expedient also to effect the conquest of German South-West Africa, and the Union ministry undertook that task. A special session of parliament was convened to meet on the 9th of September, to provide the necessary funds, and preparations for the undertaking were made without delay. The Germans were chal- lenging an attack, as on two occasions raiding parties entered British territory from Nakob, on their south-eastern border, and they sent troops to Raman's drift, on the Orange river, to occupy that door of communication with the Cape province. It is now known that their object was to attract dis- affected individuals in the Union to join them, but this was not suspected at the time. this was not suspected at the time. To check them a strong body of South African troops, with horses and stores of all kinds, was sent by sea to Port Nolloth, where they arrived early in September. (LATE) GERMAN SOUTH-WEST AFRICA. It took some time to make the neces- sary arrangements, when the troops were forwarded by the Copper Company's railway to the nearest point of the line to Raman's drift, and from that place the fourth regiment of mounted riflemen marched in two days through the desert country to the drift, defeated the enemy there, and on the 15th of September obtained possession of the post. COURSE OF THE WA 495 The remainder of the column followed as quickly as possible, and a strong detachment was sent forward in pursuit of the Germans. Colonel Grant, of the mounted rifles, who was in command of the detach- ment, followed the enemy to a place named Sand- fontein, where he found he had been drawn into a trap and was surrounded by a greatly superior force. He defended himself stubbornly until the two field guns he had with him were made useless by the opposing artillery, he himself was wounded, and all hope of successful resistance was gone, when he was obliged to surrender unconditionally, 26th of September 1914. It was the greatest disaster in the course of the war, for not only were the casual- ties heavy, but the remainder of the column was paralysed by the blow and retired from Raman's drift. Another column left Table Bay a few days after the one just mentioned, and on the 19th of September landed at Angra Pequena, which the Germans did not attempt to defend. The column took possession of the town, but did not follow the retreating German troops, who destroyed the railway line behind them as they retired towards the interior. as they retired towards the interior. A third column was on the way to Swakopmund when information reached Sir Duncan Mackenzie, who commanded it, that a rebellion had broken out in the Union, and that until it was suppressed further operations against German South-West Africa could not be undertaken. He therefore joined the column at Angra Pequena, where he assumed the chief command, but did nothing more (LATE) GERMAN SOUTH-WEST AER1CA. 496 than hole! that town and repair the railway line for some distance with materials forwarded from Capetown. Capetown. As the Germans feared an attack by a man-of- war upon Swakopmund, on the 17th of August they evacuated that town and removed as much of the loose property there as could be taken away. (LATE) GERMAN SOUTH-WEST AFRICA. Hut as no attack was made they soon returned, and then took possession of the Union port of Walvis Bay, which was not far off to the southward. When parliament assembled in Capetown General Botha asked for approval of his project of operations against the German territory and for the money needed for the purpose, on the ground that as the British empire was at war the Union, which formed part of it, was at war also, and it was the duty of its people to do all they possibly could to assist against the enemy. He was warmly supported by General Smuts, but was opposed by General Hertzog, whose motion of disapproval, however, was lost in the house of assembly by ninety-two votes against twelve. of assembly by ninety-two votes against twelve. Parliament by an overwhelming majority in both houses approved of General Botha's proposals, but now a big difficulty arose. There was a party in the Transvaal province and Orange Free State that was opposed to his policy of common action with the English section of the community, because they feared the loss of their nationality."TThey wished to remain, like the French in Canada, a separate people, and dreaded absorption Jlike that which took place after the conquest of New Netherlands by the English in the seventeenth century. THE REBELL 497 There were a few — fortunately a very few — who hoped to recover independence by alliance with the Germans, and there were a good many who were indisposed to fight what they regarded as England's battles, not theirs. They certainly did not realise what the consequences to themselves would be in the event of Germany being the victor in her struggle for the dominance of the world. They rose in what they termed an armed protest, but which was actual rebellion. General Beyers, who was in chief command of the Union forces, resigned his post as soon as the de- cision of parliament was known, and this of course prevented immediate action. The prime minister, General Botha, announced his intention of taking the field in person, but before anything further could be done Colonel Maritz, who was in command of a column directed to enter German territory by way of Nakob, betrayed his trust, was the means of causing a portion of his force to be made prisoners, and with the remainder openly joined the enemy. (LATE) GERMAN SOUTH-WEST AFRICA. General Beyers and Christian de Wet, the famous guerrilla leader in the war of 1 899-1902, also took up arms on the rebel side, and the insurrection assumed formidable propor- tions. On the 1 2th of October martial law was proclaimed throughout the Union. A sufficient force, comprising both Dutch and English speakers, was speedily assembled, and operations against the rebels were undertaken. Some of them fought stubbornly, but many were captured and others lost heart. General Botha 33 33 33 {LATE) GERMAN SOUTH-WEST AFRICA. 49$ issued a proclamation offering an amnesty to all the rank and file who would surrender before the 2 1 st of November, and this had the effect of causing many of them to give themselves up. On the ist of December De Wet was captured, and a few days later Beyers was drowned when attempting to escape across the Vaal river, so that on the ioth of that month General Botha thought he could safely announce that the rebellion was practically suppressed. This turned out to be premature, however, for until the middle of February 191 5 the insurgents with their German allies not only occupied a considerable area of Union territory, but attacked Union forces and made prisoners at several places, notably at Nous in Little Namaqua- land, from which post they compelled a strong gar- rison to retire. But in February 191 5 some six hundred of them surrendered, and Maritz, their ablest leader, fled to Angola, when the disturbance was finally quelled. The government inflicted the lightest of punishments upon the insurgents, and only one individual — Captain Fourie, who was tried by court martial — suffered the death penalty. His conduct had been similar to that of Colonel Maritz. Arrangements for the invasion of the German ter- ritory were then completed. Already a strong force had been sent by sea from Capetown to Walvis Bay, and on its approach the enemy had abandoned that place, so that it landed without opposition. A recon- noitring party that^went to make observations at Swakopmund found that town deserted also, not INVASION OF GERMAN TERRITORY 499 only by the German troops but by the civil inhabi- tants, and the public property much damaged, so it was taken in possession, 14th of January 191 5. (LATE) GERMAN SOUTH-WEST AFRICA. This column then made preparations for an advance along the railway eastward as soon as reinforcements should arrive with General Botha, who was to assume the chief command. Meantime General Mackenzie had occupied posi- tions along the line connecting Angra Pequena with the interior, and had repaired the damage to the railway through the sand belt, so that he also was only waiting the order to advance. The Germans had adopted the habits of Bushmen, by laying con- cealed mines and poisoning the water in places that they abandoned, so that the greatest care had to be taken when moving about. taken when moving about. From the southern side two columns advanced. One of them crossed the Orange at Raman's drift. and the other at Schuit drift, a long way farther up. From the south-eastern side there were also two columns of invasion, one by Nakob, whose object was to get possession of the military railway that terminated near this place, and the other by Riet- fontein, almost due east of Angra Pequena. The last of these had to traverse a desert country, where in one part of the route no water was to be had for a hundred and twenty miles, but it overcame this difficulty by using motor-cars, by which supplies for men and horses were forwarded. The other three southern columns had also to undergo many hardships and privations in marching through a dry and desolate country, where water was obtainable {LATE) GERMAN SOUTH-WEST AFRICA. 500 only at long intervals. General Smuts was in chief command of the southern forces, and upon him rested the duty of making the arrangements. rested the duty of making the arrangements. To facilitate the attacks on the eastern side a railway was laid down from Kenhart to Upfngton on the Orange river. The country was flat, so there were no obstacles, and the rails were laid at a rate of over two miles a day. This was of very great service, and it has since been continued from Uping- ton to Kalkfontein on the German line, so that now one can go by train from any part of the Union to any part of what was German South-West /\frica. But there was not time to construct the eighty-eight miles onward from Upington before the final advance was made. (LATE) GERMAN SOUTH-WEST AFRICA. The invading columns were opposed, though not so undauntedly as they had anticipated, and on the fall of Nabas, the strongest military post in the south- eastern part of the territory, which was taken by storm on the 7th of March, the Germans retreated to the central railway. They were greatly out- numbered by the Union forces, which were ad- vancing against them from different directions, and so they adopted the plan of falling back always after skirmishes and putting as many obstacles as possible in the way of pursuers. While the southern and eastern columns were closing in on the central rail- way, General Mackenzie was advancing from the west. His supplies were forwarded by sea from Capetown to Angra Pequena, and then by the rail- wax- that ran eastward to Seeheim, where it joined the central line running to the north. THE HON. LIEUTENANT-GENERAL J. C. SMUTS. THE HON. LIEUTENANT-GENERAL J. C. SMUTS. THE HON. LIEUTENANT-GENERAL J. C. SMUTS. {LATE) GERMAN SOUTH-WEST AFRICA. 502 With his cavalry he rode north-eastward from Aus, and reached the central line before the other columns. He found the Germans had got past him, so instead of intercepting them as he had hoped to do, he followed them up, and at Gibeon, about a hundred and twenty miles north of Seeheim, he overtook them. There they made a stand, and on the 26th of April a sharp engagement took place, in which they were defeated with a loss GERMAN RESERVIST PRISONERS. GERMAN RESERVIST PRISONERS. of eight killed, thirty wounded, and one hundred and sixty-six made prisoners. Having an unbroken railway behind them, the remainder managed to escape to the north, but they left six guns and a large quantity of war material behind, which fell into the hands of the Union troops. Mackenzie's casualty list was a heavy one, but he had the satisfaction of knowing that the battle of Gibeon had completed the conquest of Great Namaqualand. of eight killed, thirty wounded, and one hundred and sixty-six made prisoners. Having an unbroken railway behind them, the remainder managed to escape to the north, but they left six guns and a large quantity of war material behind, which fell into the hands of the Union troops. Mackenzie's casualty list was a heavy one, but he had the satisfaction of knowing that the battle of Gibeon had completed the conquest of Great Namaqualand. (LATE) GERMAN SOUTH-WEST AFRICA. GENERAL BOTHA TAKES COMMAND. 503 On the nth of February General Botha reached Swakopmund, and took command of the forces there. The sea was open, owing to Admiral Sturdee's destruction on the 8th of December 19 14 of the German squadron at the Falkland islands which was on its way to South Africa, and steamers could safely convey troops and stores from Cape- town to Walvis Bay. Strong reinforcements of men and horses were sent on, and arrived at their destina- KLOWN-UP BRIDGE AT EPAK.O. KLOWN-UP BRIDGE AT EPAK.O. tion a few days after the general. Among them was the first regiment of Rhodesian volunteers, that had offered its services to assist the Union. It took some time to make the necessary arrange- ments for an advance, so that it was the 20th of March before the nearest posts occupied by the Germans to the eastward could be attacked. On that day three stations were taken and over two hundred prisoners were made. The enemy when (LATE) GERMAN SOUTH-WF.ST AFRICA. 504 retreating destroyed the railway behind, and it could only be relaid slowly, as the material was received from Capetown, so General Botha aban- doned it, and marched up the bed of the Swakop nearly to Otjimbingue. Then he turned to the north, and on the 5th of May occupied Karibib, the junction of the line from the south with that from Swakopmund to Tsumeb. The retreating- Germans were already beyond that place. They had allowed many of their reserve men to return to their farms, and had left Windhoek, their capital, to the mercy of the invaders. to the mercy of the invaders. From Karibib a strong detachment of cavalry was sent to Windhoek, which General Botha accom- panied in a motor-car. Xo resistance of any kind was offered, and on the 12th of May the town was surrendered by the municipal authorities, when a garrison of Union troops was stationed in it. Martial law was proclaimed, but the civil residents were not disturbed. The general then returned to Karibib, and shortly the pursuit of the retreating Germans was com- menced. It was an easy matter for them to move on by rail, but it was a very different matter for the Union forces to follow them. (LATE) GERMAN SOUTH-WEST AFRICA. General Botha adopted a plan somewhat similar to that of the Zulus in their early wars, only his line was far more extended than theirs. His infantry formed the centre, correspond- ing to the Zulu breast. His cavalry formed the horns, but they rode far out and ahead of each of the infantry wings. Only men capable of enduring great hardships and of existing with very little food, END OF GERMAN RUL 505 and that of the coarsest kind, could have gone on as these men did day after day until, when the end of the railway was almost reached, they rounded in on the Germans and brought them to bay. on the Germans and brought them to bay. There was now no way of escape, and on the 9th of July 191 5 the German governor Dr. Seitz, and the military commander Francke were obliged to surrender with 3,947 officers and men, thirty-seven pieces of artillery, twenty-two machine guns, and all the stores that they had not destroyed. Very liberal terms of capitulation were accorded to them. The Union prisoners who were at Tsumeb, and who had been scantily fed and often harshly treated, were of course immediately released. Some of them, with Colonel Grant, had been nearly ten months in confinement. So ended German rule in South-West Africa. A strong garrison of Union troops was left in the country. British officials took the places of those appointed by the kaiser, and Generals Botha and Smuts with most of their men returned to their ordinary duties. Very shortly, however, a large proportion of the young men went to Europe to serve the cause of civilisation there, and the others, under the leadership of General Smuts, while this is being written (June 19 16), are fighting in another part of the African continent for the honour of the Union and the liberty of many nations. CHRONOLOGICAL TABLE OF EVENTS A.D. 1487. Discovery of the Cape of Good Hope by Dias. 1487. Discovery of the Cape of Good Hope by Dias. 1497. Doubling of the African continent by Vasco da Gama. 1503. Discovery of Table Bay by Antonio de Saldanha. 1503. Discovery of Table Bay by Antonio de Sald 1505. Occupation of Sofala by the Portuguese. 1505. Occupation of Sofala by the Portuguese. 1 5 10. Defeat of Portuguese by Hottentots near Table Valley. 1569. Disastrous expedition under Francisca Barreto against Bantu of South Africa. 1 570-1600. Terrible destruction caused by Bantu invaders from the country beyond the Zambesi. 1 570-1600. Terrible destruction caused by Bantu invaders from the country beyond the Zambesi. country beyond the Zambesi. 1 59 1. First visit of English ships to Table Bay. 1595. First voyage of the Dutch to India. 1595. First voyage of the Dutch to India. 1602. Formation of the Dutch East India Company. 1652. Commencement of the Dutch settlement in South Africa. 1655. Introduction of the vine. 1655. Introduction of the vine. 1657. Discovery of the Berg river. 1657. Discovery of the Berg river. 1658. Introduction of slaves. 1658. Introduction of slaves. 1659. First Hottentot war. 1659. First Hottentot war. 1660. Discovery of the Elephant river. 1672. Purchase of territory from Hottentot chiefs. 1673. Commencement of second Hottentot war. 1679. Foundation of Stellenbosch. 1679. Foundation of Stellenbosch. 1685. Discovery of the copper mines of Namaqualand. 1688. Arrival of the first Huguenot settlers. 1700. First occupation of land on the second plateau. 17 1 3. First outbreak of small-pox. 17 1 3. First outbreak of small-pox. 17 1 3. First outbreak of small-pox. 1722. Great loss of life by gale in Table Bay. 1722. Great loss of life by gale in Table Bay. CHRONOLOGICAL TABLE OF EVENTS. 508 [737. Wreck of another fleet in Table Bay. [737. Wreck of another fleet in Table Bay. 1742. First use of Simon's Bay in winter months. 1742. First use of Simon's Bay in 1740. Foundation of Swellendam. 1742. First use of Simon's Bay in winter months. 1740. Foundation of Swellendam. 1 7 .9- • Exploration of the country eastward to the Kei. 1 7 .9- • Exploration of the country eastwa 1755. Second outbreak of small-pox. 1 7 .9- • Exploration of the country eastward to the Kei. 1755. Second outbreak of small-pox. 1755. Second outbreak of small-pox. 1701. CHRONOLOGICAL TABLE OF EVENTS Exploration of southern part of Great Namaqualand, 1701. Exploration of southern part of Great Namaqualand, 17O7. Third outbreak of small-pox. 17O7. Third outbreak of small-pox. 1 779- Commencement of first Kaffir war. 17O7. Third outbreak of small-pox. 1 779- Commencement of first Kaffir war. 1781. Arrival of French troops to defend the Cape Colony against the English. the English. 1780. Foundation of Graaff-Reinet. the English. 1780. Foundation of Graaff-Reinet. 1780. Foundation of Graaff-Reinet. [789. Commencement of second Kaffir war. 1780. Foundation of Graaff-Reinet. [789. Commencement of second Kaffir war. [789. Commencement of second Kaffir war. 1792. Commencement of the Moravian mission. 1705. Surrender of the Cape Colony to the English. 1700. Commencement of the London Society's mission. 1700. Commencement of the London Societ Commencement of third Kaffir war. 1S01. Exploration of the southern part of Betshuanaland. [803. Restoration of the Cape Colon)' to the Dutch. [803. Restoration of the Cape Colon)' to the Dutch. [806. Second surrender of the Cape Colony to the English. 1809. Subjection of all Hottentot inhabitants to colonial laws. 181 2. fourth Kaffir war. 181 2. fourth Kaffir war. 1815. Slachter's Nek insurrection. 1815. Slachter's Nek insurrection. 1815. Slachter's Nek insurrection. 1815. Slachter's Nek insurrection. [818. Commencement of fifth Kaffir war. 1815. Slachter's Nek insurrection. [818. Commencement of fifth Kaffir war. 1S20. Arrival of large body of British settlers. 1822. Commencement of Zulu wars of extermination 1824. Erection of first lighthouse on South African coast. [830. Settlement of the Matabele in the valley of the Marikwa. [834. Emancipation of the slaves in the Cape Colony. [834. Emancipation of the slaves in the Cape Colony. [835. Sixth Kaffir war. [835. Sixth Kaffir war. [S36. beginning of great emigration from the Cape Colony. '•sv- flight of the Matabele to the country north of the Limpopo. 1838. Dreadful massacres of Europeans by Zulus. '•sv- flight of the Matabele to the country north of the Limpopo. 1838. Dreadful massacres of Europeans by Zulus. [840. Subjection of the Zulu tribe to the emigrant farmers. [840. Subjection of the Zulu tribe to the emigrant farmers. 1842. Occupation of Natal by a British military force. 1842. Occupation of Natal by a British military force. 1843. Creation by the British government of Griqua and Basuto treaty states. 1843. Creation by the British government of Griqua and Basuto treaty states. CHRONOLOGICAL TABLE OF EVENTS. 509 A.D. 1844. Creation of Pondo treaty state. 1844. CHRONOLOGICAL TABLE OF EVENTS Creation of Pondo treaty state. 184'. Commencement of seventh Kaffir war. 184'. Commencement of seventh Kaffir war. 1847- Creation of the province of British Kaffraria. 1845. Proclamation of British sovereignty over the territory between the Orange and Vaal rivers. 1845. Proclamation of British sovereignty over the territory between the Orange and Vaal rivers. the Orange and Vaal rivers. 1850. Commencement of eighth Kaffir war. 1850. Commencement of eighth Kaffir war. 85 1 . Commencement of first Basuto war. 1850. Commencement of eighth Kaffir war. 1 85 1 . Commencement of first Basuto war. 1852. Acknowledgment by Great Britain of the independence of the South African Republic. South African Republic. 1854. Abandonment by Great Britain of the Orange River Sovereignty. South African Republic. 1854. Abandonment by Great Britain of the Orange River Sovereignty. ■ Establishment of the Orange free State. South African Republic. 1854. Abandonment by Great Britain of the Orange River Sovereignty. ■ Establishment of the Orange free State. ■ Establishment of the Orange free State. Introduction of a representative legislature in the Cape Colon)-. ■ Establishment of the Orange free State. Introduction of a representative legislature in the Cape Colon)-. Colon)-. 1858. Second Basuto war (first with the Orange Free State). 1858. Second Basuto war (first with the Ora 1865. Commencement of third Basuto war. 1865. Commencement of third Basuto war. Annexation of British Kaffraria to the Cape Colony. Annexation of British Kaffraria to the C 1S67. Commencement of fourth Basuto war. 186S. Annexation of Basutoland to the British Empire. 186S. Annexation of Basutoland to the British 1869. Discovery of diamonds in South Africa. 1871. Creation of the province of Griqualand West. 1871. Creation of the province of Griqualand West. 1872. Introduction of responsible government in the Cape Colony. 1872. Introduction of responsible government in the Cape Colony. 1S77. Annexation of the South African Republic to the British Empire. 1S77. Annexation of the South African Republic to the British Empire. Empire. Commencement of ninth Kaffir war. Empire. Commencement of ninth Kaffir war. 1879. British conquest of Zululand. 1879. British conquest of Zululand. 1879. British conquest of Zululand. 1S80. Annexation of Griqualand West to the Cape Colony. 1S80. Annexation of Griqualand West to the Cape Colony. Commencement of fifth Basuto war. 1S80. Annexation of Griqualand West to th Commencement of fifth Basuto war. 1881. Recovery of independence by the South African Republic. 1884. CHRONOLOGICAL TABLE OF EVENTS Commencement of the German Protectorate of the western coast belt north of the Orange river. 1884. Commencement of the German Protectorate of the western coast belt north of the Orange river. 1885. Creation of the province of British Betshuanaland. 1S86. Opening of extensive gold-fields in the South African Republic. 1S86. Opening of extensive gold-fields in the South African Republic. 1887. Annexation of Zululand to the British Empire. 1889. Grant of a charter to the British South Africa Company. 1S93. Introduction of responsible government in Natal. Defeat of the Matabele by the British South Africa Company. Defeat of the Matabele by the British South Africa Company. CHRONOLOGICAL TABLE OF EVENTS. 5IO A.D. 1894. Annexation of Pondoland to the Cape Colony. 1895. Annexation of British Betshuanaland to the Cape Colony. 1896. Year of troubles. Insurrection at Johannesburg. Invasion of the South African Republic by the British South Africa Company's forces under Dr. Jameson. Terrible loss of life and property by explosion of dynamite at Johannesburg. Insurrection in Rhodesia. Great distress in many localities from drought and ravages of locusts. Almost complete destruction of cattle by rinderpest in a vast extent of territory. Foundering at sea of a passenger steamer from the Cape to England, and loss of all on board except three individuals. 1899 (nth of October). Commencement of war between Great Britain and the two republics. 1899 (nth of October). Commencement of war between Great Britain and the two republics. 1902 (31st of May). Restoration of peace by the Treaty of Vereeniging. The former republics become British colonies. 1903 to 1909. Years of great depression throughout South Africa. Vereeniging. The former republics become British colonies. 1903 to 1909. Years of great depression throughout South Africa. 1 910. Formation of the Union of South Africa. 1 910. Formation of the Union of South Africa. 1914 and 1915. Conquest of German South-West Africa by forces of the Union. 1914 and 1915. Conquest of German South-West Africa by forces of the Union. INDEX Bangwaketse tribe : account of, Adam Kok : particulars con- cerning, 226, 229, 230, 231, 232, 233, 234, 244, 245, 309, and 367 45 Bantu : description of, 5 ; suffer severely from small-pox, 82. See Angoni, Bambata, Bapedi, Baramapulana, Basuto, Bom- vana, Damara, Dingana, Dini- zulu, Faku, Fingos, Gaika, Gangelizwe, Herero, Hintsa, Hlubi, Kaffir, Khama, Kreli, Langalibalele, Lewanika, Lo- bengula, Makana, Manikusa, Mantati, Matabele, Matshan- gana, Matsheng, Moselekatse, Moshesh, Ndlambe, Ovambo, Panda, Sandile, Sekhomi,Set- sheli, Tshaka, Umtasa, Urn- zila, and Zulu Afrikander Bond : formation of, 290 290 d'Almeida, Francisco : is killed near Table Valley, 15 near Table Valley, 15 Angoni tribe : particulars con- cerning, 470 Angora hair : export of, 431 Angora hair : export of, 431 Angra Pequena : discovery of, 8 Angra Pequena : discovery of, 8 Anti-convict agitation : account of, 248 and 249 of, 248 and 249 Arabs : occupation of the East African coast by, 465 Art Gallery in Capetown : men- tion of, 85 Asiatics : introduction into Cape Colony, 35 ; introduction into Natal, 338 B Bakatla tribe : account of, 459 Bakwena tribe : account of, 459 Bamangwato tribe : account of, 459 Bambata : rebellion in 1906 of, 359 of, 248 and 249 Arabs : occupation of the East African coast by, 465 Art Gallery in Capetown : men- tion of, 85 zila, and Zulu Bantu in Natal : dealings with, 337 Asiatics : introduction into Cape Colony, 35 ; introduction into Natal, 338 337 Bapedi rebellion : account 0^383 Baramapulana tribe : revolt of, 379 ; are brought under sub- jection, 398 B Barberton : foundation of, 395 Barreto, Francisco : expedition of, 468 history of, 455 ; area of, 457 ; population of, ib. history of, 455 ; area of, 457 ; population of, ib. IXDEX Blueberg : battle of, 134 Blueberg : battle of, 134 Blythswood missionary institu- tion : mention of, 32 1 Basuto ii ibe : origin of, 171: rebellion against the Cape Colony of, 456 Bomvana clan : account of, 307 Boomplaats : battle of, 247 Boomplaats : battle of, 247 Boshof, Jacobus Nicolaas : is second president of the Orange Krce State, 364 Basuto wars : account of the war with the Orange River Sove- reignty, 257 : cause of the war of 1858 with the Orange Free State, 365 ; account of this war, 366 : account of the war of 1865 6 with the Orange Free Stale, 369 ; and of the war of 1867-8, 370 Botha, Hon. General Louis: mention of, 414, 416, 420, 426, 496, 499, 503, and 504 426, 496, 499, 503, and 504 brand, Advocate Jan Hendrik : is fourth president of the Orange Free Stale, 368 war of 1867-8, 370 Batavian Republic : constitution of the Cape Colony under, 129 Orange Free Stale, 368 British Ivaffraria: creation of tin- province, 242 ; is annexed to the Cape Colony, 287 Beaufort West : foundation of, 150 British settlers of 1820 : account of, 156 Beira : foundation of the town of, 473 : description of, 475 Berea : battle of, 263 British South Africa: population of, 464 British South Africa Company : formation of, 433 ; war with the Portuguese, 474 Berea : battle of, 263 Berg Damaras : particulars con- cerning, 482 Bronkhorst Spruit : action at, cerning, 482 Bethelsdorp mission station : foundation of, 130 Betshuanaland south of the Mo- lopo : becomes British terri- tory, 300: is annexed to the Cape Colony, 301 Bulawayo : foundation of the town of, 450 390 Burgers, Rev. Thomas Fran- cois : is the second president of the South African Repub- lic, 382 '75 Betshuanaland Protectorate : ac- count of, 457 : area of, 462 : population of, ib. : form of government of, ib. B Burgher Senate, Cape Colony : creation of, j 1 7 ; abolition of, Burgher Senate, Cape Colony : creation of, j 1 7 ; abolition of, Bushmen : description of, 1 ; mention of, 44, 63, 73, 83, 88, 461, and 492 government of, ib. Betshuana clans : dissensions among, 299 Bushmen : description of, 1 ; mention of, 44, 63, 73, 83, 88, 461, and 492 Birkenhead: wreck of the, 252 Black circuit : account of, 140 Bloemfontein : foundation of, Bloemh Caledon, Lord : administration of. 139 to 142 Caledon, Lord : administration of. 139 to 142 INDEX 3 Cape Colony : foundation of, 25 ; first surrender to the English, no : restoration to the Dutch. 128; second surrender to the English, 133 ; is ceded to Great Britain, 146 : extent in 1854, 267 ; industries of, 317 ; area of, ib. Cape of Good Hope : discovery of, 10 Capetown : descriptions of, 55, 83, and 94 : is the Legislative capita] of the Union, 423 capita] of the Union, 423 Cascades : mention of, 304 Castle of Good Hope: erection of, 40 Defence Force Act : particular- concerning. 42') Cathcarl. Sir George : adminis- tration of, 233 et : 1) concerning. 42') Delagoa Bay : references to. 74, 196, 225. 431, 407. 470. and 1) concerning. 42') Delagoa Bay : references to. 74, 196, 225. 431, 407. 470. and Cattle diseases : mention of, 320 and 430 Cattle farmers: origin of, 49 155 47i Depopulation of the territory between the Orange and Lim- popo rivers : account of, 165 Destruction of all their property by the Xosas : account of. 277 et seq. 47i Depopulation of the territory between the Orange and Lim- popo rivers : account of, 165 Census of the Cape Colon}-: in 1700, 52; m 1792, 1805, 132 ; in 1819, 97 \ 155 Chinese labourers : introduction of. 420 ; repatriation of, ib. Census of the Cape Colon}-: in 1700, 52; m 1792, 1805, 132 ; in 1819, 97 \ 155 1805, 132 ; in 1819, Chinese labourers : introduction of. 420 ; repatriation of, ib. 47i popo rivers : account of, 165 Destruction of all their property by the Xosas : account of. 277 et seq. of. 420 ; repatriation of, ib. Churches : particulars concern- ing. 39- 53- 79, 87, 101. 102, 147, 160, 233. Dias. Bartholomeu : voyage of, 8 B 323, 339, 381, 396, and 430 Coal : abundance of in Natal, 336 ; and in the Transvaal Province, 402 Cole, Sir Lowry : administration of, 17S Colley. Sir George: death of. 392 Colonists : particulars concern- ing the hist. ^2 Complaints of the burghers against the government of the Cape Colony, 9 3 Churches : particulars concern- ing. 39- 53- 79, 87, 101. 102, 147, 160, 233. 323, 339, 381, 396, and 430 Diamonds : discovery on the Vaal river ^\. 203 ; exporta- tion ii^, 318 : discovery in Gxeat Namaqualand of, 401 Diamonds : discovery on the Vaal river ^\. 203 ; exporta- tion ii^, 318 : discovery in Gxeat Namaqualand of, 401 Diamond fields : particulars con- cerning, 294 Dias. Bartholomeu : voyage of, 8 Gxeat Namaqualand of, 401 Diamond fields : particulars con- cerning, 294 Dias. Bartholomeu : voyage of, 8 Cole, Sir Lowry : administration of, 17S Diugana, Zulu chief: references to. 167, 171, 204, 203. 206, 2ir, 212. 213. '214. and 213 Dinizulu. Zulu chief: career pf, 332 and 354 Domestic animals : of the Bush- men, 2 ; of the Hottentots, ib 4 Diugana, Zulu chief: references to. 167, 171, 204, 203. 206, 2ir, 212. 213. '214. and 213 Dinizulu. Zulu chief: career pf, 332 and 354 34 /XDF.X 514 Dominican mission : account of, 468 Dorthesia : ravages of, 289 Drakenstein : settlement of, 51 Droughts damage caused by, 319 D'Urban, Sir Benjamin: ad- ministration of, 178 to 193 Durban, town of: importance of, 341 and 431 ; description of, any Exploration of South Africa : particulars concerning, 30, 44, 57, and 83 Exports of the Union in 10,13, 431 Exploration of South Africa : particulars concerning, 30, 44, 57, and 83 57, and 83 Exports of the Union in 10,13, 431 57, and 83 Exports of the Union in 10,13, 431 F minis 1 to 19 Durban, town of: importance of, 341 and 431 ; description of, any Faku, Pondo chief: references to, 223, 228, 308, 309, and 356 Dutch East India Company : formation of, 19 ; forms a refreshment station in Table Valley, 23 : decline of, 90 ; insolvency of, 1 1 1 age ribed Fifth Kaffir war: account of, 152 342 Fingos : origin of, 164 ; par- ticulars concerning, 190, 253, 306, and 307 257 First Basuto war : account of, i of 1 1 1 Dutch language : is proscribed in public offices in the Cape Colony, 1 58 ; is restored to official equality with English, 273 First Hottentot war: account of, st tentot : ount 37 First Kaffir war : account of, 89 First Matabele war with the Chartered Company, 441 etseq. Fish River : after 1 780 is the eastern boundary of the colony, 88 Dutch reformed church : see Churches Dutch ships : first voyage to India of, 18 88 Form of government of the Cape Form of government of the Cape Colony: before 1759, 53; in 1854, 269 ; in 1872, 272 Eighth Kaffir war, account of, 251 Ivok : creation of, 228 ; de- struction of, 245 Germany : takes possession of South-West Africa, 487 ; has destructive wars with the Hottentots and Hereros, 490 Germany : takes possession of South-West Africa, 487 ; has destructive wars with the Hottentots and Hereros, 490 (late) German South-West Af- rica : description of, 480 ; area of, id. ; condition before the German occupation, 481 ; industries of, 489 ; exports, 491 ; population, 492 ; form of government of, ib.\ is con- quered by Union forces, 494 et seq. ; mode of government after the surrender, 505 Gibeon : encounter at, 502 Ginginhlovu : battle of, 349 Griquas : origin of, 226 Griquas : origin of, 226 Griquas and emigrant farmers, war between, 230 Guano Islands : are annexed to the Cape Colony, 292 the Cape Colony, 292 Gungunhana, chief of the Mat- shangana : overthrow of, 478 Gwelo : foundation of, 450 Gungunhana, chief of the Mat- shangana : overthrow of, 478 Gwelo : foundation of, 450 Gungunhana, chief of the Mat- Gungunhana, chief of the Mat- shangana : overthrow of, 478 Gwelo : foundation of, 450 Eighth Kaffir war, account of, 251 Eighth Kaffir war, account of, 251 251 English, the : attempt to seize the Cape Colony in 1781, but fail, 91 ; conquer it in 1795, 105 ; in 1803 restore it to the Dutch, 128; in 1806 conquer it again, 135 37o H3 ' Franchise : in the Orange Free State, 363 ; in the South African Republic, 397 it again, 135 English ships : first visit to South Africa of, 17 French, the : take possession of Saldanha Bay, but do not keep it, 43 ; in 1 781-3 defend the Cape Colony against the English, 91 Etshowe : relief of, 349 Executive council, Cape Colony : creation of, 179 Expansion of the Cape Colony : account of. Frere, Sir Bartle : mention of, 344 and 484 Expansion of the Cape Colony : account of. Frere, Sir Bartle : mention of, 344 and 484 Expansion of the Cape Colony : account of. Frere, Sir Bartle : mention of, 344 and 484 tNDUX 5> G Grahamstown : foundation of, 145 ; is attacked by Kaffirs, Grahamstown : foundation of, 145 ; is attacked by Kaffirs, Gaika, Xosa chief : references to, 99, 123, 131, 151, 152, 153, 154, 187, and 18S ; death of, 187 Great emigration from the Cape Colony: causes of, 195; ac- count of, ib. et seq. da Gama, Vasco : voyage of, 10 da Gama, Vasco : voyage of, 10 Game : abundance of in early days, 46 ; destruction of, 318 Gangelizwe, Tembu chief: refer- ence to, 307 Genadendal : mention of, 322 German immigrants: account of, 285 Grey, Sir George : administra- tion of, 277 : mention of, 366 and 367 da Gama, Vasco : voyage of, 10 Game : abundance of in early days, 46 ; destruction of, 318 days, 46 ; destruction of, 318 Gangelizwe, Tembu chief: refer- ence to, 307 Griqualand East : account of, 309 Genadendal : mention of, 322 Griqualand West : is made Brit- ish territory, 297 ; is annexed to the Cape Colony, 299 Genadendal : mention of, 322 German immigrants: account of, 285 Genadendal : mention of, 322 German immigrants: account of, 285 German immigrants: account of, 285 Griqua treat)- state under Adam. Ivok : creation of, 228 ; de- struction of, 245 Griquas : origin of, 226 Griquas and emigrant farmers, war between, 230 Griqua treat)- state under Adam. II 73, 81, 87, 103, in, 121, 122, 124, 126, 130, 132, 140, 172, 177, 178, 239, 252, 305, and 322 Hottentots: purchase of terri- • tory from, 42 Hottentot settlement at the Kat river : account of, 178 Hottentot wars : account of, }7 and 45 Huguenots: arrival of, 51 I Imbembesi : battle of, 445 Immigration from Great Britain : account of, 155 and 235 Imports of the Union in 1913, 431 Indians : introduction into Natal of, 338 Ingogo : action at, 39 r Inhambane : particulars concern- ing, 467, 470, and 477 Insect life : abundance of, 320 Inyesane : battle of, 348 Isandlwana : terrible disaster at, 346' J Jameson, Dr. "Leander Starr : • raid into the Transvaal of, 399 ; further mention of, 434, 437, 444, and 452 first intercourse with Euro- peans, 10 ; particulars con- cerning, 25, 26, 27, 29,36, 38, 44, 59, 72. 73, 81, 87, 103, in, 121, 122, 124, 126, 130, 132, 140, 172, 177, 178, 239, 252, 305, and 322 Hottentots: purchase of terri- • tory from, 42 Hottentot settlement at the Kat river : account of, 178 Hottentot wars : account of, }7 and 45 Huguenots: arrival of, 51 I Imbembesi : battle of, 445 Immigration from Great Britain : account of, 155 and 235 Imports of the Union in 1913, 431 Indians : introduction into Natal of, 338 Ingogo : action at, 39 r Inhambane : particulars concern- ing, 467, 470, and 477 Insect life : abundance of, 320 Inyesane : battle of, 348 Isandlwana : terrible disaster at, 346' J Jameson, Dr. II Haarlem, the : is wrecked in Table Bay, 21 Gibeon : encounter at, 502 Ginginhlovu : battle of, 349 Table Bay, 21 Harbour works : particulars concerning, 288, 325, 341, 355, and 430 Glenelg, Lord : treatment of South Africa by, 192 Gold : discovery of, 395 ; pro- duction of in 191 6, 396 ; pro- duction of Rhodesia, 454 355, and 430 Herero tribe : particulars con- cerning, 482 355, and 430 Herero tribe : particulars con- cerning, 482 Hereros and Hottentots : war between, 482 Hereros and Hottentots : war between, 482 Hereros and Hottentots : war between, 482 Government of the Dutch East India Company : particulars concerning, 75 Government of the Dutch East India Company : particulars concerning, 75 van de Graaff, C. J. : administra- tion of, 96 and 97 Herero war of independence, account of, 483 189 Herero war of independence, account of, 483 189 van de Graaff, C. J. : administra- tion of, 96 and 97 Hintsa, paramount Xosa chief : references to, 152, 188, and Graaff- Reinet : foundation of, 87 ; rebellions of the people of, 104 and 121 Hlobane : disaster at, 348 Hlobane : disaster at, 348 tNbEX 5»6 [anssens, General J. W. : admin- istration of, 129 et seq. Jesuit mission : account of, 468 Johannesburg : description of, 396 ; insurrection of, 400 Justice : administration of in the Cape Colony, 324 5» 1 llubi tribe : particulars concern- ing, 333 Hoffman, Josias : is ihc first president of the Orange Free State, 364 Hottentots : description of, 2 ; first intercourse with Euro- peans, 10 ; particulars con- cerning, 25, 26, 27, 29,36, 38, 44, 59, 72. 73, 81, 87, 103, in, 121, 122, 124, 126, 130, 132, 140, 172, 177, 178, 239, 252, 305, and 322 Hottentots : description of, 2 ; first intercourse with Euro- peans, 10 ; particulars con- cerning, 25, 26, 27, 29,36, 38, 44, 59, 72. II "Leander Starr : • raid into the Transvaal of, 399 ; further mention of, 434, 437, 444, and 452 Kaffir wars : account of, 89, 99, 123, 142, 152, 187, 237, 251, and 312 Kaffraria : description of, 302 ; annexation of to the Cape Colony, 305 to 312 ; rebellion of 1880 in, 314 252, 305, and 322 Hottentots: purchase of terri- • tory from, 42 252, 305, and 322 Hottentots: purchase of terri- • tory from, 42 • tory from, 42 Hottentot settlement at the Kat river : account of, 178 • tory from, 42 Hottentot settlement at the Kat river : account of, 178 Kakamas labour colony : ac- count of, 291 Kambula : defence of, 349 Keate award : particulars con- cerning, 297 and 382 cerning, 297 and 382 Kentani : battle of, 314 I Ketshwayo, Zulu chief: refer- ences to, 343, 344, and 386 ; career of after the battle of Ulundi, 351 Imbembesi : battle of, 445 Immigration from Great Britain : account of, 155 and 235 Khama, chief of the Bamangwato: particulars concerning, 460 King, Richard : famous ride of, 221 and 222 Ingogo : action at, 39 r Inhambane : particulars concern- ing, 467, 470, and 477 Insect life : abundance of, 320 Inyesane : battle of, 348 Isandlwana : terrible disaster at, King-Williamstown : foundation of, 190 Kitchener, Lord : mention of, Kitchener, Lord : mention of, 412, 414, 415, 416, and 417 Kitchener, Lord : mention of, 412, 414, 415, 416, and 417 Kreli, paramount Xosa chief: references to, 189, 255, 279, 305. 3°r>> 307, 33> and 34 Kruger, S. J. Paul : mention of, 379, 380, 386, 387, 388, 393. 394, 401, 408, 414, and 437 ; is the third president of the South African Republic, '402 412, 414, 415, 416, and 417 412, 414, 415, 416, and 417 Kreli, paramount Xosa chief: references to, 189, 255, 279, 305. 3°r>> 307, 33> and 34 305. 3°r>> 307, 33> and 34 Kruger, S. J. Paul : mention of, 379, 380, 386, 387, 388, 393. 394, 401, 408, 414, and 437 ; is the third president of the South African Republic, '402 346' J Jameson, Dr. "Leander Starr : • raid into the Transvaal of, 399 ; further mention of, 434, 437, 444, and 452 IN BEX 517 Luderitz, German merchant: action in South Africa of, 485 Lutheran church in Capetown : establishment of, 101 Lydenburg : foundation of the town of, 225 Labour tax : mention of, y]1 Landdrost and heemraden : Labour tax : mention of, y]1 Landdrost and heemraden : powers and duties of, 53 Langalibalele. Hlubi chief: bad conduct of in Natal, 333 : rebellion of. 334 ; subsequent career of, 336 and n~] Landdrost and heemraden : powers and duties of, 53 powers and duties of, 53 Langalibalele. Hlubi chief: bad conduct of in Natal, 333 : rebellion of. 334 ; subsequent career of, 336 and n~] 461 434 Matabeleland : conquest of, 443 et seq. Moroko, Barolong chief: death of, 372 Ohrigstad: foundation of, 225 Orange Free State : creation of. 267 ; condition in 1854 of, 362 : form of government of, 363 ; industries in. 374 267 of, 372 Morosi, Baputi chief: rebellion of, 292 Moselekatse, chief of the Mata bele : references to, 167, 169, 170, 171, 198, 199, and 201 Moselekatse, chief of the Mata bele : references to, 167, 169, 363 ; industries in. 374 Orange River Sovereignty : crea- tion of, 246 ; abandonment of, 363 ; industries in. 374 Orange River Sovereignty : crea- tion of, 246 ; abandonment of, 170, 171, 198, 199, and 201 Moshesh, founder of the Basuto tribe: references to, 171, 172, 226, 228, 229, 232, 234, 244, 246, 247, 256, 257, 258, 260, 261, 264, 265, 310, 364, 365, 368, 370, and 455 Orphan chamber : duties of, 54 Ostrich farming: particulars concerning, 290 226, 228, 229, 232, 234, 244, 246, 247, 256, 257, 258, 260, Ovambo tribes: mention of, 482 261, 264, 265, 310, 364, 365, 261, 264, 265, 310, 364, 365, 368, 370, and 455 M M career of, 336 and n~] Lang's Nek : action at, 391 Macartney, Lord : administra- tion of, 120 Macartney, Lord : administra- tion of, 120 Mackenzie, Colonel (later Sir Duncan) : mention of, 359 and 495 Lang's Nek : action at, 391 Lanyon, Sir Owen : mention of, 387 Lang's Nek : action at, 391 Lanyon, Sir Owen : mention of, 387 Legislative council. Cape Colony: creation of, 179 387 Legislative council. Cape Colony: creation of, 179 Mackenzie, Colonel (later Sir Duncan) : mention of, 359 and 495 Lewanika. Barotse chief: men- tion of, 439 Maitland, Sir Peregrine : ad- ministration of, 230 to 240 Majuba hill : action at. 391 Makana, Xosa seer : references to, 151, 153, and 154 Maitland, Sir Peregrine : ad- ministration of, 230 to 240 Majuba hill : action at. 391 Lighthouse at Green Point : erec- tion of, 159 Lighthouses : mention of, 325 Majuba hill : action at. 391 Makana, Xosa seer : references to, 151, 153, and 154 Lighthouses : mention of, 325 Lintshwe, Bakatla chief: men- tion of, 459 Makaranga tribe : particulars concerning, 466 Livingstone, Rev. Dr. : refer- ence to, 378 Makoma, Xosa chief: references to, 186 and 187 Makoma, Xosa chief: references to, 186 and 187 Malays : particulars concerning, Malmesbury : foundation of, 79 Loan bank : creation of, 98 Malays : particulars concerning, Lobengula, chief o( the Mata- bele : references to, 433, 434, 437< 43S; 439- 44Q, 442, 443, 445, 446, and 44S ; death of, • 449 Malmesbury : foundation of, 79 322 Manikusa, chief of the Mat- shangana : mention of, 470 and 471 Loch, Sir Henry: mention of, 440 322 Mantati horde : account of. 165 London missionary society : com- mences work in South Africa, 126 ; assumes a hostile atti- tude towards the colonists, 145 ; is forsworn by the emi- grant farmers, 200 Maritz,Gerrit : reference to, 199 Masarwa : mention of, 458 and Mashonaland : occupation of, grant farmers, 200 Lourenco Marques : description of the town of, 472 Lovedale missionary institution : mention of, 277 and 321 Matabele tribe : origin of, 169 ; references to, 198, 199, 201, 202, and 218. M See Lobengula, Moselekatse, and First and Second Matabele war Lucas, Engelbertus : surrenders a fleet of nine ships of war to the English, 119 sis INDEX N Natal : discovery of, 12 ; depopu- lation of, 164 ; is occupied by the emigrant farmers, 205 ; extent of the republic. 216; mode of government by the emigrant farmers, 217 ; is taken in possession by British troops, 223 ; occupation by Bantu of, 331 ; extent in~i854, 267 ; government of as a Brit- ish colony, 341 and 357 : area, //>. ; franchise in, 358 ; indus- tries in, 360 concerning. 470 and 471 Mauritius : references to, 39 and Maurit 71 Methlokazulu : rebellion in 1906 of, 359 71 of, 359 Milner, Lord : mention of, 408, Milner, Lord : mention of, 408, 416, 417, and 419 416, 417, and 419 Mission societies: localities of labour, 101 Missionary efforts : result of, 4, 185, 320, 331, 422, 457, and Ndlambe, Xosa chief: references to, 99, 123, 130, 131, 143. I5°> I5I> J52, and 187 Ndlambe, Xosa chief: references to, 99, 123, 130, 131, 143. I5°> I5I> J52, and 187 461 de Mist, J. A. : references to, 129 and 131 129 and 131 Moffat, Rev. Dr.: reference to, 170 I5°> I5I> J52, and 187 Xeder burgh and Frykenius : proceedings of, 97 to 103 170 Monomotapa : mention of, 466 proceedings of, 97 to 103 Ninth Kaffir war, account of, 312 Monomotapa : mention of, 466 Montsiwa, Barolong chief: re- ference to, 381 Northern border war of 1879 : account of, 291 Northern border war of 1879 : account of, 291 Moravian mission : foundation in South Africa of, 101 P 368, 370, and 455 Mossamedes : migration of far- mers to, 383 and 483 I'aarl : description of, 85 Palgrave, W. C. : missions to Mereroland of, 484 and 486 Mozambique Company : account of, 476 Mozambiqu of, 476 INDEX 519 Panda, Zulu chief : references to. 213, 215, 216, 223, 331, and 343 Pretorius. Marthinus Wessel : is the third president of the Orange Free State, 367 : is the first president of the South African Republic, 379 ; refer- ences to. 296 and 388 343 Paper money : issue of, 92 ; re- demption of, 159 demption of, 159 Paris evangelical mission : par- ticulars concerning, 172 Progress of South African ex- ploration in 1700 : account of, ticulars concerning, 172 Phylloxera : ravages of, 289 Phylloxera : ravages of, 289 Physical conformation of South Africa, 1 3 Pietermaritzburg : foundation of, 213 Phylloxera : ravages of, 289 Physical conformation of South Africa, 1 3 57 Province of Queen Adelaide : creation of, 190 ; abandonment of, 192 57 Province of Queen Adelaide : creation of, 190 ; abandonment of, 192 Pietermaritzburg : foundation of, 213 Public debt : payment of the old, 235 ; amount in 1916, 429 van Plettenberg, Joachim : ad- ministration of, 86 to 95 ministration of, 86 to 95 Plettenberg's Bay : naming of, 87 Plettenberg's Bay : naming of, 87 Poison : use made of by Bush- men, 2 Q men, 2 Pondo treaty state : creation of, 228 Pondo tribe : particulars con- cerning, 166 and 308 cerning, 166 and 308 Population of the Union in 19 13, 429 Railways: particulars concern- ing. 288, 301, 325, y^, 372, 398, 402, 453, 461, 471, 475- 490, 493, and 500 429 Port St. John's : description o{, 311 Portuguese discoveries in South Africa : account of, 8 et scq. 490, 493, and 500 Rebellion : in 1896 in Rhodesia, 452 : in 1906 in Natal. 359 ; in 1914-15 in the Union, 497 Africa : account of, 8 et scq. Portuguese South Africa : ac- count of, 466, boundary of, 475 Potchefstroom : foundation of, 211 Reitz, Chief Justice F. W. : is the fourth president of the Orange Free State, 374 ; men- tion of, 414 and 416 Potgieter, Hendrik : references to, 197. P 198, 199, 200, 201, 202, 208, and 225 Retief, Pieter : references to, 200, 201, 203, 204, and 205 Pottinger, Sir Henry : adminis- tration of, 240 Revenue of the Union in 1913. Pretoria : is the administrative capital of the Union, 425 43 mention of, 399, 401, and 434 Rhodesia : condition of in 1888. Pretorius, Andries : references to, 211, 212, 214, 216, 220, 221, 222, 247, and 258 432 ; area, 454 ; population, ib. ; industries in, ib. IXDEX 5±o 5±o van Riebeek, Jan : references to, 22, 24, 25, 26, 28, 30, 31, and 39 Robinson, Sir Hercules: men- tion of, 400 Rbndebosch : Is occupied and named, 31 Sena : foundation of the village of, 467 Setsheli, Bakwena chief: refer- ences to, 378 Seventh Kaffir war : account of, Seventh Kaffir war : account of, 237 ct seq. 237 ct seq. Shangani : battle of, 444 Sheikh Joseph : particulars con cerning, 36 Rorke's Drift : gallant defence of post at, 347 Sheikh Joseph : particulars con cerning, 36 Shepstone, Sir Theophilus : men- tion of, 385, 386, and 387 Saint 1 lelena Bay : is visited and . named, 10 tion of, 385, 386, and 387 Shipwrecks in Table Bay: ac- count of, 76 Shipwrecks in Table Bay: ac- count of, 76 Sibepu, Zulu chief: mention of, 351 and 352 Siginanda, Zulu chief: rebellion in 1906 of, 359 Simonstown : foundation of, 77 ; description of, 85 187 . named, 10 Saint Helena Island : discovery of, 15 Sibepu, Zulu chief: mention of, 351 and 352 de Saldanha, Antonio : voyage of, 13 Salisbury: foundation of, 434 Sandfontein : disaster at, 495 Simonstown : foundation of, 77 ; description of, 85 187 Sixth Kaffir war : account of, Sandile, Xosa chief: references to, 187, 237, 238, 251, 279, 280, 281, 306, 313. and 314 Sandile, Xosa chief: references to, 187, 237, 238, 251, 279, 280, 281, 306, 313. and 314 Sand River Convention : ac- - count of, 259 Schoemansdal : abandonment of, Slachter's Nek rebellion : ac- count of, 149 280, 281, 306, 313. and 314 Sand River Convention : ac- - count of, 259 Slaves : introduction of, ^^ ; emancipation of, 180 el seq. 380 Schools : particulars concerning, 235,289, emancipation of, 180 el seq. Small-pox : ravages of, 71 and 80 380 . P 53- 79, 86, 132, 160,235,289, 316, 323, 372, 398, 402, 405, 406, 416, and 450 Smith, Sir Harry: administra- tion of, 242 to 260 Smuts, the hon. General J. C. : mention of, 416, 426, 496, 500, and 505 Sofala : is occupied by the Por- tuguese, 465 and 466 Somerset East : foundation of, 159 Smuts, the hon. General J. C. : mention of, 416, 426, 496, 500, and 505 406, 416, and 450 Scurvy : effects of, 23 Second Basuto war: account of, cond tentot ount 500, and 505 Sofala : is occupied by the Por- tuguese, 465 and 466 159 366 Second Hottentot war : account of, 45 Second Kaffir war : account of, 99 to 10 1 Second Matabele war with the Chartered Company : account of, 452 Sekukuni, Bapedi chief: rebel- lion of, 383, 386, and 388 366 Second Hottentot war : account of, 45 tuguese, 465 and 466 Somerset East : foundation of, Second Kaffir war : account of, Second Kaffir war : account of, 99 to 10 1 Somerset, Lord Charles : ad- ministration of, 148 to 161 South African Republic : inde- pendence acknowledged by (beat Britain, 260; anarchy in, 376 ; is annexed to the tNB&X British empire, 386 : war of independence, 388 el seq. ; form of government, 398 and 403 Telegraphs : particulars concern- ing, 289, 325, 402, 430, and 521 Tembu tribe : particulars con- cerning, 306 and 307 403 Stellaland : formation of the re- public of, 300 436 Tete : foundation of the village of, 467 public of, 300 van der Stel, Simon : adminis- tration of, 50 ; mention of, 55 and 57 Thaba Ntshu : becomes part of the Orange Free State, 374 123 Third Basuto war : account of, 123 van der Stel, W. A. : oppressive administration of, 65 ; he is recalled by the directors, 70 369 Third Kaffir war : account of, recalled by the directors, 70 Stellenbosch : foundation of, 50 ; description of, 85 Traffic in the seventeenth and eighteenth centuries : account of, 55 to 57 description of, 85 Steyn, Chief Justice M. T. Umbulazi, Zulu chief : reference to, 343 P : is the fifth president of the Orange Free State, 374 ; mention of, 408, 414, and 417 Treaties : with Andries Water- boer, 186; with Moshesh, 226 ; with Adam Kok, 228 and 233 ; with Faku, '228 ; of Yereeniging, 416 417 Stockenstrom, Sir Andries : re- ferences to, 191, 192, and 193 417 Stockenstrom, Sir Andries : re- ferences to, 191, 192, and 193 Tsetse fly : damage caused by, 375 204 Sugar : production of in Natal, and 375 Tshaka, Zulu chief: references to, 162, 166, 167, 171, and 338 Swaziland : account of, 462 '- area of, 464 ; population of, id. Tulbagh basin : settlement of, 61 id. Swellendam : foundation of, 79 ; description of, 85 ; rebellion of the inhabitants of, 104 Tulbagh : foundation of the village of, 79 ; formation of the district of, 132 Tulbagh : foundation of the village of, 79 ; formation of the district of, 132 Tulbagh, Ryk : administration of, 80 to 86 Tulbagh, Ryk : administration of, 80 to 86 Uitenhage : foundation of, 132 Ulundi : battle of, 350 Umbulazi, Zulu chief : reference to, 343 Umtali : foundation of the ..vil- lage of, 436 Umtali : foundation of the ..vil- lage of, 436 Table Bay: discovery of, 13; improvement of, jj ; see Har- . hour works Table Bay: discovery of, 13; improvement of, jj ; see Har- . hour works U Uitenhage : foundation of, 132 Ulundi : battle of, 350 to, 343 Table Mountain : receives its name, 13 Table Mountain : receives its name, 13 Tas, Adam : outrageous treat- ment of, 68 Tas, Adam : outrageous treat- ment of, 68 Taxation of the colonists in the olden times : system of, 55 JXDFX 522 War of 1899-1902 between Great Britain and the two republics : causes of, 405 el seq. ; account of, 411 et seq. : mode of conclusion of, 414 522 LJmtasa, chief al Manika : mention of, 473 LJmtasa, chief al Manika : mention of, 473 Union of South Africa : forma- tion of, 422 ; form of govern- ment, 423 ; area of, 429 ; population of, id. ; public debt of, id. ; exports, 431 : imports, ib. ; revenue, id. University of the Cape of Good Hope : establishment of, 289 Universities: creation of three 428 Uys, Dirk Cornelis : death of, 209 Uys, Commandant Pieter Lav ras : references to, 200, 201 and 208 ; death of, 209 Uys, Commandant Pieter Lav ras (son of the above) : death of. 349 V Vereeniging : treaty of, 416 Victoria : foundation of the town of, 436 Viervoet : battle of, 257 W Walvis Bay : annexation to the Cape Colony of, 293 War between the emigrant farmers and the Matabfle : account of, 198 and 199; between the emigrant farmers and the Zulus, 208 et seq. War between the Chartered Company and the Matabele in 1893 : account of, 437 ; and in 1896, 452 Warren, Sir Charles : expedi- tion under, 300 Wild animals : abundance of in early times, 28 ib. ; revenue, id. W W Vonge, Sir George : administra- tion of, 121 to 126 University of the Cape of Good Hope : establishment of, 289 Universities: creation of three early times, 28 Wilson, Major Allan : heroic death of with thirty-three others, 447 Winburg : foundation of the town of, 200 Winburg : foundation of the town of, 200 Windhoek : description of the town of, 488 Windhoek : description of the town of, 488 Wodehouse, Sir Philip : refer- ences to, 368 and 370 Wodehouse, Sir Philip : refer- ences to, 368 and 370 Wolseley, Sir Garnet : mention of, 349, 350, 351, and 388 Wool : production of, 185 Worcester : foundation of the town of, 159 V Vereeniging : treaty of, 416 Victoria : foundation of the town of, 436 Viervoet : battle of, 257 Vereeniging : treaty of, 416 X Xosa tribe : particulars concern- ing, 306 Vonge, Sir George : administra- tion of, 121 to 126 Walvis Bay : annexation to the Cape Colony of, 293 Walvis Bay : annexation to the Cape Colony of, 293 Cape Colony of, 293 War between the emigrant farmers and the Matabfle : account of, 198 and 199; between the emigrant farmers and the Zulus, 208 et seq. Zululand : is annexed to Natal, Zulu tribe : references to, 163, 205, 213, 218, and 331 352 205, 213, 218, and 331 Zulu wars : of devastation, 162 et seq. ; with the emigrant farmers, 208 et seq. ; with Great Britain, 345 et seq. War between the Chartered Company and the Matabele in 1893 : account of, 437 ; and in 1896, 452 Printed ill Great Britain by L'NWIN BROTHERS, LIMITED WOKING AND LONDON
https://openalex.org/W2611897930	http://www.scielo.br/pdf/bolema/v31n57/0103-636X-bolema-31-57-0380.pdf	Portuguese	null	O Processo de Construção de Abordagens Históricas na Formação Interdisciplinar do Professor de Matemática	Bolema	2,017	cc-by	14,318	The Process of Construction of Historical Approaches in Interdisciplinary Training for Mathematics Teachers Eliane Maria de Oliveira Araman* Irinéa de Lourdes Batista * Doutora em Ensino de Ciências e Educação Matemática pela Universidade Estadual de Londrina (UEL). Professora da Universidade Tecnológica Federal do Paraná (UTFPR), Cornélio Procópio, Paraná, Brasil. Endereço para correspondência: Avenida Alberto Carazzai, 1640, Bairro Centro, CEP: 86300-000, Cornélio Procópio/PR. E-mail: elianearaman@utfpr.edu.br. Abstract This paper presents the results of an investigation about the importance of the process of building historical approaches has to the process mathematics teachers’ formation, specifically in regard to the construction of interdisciplinary knowledge. The investigations show that the theoretical and methodological knowledge of history of mathematics are important for teacher education. Therefore, our objective was to analyze and show how these skills work together to structure interdisciplinary knowledge of the mathematics teacher who has experienced the process of construction and application of historical approaches in the classroom. To this end, we interviewed six teachers who had experienced this process and, by means of content analysis, we structured categories that show the knowledge developed by them and which contribute to interdisciplinary training of teachers. Keywords: History of Mathematics. Teacher’s Education. Teacher knowledge. Content Analysis. Resumo Este artigo apresenta os resultados de uma investigação a respeito da relevância que o processo de construção de abordagens históricas apresenta na formação do professor de Matemática, mais especificamente no que se refere à construção de saberes interdisciplinares. As investigações evidenciam que os conhecimentos teóricos e metodológicos da história da Matemática são importantes para a formação docente. Diante disso, nosso objetivo, neste artigo, é analisar e evidenciar como tais conhecimentos colaboram para a estruturação de saberes interdisciplinares do professor de Matemática. Para isso, entrevistamos seis professores que vivenciaram o processo de construção e aplicação de abordagens históricas em sala de aula e, por meio da análise de conteúdo, estruturamos categorias que evidenciam saberes que colaboram para a formação interdisciplinar do professor. Palavras-chave: História da Matemática. Formação de Professores. Saberes Docentes. Análise de Conteúdo. ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19  Doutora em Filosofia pela Universidade Federal de São Paulo (USP). Professora da Universidade Estadual de Londrina (UEL), Londrina, Paraná, Brasil. Endereço para correspondência: Rodovia Celso Garcia Cid, Km 380, s/n, Campus Universitário, CEP: 86051-990, Londrina/PR. E-mail: irinea@uel.br. Keywords: History of Mathematics. Teacher’s Education. Teacher knowledge. Content Analysis. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 1 Introdução * Doutora em Ensino de Ciências e Educação Matemática pela Universidade Estadual de Londrina (UEL). Professora da Universidade Tecnológica Federal do Paraná (UTFPR), Cornélio Procópio, Paraná, Brasil. Endereço para correspondência: Avenida Alberto Carazzai, 1640, Bairro Centro, CEP: 86300-000, Cornélio Procópio/PR. E-mail: elianearaman@utfpr.edu.br. p p  Doutora em Filosofia pela Universidade Federal de São Paulo (USP). Professora da Universidade Estadual de Londrina (UEL), Londrina, Paraná, Brasil. Endereço para correspondência: Rodovia Celso Garcia Cid, Km 380, s/n, Campus Universitário, CEP: 86051-990, Londrina/PR. E-mail: irinea@uel.br. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 380 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 As pesquisas que procuram compreender o processo de formação docente vêm atraindo, cada vez mais, a atenção e a preocupação da comunidade acadêmica. São várias vertentes a respeito das quais versam essas investigações, como, por exemplo, a formação inicial do professor, focada nos cursos de licenciatura, a necessidade de formação continuada, a investigação dos saberes e conhecimentos do professor necessários para sua função, a necessidade de reflexão da prática profissional, entre outros. É possível perceber um consenso entre as pesquisas no sentido de buscar compreender a complexidade de conhecimentos e saberes que os professores mobilizam visando superar os desafios diários que demandam as situações de ensino e de aprendizagem. É nesta perspectiva que a presente investigação se insere. Em nossa compreensão, os saberes docentes têm origem em muitas fontes, como a formação inicial, a própria prática, os conhecimentos teóricos das diferentes áreas, a didática, as pesquisas educacionais, entre outros. Sendo assim, nosso objetivo foi compreender e explicitar algumas relações entre os conhecimentos teóricos e metodológicos advindos da história da Matemática e a construção dos saberes interdisciplinares do professor de Matemática. Para alcançar esse objetivo, investigamos professores que vivenciaram a pesquisa, construção e aplicação de uma abordagem pedagógica, fundamentada na história da Matemática, de um dado conteúdo matemático. Por meio dos discursos desses professores e fundamentando-nos nos referenciais teóricos que subsidiam a pesquisa, procuramos estabelecer alguns elementos que evidenciam de que forma o processo de construção de abordagens históricas traz implicações para a construção dos saberes dos mesmos. A presente pesquisa apresenta uma leitura diferenciada, uma vez que analisamos os discursos de professores que vivenciaram o processo de pesquisa, construção e aplicação de uma abordagem histórica. 1 Introdução As compreensões alcançadas nesse estudo colaboram com elementos relevantes, evidenciados por meio da análise, que complementam a discussão dos saberes docentes, evidenciando o papel que o processo de construção de abordagens históricas tem no desenvolvimento de saberes interdisciplinares no professor. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 2 Saberes e conhecimentos necessários para a ação docente As atuais pesquisas a respeito da formação de professores têm chamado a atenção para a necessidade de compreensão da prática do professor, de como esse profissional desenvolve continuamente seus conhecimentos, num processo constante de ir e vir, a partir de suas experiências. As investigações que visam analisar os saberes que são mobilizados pelos 381 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 professores durante a sua prática apresentam um grau de relevância, uma vez que buscam realizar a análise numa perspectiva de contribuição para as questões de formação de professor. Para compreender as questões que envolvem os saberes docentes, realizamos um estudo teórico apoiados em pesquisas nacionais e internacionais que discutem essa temática, como Almeida e Biajone (2007); Brito e Alves (2008); Gauthier et al. (1998); Shulman (1986); Tardif (2002). As discussões versam sobre os diferentes saberes que os professores mobilizam e a importância dos mesmos para a sua atuação e desenvolvimento profissional. De acordo com Shulman (1986), as questões que envolvem os conhecimentos do professor são muito complexas, necessitando de um arcabouço teórico coerente que compreendesse tais questões. Segundo seus estudos, as pesquisas de formação docente que ocorriam até então tinham como preocupação investigar como os professores organizavam e administravam suas turmas, as atividades, o tempo, as tarefas, seus planejamentos, entre outros. Dessa forma, na visão de Shulman (apud ALMEIDA; BIAJONE, 2007, p. 287) “essas pesquisas trivializam a prática pedagógica, ignorando assim sua complexidade e reduzindo suas demandas”. Shulman apresenta algumas reflexões acerca dos conhecimentos do professor: Quais são os domínios e categorias de conhecimento do conteúdo na mente dos professores? Como, por exemplo, estão relacionados o conhecimento do conteúdo e o conhecimento pedagógico geral? De que forma são os domínios e categorias de conhecimentos representados nas mentes dos professores? Quais são as formas promissoras de aumentar a aquisição e o desenvolvimento de tal conhecimento?1 (SHULMAN, 1986, p. 9). Em sua visão, questões como essas estavam sendo ignoradas nas investigações a respeito da formação e desenvolvimento profissional do professor. Assim, Shulman (1986) e seus colaboradores tentam abordar esses questionamentos em um programa de pesquisa que visa investigar como os conhecimentos de um professor são adquiridos, revistos e combinados, compondo uma base de conhecimentos (knowledge base). 1 Oferecemos ao leitor o texto original da citação: What are the domains and categories of content knowledge in the minds of teachers? How, for example, are content knowledge and general pedagogical knowledge related? In which forms are the domains and categories of knowledge represented in the minds of teachers? What are promising ways of enhancing acquisition and development of such knowledge? Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 2 Saberes e conhecimentos necessários para a ação docente Shulman (1986) sugere uma distinção entre três categorias de conhecimentos, quando se refere ao conhecimento da disciplina para ensiná-la: o conhecimento do conteúdo da disciplina (subject matter content knowledge), o conhecimento pedagógico do conteúdo da disciplina (pedagogical content knowledge), e o conhecimento do currículo (curricular Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 382 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 knowledge). Para ele, é na articulação entre essas três categorias que se forma a base de conhecimentos do professor. Nos estudos desenvolvidos por Gauthier e seus colaboradores (1998), observamos a preocupação em considerar o contexto real em que o ensino efetivamente acontece para a construção dos saberes dos professores. Tais estudos procuraram identificar, nas pesquisas norte-americanas sobre o ensino, as convergências observadas no que se refere aos saberes mobilizados pelos professores na prática pedagógica (knowledge base)2. Os autores argumentam que é primeiramente necessário conhecer o processo de ensino para depois refletir sobre ele e que, “ao contrário de outros ofícios que desenvolveram um corpus de saberes, o ensino tarda a refletir sobre si mesmo” (GAUTHIER et al., 1998, p. 20). Em seus estudos, arrazoam que ainda temos pouca compreensão a respeito das atitudes dos professores em sala de aula e da influência que exercem na aprendizagem. Entretanto, conhecer esses aspectos “do saber profissional docente é fundamental e pode permitir que os professores exerçam o seu ofício com muito mais competência” (GAUTHIER et al., 1998, p. 20). De acordo com Brito e Alves (2008), a profissionalização do ofício do ensino pressupõe a compreensão e descrição da natureza dos saberes que fundamentam a prática profissional do professor. Dessa forma, a partir da década de 1980, aproximadamente, observamos um ímpeto nas pesquisas com relação a compreender e descrever a ação docente por meio de investigações realizadas diretamente nas salas. Nesse contexto, as questões que envolvem os saberes, as habilidades e as atitudes que os professores mobilizam durante o exercício de sua profissão começam a ser debatidas. Assim, [...] as inúmeras pesquisas realizadas nos últimos anos, com o objetivo de definir um repertório de conhecimentos para a prática pedagógica, podem ser interpretadas como uma série de incentivos para que o docente se conheça enquanto docente, como uma série de tentativas de identificar os constituintes da identidade profissional e de definir os saberes, as habilidades e as atitudes envolvidas no exercício do magistério (GAUTHIER et al., 1998, p. 18). 2 Gauthier et al. (1998) esclarecem que, em inglês, o termo knowledge base é empregado frequentemente em um sentido amplo, que engloba todos os saberes do professor, como os citados por Shulman. Em seus estudos, preferem utilizar a expressão repertório de conhecimentos para designar o que chamam de saberes da ação pedagógica. 2 Saberes e conhecimentos necessários para a ação docente Os autores entendem que a profissionalização da atividade docente deve enfrentar o desafio de identificar os saberes próprios do ensino, considerando o contexto real, complexo, no qual muitas variáveis interferem no processo de ensino e nas tomadas de decisão do professor. Propõem um ofício feito de saberes, os quais “formam uma espécie de reservatório” que possibilita ao professor se abastecer para responder à diversidade de Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 383 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 questões que envolvem o ensino (GAUTHIER et al., 1998, p. 27). Esse reservatório, nos estudos apontados por Gauthier et al. (1998), é composto por seis tipos de saberes: saberes disciplinares; saberes curriculares; saberes das ciências da educação; saberes da tradição pedagógica; saberes da experiência e saberes da ação pedagógica. Salientamos os saberes da ação pedagógica, que, segundo Gauthier et al. (1998) são os saberes da experiência quando se tornam públicos e são testados por meio das pesquisas realizadas em sala de aula. Os julgamentos que os professores fazem a respeito de suas experiências e os motivos que apoiam as suas tomadas de decisão podem ser estudados pelos pesquisadores, com a finalidade de estabelecer algumas regras de ação que podem ser conhecidas e aprendidas por outros. Os resultados de tais pesquisas podem trazer muitas contribuições para a formação docente, entretanto, o que observamos é que os saberes da ação pedagógica são pouco investigados, trazendo poucos benefícios para os programas de formação. Tardif (2002), juntamente com seus colaboradores, procurou evidenciar a necessidade de investigações que analisem o professor como produtor de saberes. Em sua compreensão, os professores, no exercício da profissão, utilizam um leque de saberes para atender às suas necessidades de ensinar. Embora isso ocorra cotidianamente, eles apresentam dificuldades em “teorizar a sua prática e formalizar seus saberes” (TARDIF, 2002, p. 274). Como consequência, tais saberes são pouco explicitados e estudados, tornando-se pessoais, tácitos e intuitivos. No entendimento de Tardif (2002), o professor, no exercício de sua profissão, utiliza diversos saberes em sua prática diária, tais como os disciplinares; os curriculares; os profissionais; os pedagógicos e os saberes. Os saberes docentes, como salientam Rocha e Fiorentini (2006, p. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 2 Saberes e conhecimentos necessários para a ação docente 156), são mobilizados no contexto da prática, “a partir dos problemas e desafios da prática – mediante mobilização e (re)significação de saberes adquiridos ao longo da vida”, compreendendo não somente a dimensão prática, mas também são formados por elementos teóricos e conceituais da disciplina, das ciências da educação e das concepções que o professor carrega consigo do que é ser professor. Tardif (2002) salienta que os saberes docentes podem ser caracterizados por um sincretismo, uma vez que o professor apresenta um conjunto de conhecimentos, de atitudes, de concepções “que utiliza em sua prática, em função, ao mesmo tempo, de sua realidade cotidiana e biográfica e de suas necessidades, recursos e limitações” (p. 65). A proposta de Tardif (2002) vai além de mostrar uma definição ou uma categorização dos saberes docentes. O autor advoga em favor de colocar o professor como sujeito do conhecimento e produtor de saberes, de forma que as pesquisas em formação docente devem 384 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 considerar essa problemática. Em outras palavras, “a epistemologia da prática profissional sustenta que é preciso estudar o conjunto de saberes mobilizados e utilizados pelos professores em todas as suas tarefas” (TARDIF, 2002, p. 259). As pesquisas em saberes docentes evidenciam a importância de um estudo sistematizado dos saberes mobilizados pelos professores durante a sua prática, de forma que esses saberes não fiquem restritos às experiências particulares de cada docente, mas que possam ser analisadas à luz das pesquisas científicas, e é com essa proposta que desenvolvemos nosso trabalho. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 3 A história da Matemática na formação docente É ampla a literatura que discorre a respeito da relevância dos conhecimentos da história da Matemática para a Educação Matemática. Encontramos um vasto campo de argumentos favoráveis à implementação de abordagens históricas nas aulas de Matemática, mas temos que analisar a formação do professor para tal. Autores como Bicudo (1999), Furinguetti (2007), Brito e Carvalho (2009), Bursal (2010), entre outros, consideram que os estudos históricos a respeito dos diversos tópicos de Matemática podem enriquecer o conhecimento do professor e, em consequência disso, sua atuação em sala de aula. Consideramos que a história da Matemática, com seu enfoque epistemológico e metodológico, pode ser um fator contributivo para a formação de professores de Matemática, como já nos diz a literatura, mas especificamente, pode auxiliar o professor na estruturação de seus saberes docentes (ARAMAN; BATISTA, 2013). A integração entre a história da Matemática na formação inicial e também em serviço tem sido um tema de interesse internacional nas pesquisas nos últimos anos (FAUVEL; MAANEN, 2000). O seu uso nos contextos educacionais exige reflexão didática, já que o docente precisa estar preparado para isso. De acordo com as pesquisas realizadas, a inserção de elementos históricos trazem benefícios para a formação do professor de Matemática, em diversos aspectos. Uma dessas contribuições se dá em relação à compreensão da natureza do conhecimento matemático. Em nossos estudos teóricos – Bicudo (1999), Charalambous, Panaoura e Philippou (2009), Barbin (2000), Bursal (2010) – pudemos perceber que muitos professores não apresentam uma compreensão adequada da sua ciência, no caso, a Matemática. Apresentam a noção de um corpo de conhecimentos pronto, acabado, no qual não há revisões a serem feitas. Apresentam também a noção de uma ciência de caráter empírico, em que as interpretações são feitas 385 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 apoiadas nas observações. Também têm a concepção de que os conteúdos, teorias, leis, entre outros, são “descobertos” por pessoas geniais, com pouca colaboração entre os pares. Além disso, concebem uma ciência livre de influências sociais, culturais e políticas. Outro aspecto salientado pelas pesquisas é que a história da Matemática pode contribuir para a compreensão dos conteúdos matemáticos. 3 Segundo Lavaqui e Batista (2007), a conceituação de interdisciplinaridade não apresenta uma definição estável, associada a diferentes concepções epistemológicas. Salientamos que, nessa pesquisa, a compreensão que temos de interdisciplinaridade refere-se às interações possíveis e existentes entre duas ou mais disciplinas, cuja interação pode ir desde a simples comunicação de ideias até a integração mútua de conceitos, promovendo enriquecimentos mútuos. undo Lavaqui e Batista (2007), a conceituação de interdisciplinaridade não apresenta uma definição estáve Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 3 A história da Matemática na formação docente Ao estudar um determinado conceito, a partir de uma abordagem histórica, o professor pode caminhar para uma compreensão de como aquele conceito foi sendo desenvolvido, quais os elementos conceituais necessários para a sua compreensão, quais são os pontos de maior dificuldade, por que eles foram importantes naquela época, por que são importantes hoje, quais eram as necessidades para o desenvolvimento daquele dado conceito, entre outros – Batista e Luccas (2004), Miguel (2005), Matthews (1995), Bursal (2010), Brito e Carvalho (2009), Furinguetti (2007). Essa compreensão extrapola aquela recebida durante a sua formação, ocasionando um entendimento mais amplo e significativo do conteúdo matemático, o que trará benefícios para suas aulas. Ao desenvolver um conteúdo com seus alunos, o professor pode oferecer muito mais do que fórmulas e exercícios, desde que ele tenha conhecimento para isso. Encontramos ainda pesquisas que indicam que a história da Matemática contribui para formação metodológica do professor. Ao elaborar uma abordagem histórica para ensinar algum conteúdo matemático, o professor precisa ter cuidados metodológicos, só que de caráter pedagógico, como adequar o material histórico ao nível de desenvolvimento dos seus alunos, ao tempo disponível para tal, certificar-se de que a proposta colabore efetivamente para a aprendizagem, entre outros – Dass (2005), Fried (2008), Cury e Motta (2008), Carvalho (2000). Ao se propor a desenvolver abordagens históricas com seus alunos, o professor utiliza conhecimentos que vão além dos históricos ou dos conceituais relacionados ao conteúdo. Ele utiliza também conhecimentos pedagógicos vindos de estudos teóricos e também de sua prática, a fim de tornar factível o uso daquelas informações históricas em sala de aula. Outra discussão que encontramos indica que a história da Matemática colabora com a visão interdisciplinar do professor. A questão da interdisciplinaridade3 vem sendo muito debatida na comunidade acadêmica, que ressalta a necessidade da superação daquela visão compartimentada das áreas do conhecimento. A história da Matemática já guarda em si esse Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 386 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 caráter interdisciplinar. Temos, num eixo, o estudo histórico e suas especificidades; em outro encontramos as questões filosóficas; e ainda temos o eixo relativo ao conhecimento matemático e suas características, que são diferentes de outras ciências. Então, trabalhar com história da Matemática já pressupõe uma postura interdisciplinar. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 3 A história da Matemática na formação docente Além disso, os estudos históricos proporcionam uma visão mais ampla do conhecimento matemático, em contraste com a visão especializada – e por vezes compartimentada – da formação inicial. Essa visão permite que o professor, por meio dos estudos históricos, observe as relações existentes entre as várias áreas do conhecimento científico e como os conhecimentos de uma área podem contribuir para o desenvolvimento de outras. Muitas vezes, o professor não consegue perceber esse caráter interdisciplinar da ciência, e os estudos históricos podem contribuir para isso – Batista (2009), Tzanakis e Arcavi (2000), Grugnetti e Rogers (2000). Mas ainda é importante, e fundamental, salientar a interface com os conhecimentos pedagógicos do professor. Ao estudar e desenvolver uma abordagem histórica para o ensino, o professor percebe a necessidade da aproximação de todas essas áreas, como já nos referimos nas questões metodológicas. De acordo com Batista, Lavaqui e Salvi (2008), a condução de uma prática educativa interdisciplinar apresenta elementos próprios, diferentes das práticas interdisciplinares da pesquisa científica. No entendimento desses pesquisadores, as práticas interdisciplinares presentes na educação escolar “revelam alguns aspectos característicos, tais como as finalidades a que se destinam; os objetos de estudos e as consequências que produzem” (BATISTA, LAVAQUI; SALVI, 2008, p. 211- destaques dos autores). A figura que apresentamos a seguir procura sintetizar o caráter interdisciplinar da construção de uma abordagem histórica para o ensino de Matemática, que se dá por meio da articulação e integração de diversos conhecimentos oriundos da formação, cuja relação está representada pelas setas duplas: Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 387 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 ISSN 1980-4415 Figura 1: Caráter interdisciplinar da construção de uma abordagem histórica para o ensino de Matemática Fonte: Araman (2011). Conhecimento relativo à história da Matemática na Ed. Matemática Conhecimento relativo às adequações pedagógicas Conhecimento relativo à natureza do conhecimento matemático Conhecimento relativo ao conteúdo matemático Conhecimento relativo à história da Matemática na Ed. Matemática Conhecimento relativo à natureza do conhecimento matemático Figura 1: Caráter interdisciplinar da construção de uma abordagem histórica para o ensino de Matemática Fonte: Araman (2011). Sendo assim, compreendemos que a elaboração de uma abordagem pautada na história da Matemática colabora para uma prática educativa interdisciplinar, na qual elementos advindos de áreas diversas precisam ser relacionados e adaptados com a finalidade de colaborar com a aprendizagem em sala de aula. 3 A história da Matemática na formação docente Na perspectiva da presente pesquisa, consideramos que esses aspectos salientados são relevantes e contribuem para a estruturação dos diversos saberes do professor de Matemática. Entretanto, nosso foco é discutir as contribuições que o processo de construção de aplicação de abordagens históricas desempenha para o desenvolvimento dos saberes interdisciplinares, uma vez que, como já dissemos, tal processo requer a interação entre diversas áreas do saber. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia Este estudo está inserido no âmbito de pesquisa qualitativa em Educação Matemática. Na concepção de Bogdan e Biklen (1994) e de Lüdke e André (1986), esse tipo de investigação apresenta uma natureza descritiva, na qual os pesquisadores têm um interesse maior no processo e nos seus significados do que nos resultados ou produtos. A partir dos estudos teóricos que realizamos e que já explicitamos na seção anterior, conseguimos definir alguns aspectos que evidenciam a relevância da história da Matemática para a formação dos saberes interdisciplinares do professor de Matemática. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 388 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 Para a coleta dos dados empíricos, utilizamos entrevistas semiestruturadas com seis professores que vivenciaram a experiência de construir e aplicar uma abordagem pedagógica, fundamentada na história da Matemática, em sala de aula. No entendimento de Fiorentini e Lorenzato (2007, p. 120), as entrevistas são vantajosas “pois permitem ao entrevistado fazer emergir aspectos que não são normalmente contemplados por um simples questionário” e permitem “correções, esclarecimentos e adaptações que a tornam sobremaneira eficaz na obtenção de informações desejadas” (LÜDKE; ANDRÉ, 1986, p.34). As questões versaram a respeito da formação dos professores, da atuação profissional dos mesmos, do interesse pela história da Matemática e sua utilização na Educação Matemática, do processo de pesquisa, construção e aplicação da abordagem histórica em sala de aula. Nosso objetivo foi, por meio das entrevistas, identificar como ocorreu o processo de articulação e adequação de conhecimentos oriundos de diversas áreas para a construção da abordagem histórica e de como tal empreendimento contribuiu para os saberes interdisciplinares do professor. Elegemos como metodologia de análise de dados a análise de conteúdo, pois esta possibilita uma interpretação dos dados que “oscila entre os dois pólos do rigor da objetividade e da fecundidade da subjetividade” (BARDIN, 2004, p. 7). A análise de conteúdo busca a interpretação dos discursos por meio de uma análise sistematizada das comunicações. Segundo Bardin (2004), a categorização é um processo estruturado que comporta duas dimensões: isolar os elementos (construção das unidades de registro) e classificá-los com a finalidade de organizar o material analisado (agrupar os elementos por analogia). Os temas são muito utilizados nas análises e podem ser compreendidos como afirmações acerca de um assunto, ou as ideias, enunciados, proposições, que sejam portadores de significações. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia Os temas são utilizados como unidades de registro “para estudar motivações de opiniões, de atitudes, de valores, de crenças, de tendências, etc.” (BARDIN, 2004, p. 99). Dessa forma, optamos por um processo de análise categorial temático. A partir da transcrição das entrevistas, iniciamos o processo de análise dos dados à luz da análise de conteúdo, identificando unidades de registros e, a partir das similitudes dos discursos dos professores, conseguimos atingir uma compreensão da relevância que o processo de construção de abordagens históricas teve na construção dos saberes dos docentes envolvidos nesta investigação. O processo de seleção dos sujeitos da pesquisa se deu pela adequação do perfil do professor que procurávamos com os objetivos da pesquisa. Os professores sujeitos do estudo deveriam ter vivenciado a experiência de desenvolver e aplicar abordagens direcionadas à aprendizagem, fundamentadas na história da Matemática. Por meio de um levantamento 389 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 realizado em programas de Pós-Graduação na área de ensino de Ciências e Educação Matemática conseguimos localizar nos bancos de teses e dissertações 18 trabalhos com tais características, conforme Araman e Batista (2013). Estabelecemos contato com vários autores das referidas pesquisas e conseguimos realizar entrevistas com seis deles, os quais concordaram, esclarecidamente, em participar de nossa pesquisa. Os professores que participaram da coleta de dados para a pesquisa e nos concederam entrevista foram designados por P1 (professor 1), P2 (professor 2) e assim sucessivamente, com a finalidade de preservar a identidade dos mesmos. O quadro a seguir apresenta uma síntese do perfil dos sujeitos participantes: Sujei to Formação Experiência profissional Proposta desenvolvida Graduação Especiali- zação Mestrado Doutorado Conteúdo Nível de ensino P1 Licenciatura em Ciências – Hab. Matemática CESULON- PR Educação Matemática UEL-PR Ensino de Ciências e Educação Matemática UEL-PR Ensino de Ciências e Educação Matemática (em curso) UEL-PR Aproximada- mente 16 anos – Ensino Fund/Médio/ Superior Sistemas Lineares/ Determinan- tes/Matrizes 2º ano do Ensino Médio P2 Licenciatura em Ciências – Hab. 5 Apresentação e análise dos dados 4 Metodologia Observem alguns exemplares no quadro a seguir: p p g g p q g Categoria “Conhecimento relativo à História da Matemática na Educação Matemática” P1 Então, na reconstrução, havia determinadas situações em que você percebe que historicamente os matemáticos ficaram enroscados vinte anos em determinado assunto. Se os matemáticos ficaram enroscados vinte anos, é possível que no momento em que você estiver ensinando aquele conteúdo, os alunos apresentem dificuldades nele. Aprender a técnica é importante, a técnica, o método é importante, mas só isso não basta para o ensino de matemática. É importante contextualizar e a história ajuda muito. P2 Hoje eu percebo que para ensinar trigonometria é preciso saber muito mais do que as relações fundamentais, as funções trigonométricas. É preciso mostrar aos alunos a importância desse conhecimento, por que ele está presente até hoje nos currículos, como e por que ele se desenvolveu, qual a relação dele com outros conteúdos, etc. Isso a história me ajudou. [...] eu procurei inicialmente identificar o que era cada palavra daquelas, seno, cosseno, dentro do contexto histórico porque a gente sabe que na matemática principalmente vem do latim, vem do árabe, vem de outras civilizações. Então eu queria entender e mostrar para o aluno o que significava cada palavra daquela referente às funções trigonométricas, mas de forma contextualizada e não apenas só a memorização dos nomes. P3 [...] usar alguns aspectos de como foi descoberto um conceito matemático, pode facilitar o entendimento do aluno, visto que, apresentar um conceito já formalizado, pronto e acabado, pode dificultar a aprendizagem e tornar a matemática algo dogmático. Em certos momentos, a apresentação dedutiva é extremamente necessária. Mas o que não pode ocorrer é o professor ficar só nisso, não saber contextualizar o conhecimento para seu aluno, não conseguir explicar por que aquele conhecimento é importante, em que ele contribui, por que ele surgiu, e isso quase nunca é feito. A História da Matemática melhora isso, contribui para o professor justificar, contextualizar, dar significado a esses conteúdos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 391 Categoria “Conhecimento relativo à História da Matemática na Educação Matemática” P1 Então, na reconstrução, havia determinadas situações em que você percebe que historicamente os matemáticos ficaram enroscados vinte anos em determinado assunto. 4 Metodologia Se os matemáticos ficaram enroscados vinte anos, é possível que no momento em que você estiver ensinando aquele conteúdo, os alunos apresentem dificuldades nele. Aprender a técnica é importante, a técnica, o método é importante, mas só isso não basta para o ensino de matemática. É importante contextualizar e a história ajuda muito. P2 Hoje eu percebo que para ensinar trigonometria é preciso saber muito mais do que as relações fundamentais, as funções trigonométricas. É preciso mostrar aos alunos a importância desse conhecimento, por que ele está presente até hoje nos currículos, como e por que ele se desenvolveu, qual a relação dele com outros conteúdos, etc. Isso a história me ajudou. [...] eu procurei inicialmente identificar o que era cada palavra daquelas, seno, cosseno, dentro do contexto histórico porque a gente sabe que na matemática principalmente vem do latim, vem do árabe, vem de outras civilizações. Então eu queria entender e mostrar para o aluno o que significava cada palavra daquela referente às funções trigonométricas, mas de forma contextualizada e não apenas só a memorização dos nomes. P3 [...] usar alguns aspectos de como foi descoberto um conceito matemático, pode facilitar o entendimento do aluno, visto que, apresentar um conceito já formalizado, pronto e acabado, pode dificultar a aprendizagem e tornar a matemática algo dogmático. Em certos momentos, a apresentação dedutiva é extremamente necessária. Mas o que não pode ocorrer é o professor ficar só nisso, não saber contextualizar o conhecimento para seu aluno, não conseguir explicar por que aquele conhecimento é importante, em que ele contribui, por que ele surgiu, e isso quase nunca é feito. A História da Matemática melhora isso, contribui para o professor justificar, contextualizar, dar significado a esses conteúdos. Categoria “Conhecimento relativo à História da Matemática na Educação Matemática” P1 Então, na reconstrução, havia determinadas situações em que você percebe que historicamente os matemáticos ficaram enroscados vinte anos em determinado assunto. Se os matemáticos ficaram enroscados vinte anos, é possível que no momento em que você estiver ensinando aquele conteúdo, os alunos apresentem dificuldades nele. Aprender a técnica é importante, a técnica, o método é importante, mas só isso não basta para o ensino de matemática. É importante contextualizar e a história ajuda muito. P2 Hoje eu percebo que para ensinar trigonometria é preciso saber muito mais do que as relações fundamentais, as funções trigonométricas. 4 Metodologia Matemática FAFICOP- PR Educação Matemática FAFICOP- PR Ensino de Ciências e Educação Matemática UEL-PR Aproximada- mente 8 anos – Ensino Fund/Médio/ Superior Funções trigonomé- tricas 2º ano do Ensino Médio P3 Licenciatura em Matemática UFSC-SC Educação para a Ciência e o Ensino de Matemática UEM-PR Aproximada- mente 4 anos – Ensino Médio/Supe- rior/ cursos preparatórios/ tutor de EAD Conceito de função 8ª série do Ensino Fundamen- tal P4 Licenciatura em Matemática UFPB-PB Ensino de Matemáti- ca UFPB-PB Ensino de Ciências Naturais e Matemática UFRN-RN Educação Matemática (em curso) UNESP-SP Aproximada- mente 30 anos – Ensino Fund/Médio/ Superior Secções Cônicas 3º ano do Ensino Médio P5 Licenciatura em Matemática UFPA-PA Ensino de Ciências e Matemáti- ca UFPA-PA Educação UFRN-RN Educação UFRN-RN Aproximada- mente 25 anos – Ensino Fund/Médio/ Superior Trigonome- tria 1º ano do Ensino Médio P6 Licenciatura em Matemática UFRN – RN Ensino de Ciências Naturais e Matemática UFRN/RN Aproximada- mente 10 anos – Ensino Fund/Médio/ Superior/ cursos preparatórios/ EJA Números Complexos 3º ano do Ensino Médio Quadro 1 - Síntese do perfil dos professores entrevistados Fonte: Araman (2011). Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 390 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 A análise foi desenvolvida agrupando as unidades por similitudes, conforme as categorias estruturadas na fundamentação teórica da pesquisa e explicitadas na figura 1 deste artigo. Sendo assim, identificamos nas entrevistas trechos que se enquadrassem em cada uma delas. O resultado obtido pode ser observado na sequência do texto.  Categoria “Conhecimento relativo à história da Matemática na Educação Matemática”  Categoria “Conhecimento relativo à história da Matemática na Educação Matemática” Essa categoria evidencia a presença de conhecimentos relacionados às potencialidades do uso da história da Matemática na formação do professor que vivenciou o processo de construção de uma abordagem pedagógica baseada na história da Matemática. É fundamental que o professor, além de ter contato com estudos históricos a respeito do conteúdo matemático selecionado para a construção da abordagem, bem como dos fundamentos metodológicos relacionados à pesquisa histórica, como o uso de fontes, a seleção de episódios, entre outros, tenha a percepção das contribuições que os estudos históricos podem ter para a aprendizagem matemática. 4 Metodologia É preciso mostrar aos alunos a importância desse conhecimento, por que ele está presente até hoje nos currículos, como e por que ele se desenvolveu, qual a relação dele com outros conteúdos, etc. Isso a história me ajudou. [...] eu procurei inicialmente identificar o que era cada palavra daquelas, seno, cosseno, dentro do contexto histórico porque a gente sabe que na matemática principalmente vem do latim, vem do árabe, vem de outras civilizações. Então eu queria entender e mostrar para o aluno o que significava cada palavra daquela referente às funções trigonométricas, mas de forma contextualizada e não apenas só a memorização dos nomes. P3 [...] usar alguns aspectos de como foi descoberto um conceito matemático, pode facilitar o entendimento do aluno, visto que, apresentar um conceito já formalizado, pronto e acabado, pode dificultar a aprendizagem e tornar a matemática algo dogmático. Em certos momentos, a apresentação dedutiva é extremamente necessária. Mas o que não pode ocorrer é o professor ficar só nisso, não saber contextualizar o conhecimento para seu aluno, não conseguir explicar por que aquele conhecimento é importante, em que ele contribui, por que ele surgiu, e isso quase nunca é feito. A História da Matemática melhora isso, contribui para o professor justificar, contextualizar, dar significado a esses conteúdos. ategoria “Conhecimento relativo à História da Matemática na Educação Matemática” Aprender a técnica é importante, a técnica, o método é importante, mas só isso não basta para o ensino de matemática. É importante contextualizar e a história ajuda muito. Em certos momentos, a apresentação dedutiva é extremamente necessária. Mas o que não pode ocorrer é o professor ficar só nisso, não saber contextualizar o conhecimento para seu aluno, não conseguir explicar por que aquele conhecimento é importante, em que ele ontribui, por que ele surgiu, e isso quase nunca é feito. A História da Matemática melhora isso, contribui para o professor justificar, contextualizar dar significado a esses conteúdos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 391 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 P4 Boa parte das minhas aulas se diferenciavam dos meus colegas professores, como já te falei, mas mesmo assim elas se diferenciavam porque eu usava atividades dinâmicas, experimentos, investigações, etc. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia Mas ainda sentia falta de encontrar um sentido para os conteúdos, de justificar perante meus alunos por que aqueles conteúdos matemáticos eram importantes, em que contexto eles foram construídos, se os pesquisadores da época tiveram ou não dificuldades, com quais outros conhecimentos eles se relacionam, etc. Então eram dúvidas da própria matemática, quer dizer, da construção do conhecimento matemático que, às vezes, uma atividade dinâmica ou um experimento por si só não davam conta também de responder. E a história me ajudou muito nisso. [...] eu usei apenas algumas atividades históricas, mas me chamou a atenção o fato de que essas atividades históricas conseguiram contextualizar um pouco esse conhecimento matemático e o aluno entendeu um pouco disso. Quer dizer, o contexto histórico traz algumas compreensões que não são possíveis apenas por meio de atividades experimentais. P5 [...] começamos a perceber a importância que a história tem, principalmente quando começamos a entender melhor o desenvolvimento dos conceitos matemáticos, podendo dar uma explicação melhor para os alunos. [...] a história pode esclarecer conceitualmente a gente e na medida em que ela nos esclarece conceitualmente, ela nos abre a possibilidade de uma reformulação do nosso exercício docente. Um outro exemplo do que chamou a atenção foi sobre ângulos, sobre uma ideia que tem lá que fala de minuto e segundo, isso também chamou a atenção deles porque era uma curiosidade que eles relacionaram com a contagem do tempo e a medida do ângulo que era na base sessenta, então eles gostaram dessa informação, pois era um assunto que eles já conheciam, mas que foi contextualizado pela história. P6 Hoje eu compreendo que para ensinar matemática precisa muito mais do que saber o conteúdo, o professor tem que conhecer novas estratégias de ensino, precisa compreender como seu aluno aprende e a história da matemática me mostrou algumas possibilidades. Busquei na história esses elementos que mostrassem o contexto no qual os números complexos foram criados, qual era a necessidade disso, o que estavam pesquisando, como eles foram sistematizados, em que eram aplicados, etc. Buscamos uma maneira de tornar esse conteúdo contextualizado historicamente e que isso facilitasse a aprendizagem dos alunos. 4 Metodologia Quadro 2 - Discursos que remetem à categoria “Conhecimento relativo à História da Matemática na Educação Matemática” P4 Boa parte das minhas aulas se diferenciavam dos meus colegas professores, como já te falei, mas mesmo assim elas se diferenciavam porque eu usava atividades dinâmicas, experimentos, investigações, etc. Mas ainda sentia falta de encontrar um sentido para os conteúdos, de justificar perante meus alunos por que aqueles conteúdos matemáticos eram importantes, em que contexto eles foram construídos, se os pesquisadores da época tiveram ou não dificuldades, com quais outros conhecimentos eles se relacionam, etc. Então eram dúvidas da própria matemática, quer dizer, da construção do conhecimento matemático que, às vezes, uma atividade dinâmica ou um experimento por si só não davam conta também de responder. E a história me ajudou muito nisso. [...] eu usei apenas algumas atividades históricas, mas me chamou a atenção o fato de que essas atividades históricas conseguiram contextualizar um pouco esse conhecimento matemático e o aluno entendeu um pouco disso. Quer dizer, o contexto histórico traz algumas compreensões que não são possíveis apenas por meio de atividades experimentais. [...] começamos a perceber a importância que a história tem, principalmente quando começamos a entender melhor o desenvolvimento dos conceitos matemáticos, podendo dar uma explicação melhor para os alunos. Todos os professores apresentaram alguns elementos que indicam as contribuições que a pesquisa em história da Matemática trouxe para a sua formação e para a sua ação docente. Para P1, a sequência histórica de evolução dos conceitos pode ser mais adequada para o processo de ensino, pois os alunos podem ter mais facilidade e interesse se os conteúdos forem apresentados segundo a ordem histórica de seu desenvolvimento. Chama a atenção também para o fato de que os estudos históricos podem indicar pontos de dificuldades na aprendizagem e aponta, como contribuição, o fato de que, ao saber disso, o professor pode ter mais cuidado com seus alunos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 392 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 P2 esclarece que percebeu que ensinar trigonometria vai além de ensinar as relações fundamentais, as funções trigonométricas, e essa percepção foi possibilitada pelos estudos históricos. Afirma ainda que esse fato está influenciando a sua atuação em sala de aula. No entendimento de P3, a história possibilita uma alternativa de compreensão diferenciada em relação ao ensino tradicional e que pode auxiliar na compreensão dos alunos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia Para ele, essa percepção colaborou para sua formação, considerando-se um professor mais bem preparado. O discurso de P4 é muito interessante, pois este professor relata que já buscava em sua ação docente elementos diferenciados para abordar os conteúdos matemáticos, como o uso de experimentos, atividades de investigação, entre outros. No entanto, aponta que mesmo essas atividades diferenciadas não conseguiam dar algumas justificativas e explicações em relação aos conhecimentos. E indica a investigação histórica como uma contribuição para complementar a prática diferenciada que realizava. Para P5, o entendimento do conteúdo por meio da história permite ao professor dar explicações mais adequadas para seus alunos. Acrescenta, como contribuição, que a compreensão dos conteúdos possibilitada pela história permite ao professor reformular a sua ação docente. P6 discorre a respeito da possibilidade que a história oferece de colocar o aluno em uma situação similar àquela ocorrida no contexto histórico. Acrescenta também que os estudos históricos que fez sobre os números complexos permitiu que ele tivesse uma atuação mais adequada junto aos alunos. Ainda aponta a história como uma opção para o processo de ensino de Matemática. Os dados revelam que os professores atribuem à história da Matemática a capacidade de contextualizar os conteúdos matemáticos e que essa contextualização é importante para o processo de ensino e de aprendizagem. P1 compreende que a abordagem pela história auxilia a contextualização do conteúdo, colaborando para que o aluno aprenda além das técnicas. P2 aponta que a terminologia usada na Matemática pode também ser favorecida por meio da abordagem pela história, uma vez que os alunos podem conhecer os contextos nos quais os termos foram criados e não apenas memorizá-los. Além disso, coloca que a contextualização histórica colabora para a compreensão dos conceitos e para o reconhecimento da importância dos mesmos. Os trechos de P3 e P6 evidenciam a necessidade de contextualização dos conteúdos para a percepção da importância deles, das contribuições que eles trouxeram para a Matemática e para outras áreas, e que essas compreensões podem colaborar para a aprendizagem. P4 retoma a discussão a respeito das atividades experimentais, dizendo que a Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 393 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 contextualização possibilitada pela história favorece algumas compreensões que as atividades experimentais, sozinhas, não favorecem. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia E P5 nos coloca que o contexto histórico colaborou também no sentido de os alunos compreenderem algumas noções que já tinham, como a base sessenta, mas que foi contextualizada pela história na explicação sobre ângulos abordada na trigonometria. Por meio da análise que realizamos, pudemos observar que a proposição de uma abordagem de ensino diferenciada, como a pautada na história da Matemática, requer alguns cuidados metodológicos que foram observados pelos professores. Como o objetivo de qualquer abordagem de ensino é proporcionar aprendizagem, os professores mostraram essa preocupação, de que a proposta que desenvolveram colabore, com elementos consistentes, para a aprendizagem matemática. Encontramos essa discussão feita por muitos pesquisadores que evidenciam as potencialidades pedagógicas do uso da história da Matemática em sala de aula – Fauvel e Maanen (2000); Brolezzi (2003); Miguel e Miorim (2008), entre outros. Segundo eles, a abordagem dos conteúdos matemáticos via história tem potencialidades pedagógicas que devem ser aproveitadas pelos professores. Os professores desenvolveram uma abordagem com finalidades pedagógicas, ou seja, com a finalidade de ensinar determinado conteúdo ou conceito. Os conceitos são trabalhados via abordagem histórica, como uma alternativa metodológica para a Educação Matemática. Essa maneira de conceber a inclusão da história da Matemática nos processos de ensino e de aprendizagem é apontada por Fried (2008) como a estratégia da acomodação. Essa compreensão, a nosso ver, foi possibilitada pelo fato de os professores terem contato com os aportes teóricos da história da Matemática na Educação Matemática. Esse entendimento pode ter sido proporcionado por meio dos estudos teóricos que realizaram. Vimos que as pesquisas que envolvem as discussões a respeito das potencialidades pedagógicas da história da Matemática atribuem a ela a capacidade de contextualizar os conteúdos, uma vez que realizar a contextualização não é uma tarefa trivial para os professores. Segundo Matthews (1995), a história pode colaborar para a “superação do ‘mar de falta de significação’ que se diz ter inundado as salas de aula de ciências, onde fórmulas e equações são recitadas sem que muitos cheguem a saber o que significam” (MATTHEWS, 1995, p. 165). Bicudo (1999), Miguel e Miorim (2008), Brito e Carvalho (2009), entre outros, consideram que a formação inicial recebida pelos professores muitas vezes não favorece a 394 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 capacidade de justificar e contextualizar os conteúdos matemáticos em sala de aula. Os estudos históricos podem contribuir para minimizar essa dificuldade dos professores. 4 Metodologia Esse fato foi evidenciado também pelos sujeitos, que perceberam que a história da Matemática colabora para a compreensão contextualizada dos conteúdos trabalhados. Pelos resultados obtidos nesta categoria, consideramos que os professores que tiveram contato com as pesquisas na área de história da Matemática na Educação Matemática, tiveram a oportunidade de desenvolver o entendimento das potencialidades que ela pode ter no processo de ensino e de aprendizagem, que vão além da abordagem da história como curiosidade ou motivação.  Categoria “Conhecimento relativo à natureza do conhecimento matemático” Já argumentamos, apoiadas nas pesquisas científicas, que o entendimento dos professores acerca da natureza do conhecimento matemático precisa ser melhorado e que os estudos históricos podem ser relevantes para isso – Batista e Luccas (2004); Matthews (1995); Martins (2007); Liu (2009). Sendo assim, considerando que os sujeitos desta pesquisa tiveram a oportunidade de estudar o desenvolvimento histórico de um conteúdo matemático, nossa intenção foi analisar, nesta categoria, se os mesmos explicitam, de alguma forma, contribuições da história da Matemática para a compreensão da natureza do conhecimento matemático. Os resultados encontrados podem ser observados a seguir: Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 395 Categoria “Conhecimento relativo à natureza do conhecimento matemático” P1 [...] eu mudei radicalmente de uma visão passiva da matemática para uma visão completamente dinâmica. E a história faz com que a gente perceba isso, que para um conceito chegar aonde ele chegou foi necessário muito trabalho, muita dedicação, colaboração entre muitas pessoas, pessoas reais, que erram, que acertam. Foi bem interessante, para mim foi muito interessante[...]. Então isso muda aquela visão de mito, pois muita gente trabalha e contribui para que aquela teoria fique cada vez mais concisa, consistente. P2 A reconstrução histórica foi me mostrando, a cada dia que eu achava algo novo nos livros, nas fontes, que a matemática não estava pronta e acabada, mas que ela tinha uma evolução [...]. [...] a reconstrução histórica me ajudou a entender essa evolução da ciência, do desenvolvimento da matemática, da trigonometria. P3 A pesquisa histórica nos faz perceber que o conhecimento matemático não é pronto e acabado, mas que se desenvolve em um processo dinâmico. Isso mudou, percebi que o caminho é o caminho da investigação, das tentativas, do uso dos conhecimentos já estruturados por outros na formação dos novos conhecimentos [...]. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 4 Metodologia Também tem a questão da criação de mitos, que a história me ajudou a compreender que os Categoria “Conhecimento relativo à natureza do conhecimento matemático” Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 395 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 conhecimentos matemáticos são frutos de erros, acertos, de muito trabalho, de anos de pesquisas, de dedicação. P4 [...] a pesquisa histórica me auxiliou nessa compreensão, quer dizer, pude viajar até o ano 600 a.C. e vir pouco a pouco montando uma história que, especificamente, desconhecia. Essa pesquisa contribuiu para que eu compreendesse o desenvolvimento da matemática de uma outra forma, de um modo dinâmico, com acertos, erros, avanços, etc. Tinha aquela noção de mito que é passada para nós durante toda a nossa vida escolar, lei de fulano, teorema de sicrano, essa visão vai mudando quando estudamos a história. E isso foi importante para a minha compreensão e também para não dar uma compreensão errada para meus alunos. P5 A matemática não é pronta e acabada pelo seguinte: se fosse, os matemáticos não estariam mais produzindo mais matemática. Agora, essa consciência a gente só vai adquirindo na medida em que a gente lê, a gente discute com outras pessoas, a gente ouve e a história me ajudou muito nessa compreensão. [...] a pesquisa histórica ajuda a ver que não é bem assim, um conhecimento para ser posto, aceito, precisa passar por muitas contribuições, não é tarefa de um único ser humano extraordinário, fantástico. Isso a pesquisa possibilitou. P6 Hoje não, hoje eu tenho uma compreensão do conhecimento matemático bem diferente de antes, e isso eu acho que contribuiu para a minha prática profissional. E isso foi possível graças à pesquisa histórica. [...] a história me fez perceber os fatos e os contextos em que os conceitos matemáticos foram criados, a necessidade por que eles foram criados e ainda mais desmistificando aquela velha visão de que os conceitos matemáticos nascem do nada, ou seja, me fez enxergar o quão árdua e penosa é a criação de um conceito dessa natureza e que essa aquisição é um esforço conjunto de várias pessoas ao longo de um vasto intervalo de tempo. Quadro 3 - Discursos que remetem à categoria “Conhecimento relativo à natureza do conhecimento matemático” Fonte: Araman (2011). hecimentos matemáticos são frutos de erros, acertos, de muito trabalho, de anos de quisas, de dedicação. 4 Metodologia [...] a pesquisa histórica me auxiliou nessa compreensão, quer dizer, pude viajar até o ano 600 a.C. e vir pouco a pouco montando uma história que, especificamente, desconhecia. Essa pesquisa contribuiu para que eu compreendesse o desenvolvimento da matemática de uma outra forma, de um modo dinâmico, com acertos, erros, avanços, etc. Tinha aquela noção de mito que é passada para nós durante toda a nossa vida escolar, lei de ulano, teorema de sicrano, essa visão vai mudando quando estudamos a história. E isso foi mportante para a minha compreensão e também para não dar uma compreensão errada para meus alunos. [...] a pesquisa histórica ajuda a ver que não é bem assim, um conhecimento para ser posto aceito, precisa passar por muitas contribuições, não é tarefa de um único ser humano extraordinário, fantástico. Isso a pesquisa possibilitou. Pelos depoimentos dos professores, podemos observar que o contato que tiveram com a história da Matemática colaborou no sentido de proporcionar uma compreensão do conhecimento matemático como um processo dinâmico e em constante evolução. Todos os professores ouvidos afirmaram que esta visão de Matemática foi possível após os estudos históricos realizados por eles. Na compreensão de Liu (2009), a Matemática comumente é vista como um corpo absoluto e rígido de conhecimentos, e a compreensão da natureza da Matemática pode ser impulsionada por meio de estudos históricos. Nessa mesma perspectiva, Batista e Luccas (2004), Matthews (1995), Martins (2007), entre outros, apontam que os componentes históricos são relevantes para a formação docente, uma vez que podem colaborar para uma visão mais adequada do conhecimento científico. Nossa análise nos permite afirmar que, para este grupo de professores, os estudos históricos proporcionaram o entendimento da Matemática como um corpo de conhecimento dinâmico, cuja evolução se faz por meio da colaboração entre muitos pesquisadores, ao longo de muitos Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 396 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 anos de dedicação, possibilitando novos e relevantes conhecimentos acerca da natureza do conhecimento matemático. Para Liang et al. (2009), desenvolver uma compreensão adequada, por parte dos professores, acerca da natureza da ciência é uma tendência mundial. Assim, consideramos que o processo de construção de abordagens históricas vivenciado pelos professores, que envolveu estudos históricos, colaborou para o desenvolvimento de uma visão adequada da natureza do conhecimento matemático por parte dos mesmos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 Essa compreensão mais profunda é discutida por Brito e Carvalho que concordam que os estudos referentes à história da Matemática podem colaborar para esse entendimento: Essa compreensão mais profunda é discutida por Brito e Carvalho que concordam que os estudos referentes à história da Matemática podem colaborar para esse entendimento: Essa compreensão mais profunda é discutida por Brito e Carvalho que concordam que os estudos referentes à história da Matemática podem colaborar para esse entendimento: história da Matemática podem colaborar para esse entendimento: Entendemos “saber profundamente” em um sentido que vai além de o professor demonstrar com exatidão teoremas, lidar com linguagem matemática de um modo mecânico e ter algum conhecimento sobre quem e em que época tal teorema ou propriedade matemática “foi descoberta”. Para nós o professor “saber profundamente Matemática” significa que além de conhecer teoremas, consegue relacionar diferentes campos desse conhecimento, refletir sobre os fundamentos da Matemática, perceber seu dinamismo interno e suas relações com outros campos do saber, transitar nos diferentes sistemas de registro de representação e, principalmente, entender o conhecimento matemático como um saber que coloca problemas e não apenas soluções (BRITO; CARVALHO, 2009, p. 15-16). Dessa forma, os resultados da análise desta categoria podem ser observados no seguinte quadro: Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 398 Dessa forma, os resultados da análise desta categoria podem ser observados no seguinte quadro: Categoria “Conhecimento relativo ao conteúdo matemático” P1 Eu tive que estudar muita matemática para poder desvendar principalmente a teoria do japonês (Seki Kowa), que tinha muito diagrama, muito quadrado, para saber o que significava tudo aquilo, eu tive que aprender muita coisa, inclusive técnicas, métodos matemáticos eu tive que aprender. P2 Percebi então que aumentou a minha capacidade de entendimento da matemática, do conteúdo, de entender os porquês, de entender qual foi a importância, quais foram os fatos marcantes, os impasses, o que estava acontecendo na época, que fenômenos eles estavam descobrindo, para que a trigonometria foi importante e também para quais ciências a trigonometria foi importante. P3 Então, na pesquisa histórica eu percebi que primeiro foi necessário a compreensão das relações qualitativas de dependência, depois o uso de tabelas e da observação de regularidades, as variáveis, somente depois disso veio a representação gráfica, a introdução da linguagem algébrica e a representação analítica. P4 Então, como o tema escolhido para ser desenvolvido foram as secções cônicas, eu precisei de uma fundamentação histórica sobre tal. E foi muito bom fazer a parte histórica, me esclareceu muitas dúvidas com relação a esse conteúdo. 4 Metodologia Conforme Charalambous, Panaoura e Philippou (2009), a forma de o professor conceber a Matemática influencia a maneira como ele compreende o processo de ensino e de aprendizagem e, consequentemente, a sua ação pedagógica. Segundo Dass (2005), existe uma relação entre a compreensão da natureza da ciência que o professor possui e sua atuação em sala de aula. Para Barbin (2000), a história pode colaborar para alterar a compreensão do próprio professor a respeito da Matemática. Esse fato pode influenciar no modo como ele ensina Matemática e, consequentemente, no modo como o aluno a compreende. Em suma, a análise desta categoria nos permite perceber que a pesquisa realizada pelos professores colaborou com alguns elementos teóricos para a compreensão da natureza do conhecimento matemático, colaborando para a estruturação de saberes interdisciplinares. Essa compreensão, além de proporcionar ao professor um entendimento maior a respeito da área de conhecimento com a qual atua, pode trazer implicações para a sua atuação pedagógica.  Categoria “Conhecimento relativo ao conteúdo matemático” Encontramos na literatura discussões a respeito da formação conceitual do professor, que muitas vezes não é suficiente ou adequada para responder às demandas de uma sala de aula – Miguel (2005); Matthews (1995); Bursal (2010); Brito e Carvalho (2009); Furinguetti (2007). Nesse sentido, nesta categoria consideramos as evidências apresentadas pelos professores de que a investigação histórica de um conteúdo matemático proporcionou um entendimento mais “profundo” do mesmo, uma compreensão voltada às justificativas, à explicitação dos porquês, dos debates, etc, que não tinham antes de realizar a pesquisa. Todos os professores ouvidos alegaram que o contato que tiveram com o desenvolvimento histórico de dado conteúdo matemático proporcionou um tipo de compreensão diferente daquele que tinham até então. A compreensão a que se referem é aquela que extrapola a axiomatização e a aplicação mecânica dos conteúdos. 397 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 Essa compreensão mais profunda é discutida por Brito e Carvalho que concordam que os estudos referentes à história da Matemática podem colaborar para esse entendimento: Eu compreendi melhor os conceitos, nem tanto nos aspectos estruturais, do conhecimento matemático em si, pois já o dominava, a estrutura eu já dominava, mas compreendi, por exemplo, por que, historicamente, foi necessário realizar esses estudos, quais os problemas que os matemáticos enfrentavam na época, quais eram os motivos que os moviam, etc. [...] conheci alguns métodos de traçados utilizados ao longo da história, como traçados de figuras cônicas em quadro perfurado utilizando o método do jardineiro (séc. VI), o método de Kepler (usado já também no séc. VI). Então esse novo conjunto de compreensões que eu tive sobre o assunto de secções cônicas foi a história que me proporcionou. P5 A história me possibilitou entender e explicar isso muito bem para o aluno, da ideia de cosseno como seno reverso, da ideia de complementaridade entre seno e cosseno para corresponder exatamente ao triângulo retângulo, explicar para o aluno que quando o seno aumenta, o cosseno diminui e vice-versa. Quando eu me deparei com a História mudou tudo, porque aí eu passei a entender detalhadamente, por exemplo, uma coisa importantíssima na minha formação histórica e que eu consegui passar para os meus alunos, que seno e cosseno são fragmentos do raio, todos Categoria “Conhecimento relativo ao conteúdo matemático” A história me possibilitou entender e explicar isso muito bem para o aluno, da ideia de cosseno como seno reverso, da ideia de complementaridade entre seno e cosseno para corresponder exatamente ao triângulo retângulo, explicar para o aluno que quando o seno aumenta, o cosseno diminui e vice-versa. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 398 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 aqueles valores da tabela correspondem a fragmentos do raio, que são exatamente as projeções do raio nos eixos. P6 Logo, passei a encontrar algumas respostas bem razoáveis para esses questionamentos exatamente com o uso da história da matemática, quer dizer, a história melhorou a minha compreensão a respeito dos números complexos e isso me ajudou a dar algumas respostas para os alunos. [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 Essa compreensão mais profunda é discutida por Brito e Carvalho que concordam que os estudos referentes à história da Matemática podem colaborar para esse entendimento: A representação no plano cartesiano também foi bem interessante, porque sem a história parece meio que uma regra meio que mecânica, mas só observando a formação desse processo histórico é que vemos toda a dedução desse processo de formação que já vem de outros conhecimentos mais consolidados como vetores o que também gera um maior poder de compreensão. Quadro 4 - Discursos que remetem à categoria “Conhecimento relativo ao conteúdo matemático” Fonte: Araman (2011). [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! O corpus analisado salienta que todos os professores perceberam que o contato que tiveram com desenvolvimento histórico dos conteúdos estudados por eles colaborou para a compreensão conceitual desses conteúdos e também no entendimento das razões, dos motivos pelos quais eles foram se desenvolvendo, da importância que os mesmos tiveram na época, da relação com outros conceitos, entre outros. Ou seja, uma compreensão que extrapola os aspectos estruturais e axiomáticos dos conteúdos em questão. P1 esclarece que precisou de muitos estudos para compreender o raciocínio e as técnicas utilizadas na época e que, embora já conhecesse algumas delas, conseguiu ter uma compreensão de como foram estruturadas, qual era o raciocínio implícito em seu desenvolvimento e da importância delas no contexto de seu desenvolvimento e também no contexto atual. Esses aspectos foram lembrados também por P2, que alega que a pesquisa histórica proporcionou um aumento em sua capacidade de entendimento da trigonometria. P3 salientou que esse estudo colaborou no sentido de demonstrar quais foram as etapas necessárias no desenvolvimento do conceito de função até termos hoje a linguagem algébrica e a representação analítica. P4 aponta que a pesquisa histórica esclareceu, em relação ao conteúdo estudado, muitas dúvidas que extrapolam os aspectos estruturais, trazendo “um novo conjunto de compreensões” possibilitado pela história. O relato de P5 levanta um ponto importante, que é a relação entre a sua compreensão de questões relacionadas à trigonometria e as implicações dessa compreensão para seus alunos. Segundo ele, a história foi “decisiva” para o entendimento de várias relações presentes no estudo de trigonometria. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 399 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 P6 aponta a compreensão, possibilitada pelos estudos históricos, da representação no plano cartesiano dos números complexos, que, de acordo com ele, sem a história, configura-se apenas como uma regra mecânica. Além disso, alega que tal entendimento possibilitou dar outras explicações para seus alunos. De acordo com os relatos, a pesquisa histórica proporcionou aos professores uma compreensão mais profunda dos conteúdos pesquisados. Isso ocorreu com todos os professores. Essa compreensão, além de referir-se aos conteúdos matemáticos propriamente ditos, como a trigonometria, as secções cônicas, números complexos, entre outros, possibilitou o entendimento dos porquês, dos fatos marcantes, dos impasses, e o encontro de algumas respostas que antes não tinham. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! A compreensão do processo de construção do conhecimento, e não apenas do produto final, foi evidenciada nas falas dos sujeitos, que acrescentam que o estudo do processo pode minimizar a mera aplicação mecânica da Matemática. Segundo alguns relatos, esse entendimento mais profundo dos conteúdos matemáticos influencia a prática pedagógica do professor, uma vez que ele se sente mais preparado para responder aos questionamentos dos seus alunos e com mais autonomia frente aos conteúdos. Carvalho (2000) alega que a perspectiva histórica permite aos professores perceber que a maneira como um conceito é apresentado hoje é consequência das ideias anteriores, possibilitando observar a Matemática atual e a do passado, as diferenças nos usos das notações, terminologias, entre outros, proporcionando ao professor uma melhor compreensão conceitual. Os dados evidenciaram que os professores sentiam falta de compreenderem mais a Matemática, os conteúdos matemáticos com os quais trabalham para poderem abordá-los com mais segurança na sala de aula. A experiência vivenciada nos estudos históricos do conhecimento matemático, numa formação complementar, incorporou alguns elementos relevantes para suprir as necessidades formativas dos professores envolvidos.  Categoria “Conhecimento relativo às adequações pedagógicas”  Categoria “Conhecimento relativo às adequações pedagógicas” Esta categoria tem como objetivo analisar a necessidade de adequações pedagógicas aos estudos históricos realizados, com o intuito de possibilitar o uso da história da Matemática em sala de aula, ou seja, a necessidade de concatenar os vários aspectos oriundos da pesquisa histórica desenvolvida com elementos próprios da Pedagogia (BATISTA, 2009). B l Ri Cl (SP) 31 57 380 407 b 2017 400 Em nossa compreensão, o processo de elaboração de uma abordagem pedagógica fundamentada na história da Matemática proporciona a integração entre o conteúdo 400 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 matemático a ser abordado, os elementos históricos desse conteúdo e há a necessidade de várias adequações pedagógicas por parte do professor para tornar a abordagem adequada à realidade de uma sala de aula, colaborando para sua formação interdisciplinar. [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! Na análise do corpus, encontramos em todos os professores elementos que indicam a necessidade que os mesmos tiveram de realizar adequações pedagógicas para a implementação da abordagem em sala de aula, e os resultados apresentamos no quadro abaixo: q q ç p g g p implementação da abordagem em sala de aula, e os resultados apresentamos no quadro abaixo: Categoria “Conhecimento relativo às adequações pedagógicas” P1 Mas uma coisa é a pesquisa histórica, é você fazer aquela pesquisa teórica, outra coisa é fazer a transposição, a adequação didática dessa pesquisa possível de ser colocada em sala de aula. Qual é a adequação que você tem que fazer desse conteúdo, quais são os cuidados, o que é essencial para sua pesquisa, para você adaptar de forma coerente, adequada para você levar para a sala de aula. P2 [...] procurei sempre amarrar, fazer essa relação entre o que era história da matemática, como foi desenvolvida a trigonometria nessa história e as atividades didáticas para que o aluno também não ficasse [...] não ficasse cansativo para ele. [...] as atividades tinham que ser também dinâmicas, uma atividade que estimulasse o aluno para o aprendizado, não podia ser um texto histórico somente, tinha que ter adequações. P3 Pois eu tinha na mão muitas informações históricas, mas e aí, quais escolher? Como minha intenção era compor uma sequência de atividades para a aprendizagem de função, então isto também estava presente na investigação histórica. [...] organizar tudo isso de forma integrada é um grande desafio, é muito difícil conseguir isso. Só fui perceber como é complexo o processo de construção de uma abordagem histórica depois de fazer essa pesquisa, porque não é somente “pegar” a história da matemática e contar na sala de aula. Não é isso, é necessário montar uma proposta possível de ser desenvolvida em turmas normais, com alunos comuns que encontramos todos os anos, e montar uma proposta que contribua para a aprendizagem de conteúdos matemáticos, e não se faz isso de qualquer forma, é necessária muita pesquisa. P4 [...] a maior dificuldade é selecionar, entre todos esses materiais históricos disponíveis, aqueles que se adequam melhor aos objetivos da pesquisa. Quer dizer, o que usar? Como usar essa parte histórica de forma pedagógica? Como relacionar a história com as atividades de investigação em sala de aula? [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! Não é algo trivial de ser feito, nem pode ser feito de qualquer jeito, é preciso estudar muito. Então, em cada livro, em cada autor, eu tinha que ir procurando esses elementos históricos que trouxessem a problemática, os impasses, que pudessem ser contemplados em sala de aula. É preciso adequar toda a parte histórica com a didática, que no caso era embasada em Kemp, e cuidar para que fosse possível de ser resolvida neste nível de ensino. Se ela for fácil demais, não colabora com a aprendizagem requerida em tal nível, se for complexa demais, fica inacessível para o aluno. Quadro 5 - Discursos que remetem à categoria “Conhecimento relativo às adequações pedagógicas” Fonte: Araman (2011). ntão, em cada livro, em cada autor, eu tinha que ir procurando esses elementos histórico ue trouxessem a problemática, os impasses, que pudessem ser contemplados em sala d ula. Quadro 5 - Discursos que remetem à categoria “Conhecimento relativo às adequações pedagógicas” Fonte: Araman (2011). P1 apresenta, em sua fala, um ponto importante de ser observado, que também foi evidenciado por outros professores. Os dados, as informações provenientes da pesquisa teórica não podem simplesmente ser levadas para sala de aula. Segundo ele, são necessárias adequações coerentes dos estudos teóricos para que os mesmos possam ser usados em sala de aula. Esse ponto também é levantado por P2, que considera não ser suficiente um texto histórico, são necessárias adequações para torná-lo passível de ser usado em sala de aula. Chama a atenção para o fato de que precisou “amarrar” as informações históricas da trigonometria com atividades didáticas, que fossem dinâmicas para não se tornarem cansativas aos alunos. Para P3, que dispunha de muitas informações históricas, o desafio foi, dentre tantas opções, como proceder a seleção de forma que atendesse aos objetivos de sua pesquisa, que era a aprendizagem do conceito de funções. Evidencia, assim como os demais, que usar a história da Matemática em sala de aula vai muito além do que buscar informações históricas. Como a perspectiva de P4 era desenvolver atividades de investigação apoiadas no desenvolvimento histórico, considerou importante selecionar, diante de muitos materiais históricos, aqueles mais adequados para relacionar com as atividades de investigação, que apresentam características mais práticas. P5 utiliza o termo “transposição didática” ao referir-se à necessidade de adequar os textos históricos formalizados, acadêmicos, para serem usados nas salas de aula. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! Então não é só pegar o que os livros de história da matemática trazem, tem que selecionar os trechos relevantes e, depois, adequá-los à proposta que está sendo desenvolvida, e isso não é fácil. Então eu selecionei recortes históricos que tinham algo prático, que pudesse ser “feito” pelo aluno, como o método do jardineiro e de Kepler, que eram usados no século VI. P5 Como é que eu pego uma história que está formalizada nos textos acadêmicos e construo um texto histórico para trabalhar com os alunos de forma mais didática. Isso se eu pensar em trabalhar com algum texto. Já começa um problema aí, porque eu preciso saber fazer uma transposição didática desse texto, do acadêmico para um texto para o aluno. Categoria “Conhecimento relativo às adequações pedagógicas” [...] a maior dificuldade é selecionar, entre todos esses materiais históricos disponíveis aqueles que se adequam melhor aos objetivos da pesquisa. Quer dizer, o que usar? Como usar essa parte histórica de forma pedagógica? Como relacionar a história com as atividades de investigação em sala de aula? Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 401 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 P6 [...] foi difícil articular tudo isso, a parte histórica com a organização das atividades. Você tem que aproveitar a história, mas também tem que levar em consideração outros aspectos pedagógicos para ter aprendizagem. Tivemos que fazer várias adequações na sequência de atividades. Não é algo trivial de ser feito, nem pode ser feito de qualquer jeito, é preciso estudar muito. Então, em cada livro, em cada autor, eu tinha que ir procurando esses elementos históricos que trouxessem a problemática, os impasses, que pudessem ser contemplados em sala de aula. É preciso adequar toda a parte histórica com a didática, que no caso era embasada em Kemp, e cuidar para que fosse possível de ser resolvida neste nível de ensino. Se ela for fácil demais, não colabora com a aprendizagem requerida em tal nível, se for complexa demais, fica inacessível para o aluno. Quadro 5 - Discursos que remetem à categoria “Conhecimento relativo às adequações pedagógicas” Fonte: Araman (2011). P6 [...] foi difícil articular tudo isso, a parte histórica com a organização das atividades. Você tem que aproveitar a história, mas também tem que levar em consideração outros aspectos pedagógicos para ter aprendizagem. Tivemos que fazer várias adequações na sequência de atividades. [...] a pesquisa me ajudou muito na compreensão desse conteúdo (números complexos)! Também aponta como uma tarefa difícil selecionar pontos que sejam relevantes e provoquem discussões profícuas com os alunos. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 402 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 Na opinião de P6, fazer todas as adequações necessárias para utilizar a história da Matemática em sala de aula não é algo trivial, exige muito estudo por parte do professor. Além disso, coloca que ainda foi necessário articular essas escolhas em relação à parte histórica com o embasamento didático que escolheu, respeitando o nível de desenvolvimento de seus alunos. A construção de uma abordagem apoiada na história da Matemática evidenciou um caráter interdisciplinar, que integra e relaciona entre si conhecimentos conceituais, teóricos e metodológicos de diversas áreas. Fica evidente, por meio da análise realizada, que os professores tiveram que articular elementos relativos à área da pesquisa em história da Matemática com os relacionados aos aportes didáticos. As opções vão desde a seleção, nos estudos históricos, dos elementos relevantes e adequados para cada conteúdo, cada nível de ensino. Além disso, esses elementos devem ser adaptados, didaticamente, para atender ao objetivo de proporcionar aprendizagem. Alguns fazem a opção pela resolução de problemas, outros por atividades práticas e dinâmicas, enfim, em cada opção, em cada escolha do professor, as adequações foram necessárias. São muitos conhecimentos e saberes que os professores articulam nesse processo rico e complexo, e que, certamente, colaboram para a sua formação profissional, numa perspectiva holística, pois abrangem reflexões e tomadas de decisão sob vários aspectos. Como os próprios sujeitos evidenciaram, o processo de construção de uma abordagem pautada na história da Matemática para ser aplicada em sala de aula não é um processo trivial. Ele se dá pela interface entre vários elementos e só é possível por meio de estudos teóricos e metodológicos sistematizados, como os vivenciados pelos sujeitos de nossa investigação. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 6 O processo de construção de abordagens históricas e o desenvolvimento de saberes interdisciplinares do professor de Matemática: algumas considerações No decorrer desta investigação, procuramos bases teóricas que discutissem a importância de estudos a respeito dos saberes docentes. Os autores que estudamos defendem que o professor, no exercício de sua profissão, elabora e reelabora diversos saberes na tentativa de lidar com as situações complexas de uma sala de aula. Recorremos, ainda, a muitos estudos que evidenciam a relevância que a história da Matemática tem na formação do professor de Matemática. Muitas pesquisas salientam a importância de tais conhecimentos para essa formação, em seus vários aspectos, como já 403 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 ISSN 1980-4415 colocamos ao longo do texto. Entretanto, não encontramos pesquisas que procuram evidenciar como ocorre o processo de saberes do professor que desenvolve e aplica uma proposta histórica na Educação Matemática. É neste ponto que nossa investigação colabora com as pesquisas na área, estudando, de forma sistematizada, como se dá essa relação. Em nosso entendimento, a vivência de realizar uma pesquisa em história da Matemática na qual o objetivo seja o de compor uma abordagem pedagógica aplicável em sala de aula proporciona ao docente uma formação que envolve muitos elementos, como conceituais, metodológicos e experienciais. Isso ocorre pelo fato de que, ao se propor a construir uma abordagem histórica, o professor, além de entrar em contato com a história do conteúdo matemático pesquisado, com os referenciais a respeito das potencialidades do uso pedagógico da história da Matemática, é envolvido num processo que demanda, a todo o tempo, escolhas, reflexões, articulações entre esses elementos e os aspectos pedagógicos. É um processo criativo, que contribui para a construção de vários saberes, da autonomia do professor e da sua identidade profissional, uma vez que supera a perspectiva da racionalidade técnica, como descrita por Schön (1995), situando o professor como sujeito produtor de saberes e conhecimentos. A literatura assinala como necessária a identificação dos saberes próprios do ensino, considerados em contextos reais, complexos, em que as variáveis não podem ser controladas. Gauthier et al. (1998) propõem que os saberes docentes devem ser estudados, explicitados e legitimados pelas pesquisas, mas que tenham como base as situações reais de ensino. E este é o caso da nossa investigação, uma vez que procuramos explicitar saberes e conhecimentos produzidos pelos professores ao entrar em contato com a história da Matemática e ao construir uma abordagem pedagógica para usá-la em sala de aula. Bolema, Rio Claro (SP), v. 31, n. 57, p. 380 - 407, abr. 2017 6 O processo de construção de abordagens históricas e o desenvolvimento de saberes interdisciplinares do professor de Matemática: algumas considerações Os professores construíram e reconstruíram muitos saberes, que foram explicitados e estudados de forma sistematizada na análise dos dados de nossa pesquisa. De acordo com a análise realizada, as categorias evidenciaram que os professores desenvolveram saberes relacionados ao entendimento dos conteúdos matemáticos, às potencialidades pedagógicas do uso da história da Matemática em sala de aula, à compreensão da natureza do conhecimento matemático e à necessidade de adequações pedagógicas para a aplicação da história da Matemática em sala de aula. Exige também o conhecimento prático do professor, pois é a partir da relação entre o conhecimento prático e as diversas opções teóricas e metodológicas que ele fará suas escolhas. A figura 1 que apresentamos no início deste artigo evidencia o caráter interdisciplinar que envolve o processo de construção de uma abordagem histórica para o ensino de 404 ISSN 1980-4415 DOI: http://dx.doi.org/10.1590/1980-4415v31n57a19 Matemática. Esse processo demanda muitos conhecimentos e, principalmente, a articulação e integração entre eles. Esse processo articulador e integrador que compreendemos ser relevante para a formação interdisciplinar do professor e se constitui no saber interdisciplinar (BATISTA, 2004). O conhecimento escolar apresenta características interdisciplinares, uma vez que tal conhecimento não é uma mera simplificação do conhecimento científico disciplinar. Ao contrário, ele passa por diversas adequações que envolvem desde a elaboração do currículo até a prática pedagógica em sala de aula. Essas características interdisciplinares são similares às envolvidas no processo de construção de abordagens históricas, como já evidenciamos no texto. Sendo assim, compreendemos que o viés interdisciplinar presente na construção de abordagens pedagógicas fundamentadas na história da Matemática enriquece a base de conhecimentos do professor. 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https://openalex.org/W4287378728	https://ela.kpi.ua/bitstream/123456789/53014/1/VPSP2022-1_47-52.pdf	Ukrainian	null	FUNCTIONS OF THE INTELLECTUAL ELITE IN THE POLITICAL LIFE OF SOCIETY	Vìsnik Nacìonalʹnogo tehnìčnogo unìversitetu Ukraïni «Kiïvsʹkij polìtehnìčnij ìnstitut». Polìtologìâ, socìologìâ, pravo	2,022	cc-by	4,549	ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Білозерова Я. В., аспірант кафедри політології Одеського національного університету імені І. І. Мечникова ORCID ID: 0000-0002-3922-0671 ya.vita56@gmail.com У статті проведено аналіз функцій та ролі інтелектуальної еліти в політичному житті. Встановлено, що в умовах демократичного суспільства інтелектуальна еліта відіграє значну роль, впливаючи на всі сфери суспільного життя, в тому числі і на політичне життя, зокрема на формування політичної еліти, політичну свідомість та політичну культуру членів суспільства, визначення пріоритетів його розвитку та прогнозування суспільно-політичних трансформацій. Запропоновано поділ функцій інтелектуальної еліти на чотири блоки. До основних блоків функцій інтелектуальної еліти в політичному житті віднесені: інформування громадськості та пояснення політичних процесів та явищ, формування політичної свідомості та політичної культури, цілепокладання у розвитку суспільства в цілому та політичної системи зокрема, інтеграція суспільства та раціоналізація суспільно-політичного порядку. Визначено, що до першого блоку належать пізнавальна, консультаційна, контролююча функція, а також критична рефлексія життєво важливих сфер суспільної системи. З’ясовано, що другий блок завдань інтелектуальної еліти представлений аксіологічною функцією та функцією збагачення та розвитку інтелектуальної культури (як ресурсу для політичного розвитку суспільства). Встановлено, що третій функціональний блок складають стратегічна, управлінська та інноваційна функції. Показано, що четвертий блок представлений комунікативною, інтегративною, стабілізаційною, прогностичною функціями, а також функцією структурування нової ієрархії і нерівності та функцією раціоналізації існуючого порядку. Обґрунтовано, що основи специфічних функцій інтелектуальної еліти в Україні пов’язані із сучасною політичною ситуацією. Зокрема, визначено, що такі функції, як збереження духовних цінностей, трансляція знань та ідей, вміння служити суспільству, його інформування, допомога в оцінці подій, що відбуваються практично повсюдно, проектуються на інтелектуальну еліту. Визначено, що завдяки їх виконанню відбувається консолідація нації, що є запорукою гармонізації політичного життя. Ключові слова: еліта, інтелектуальна еліта, інтелектуалізм, функції інтелектуальної еліти, політичне життя. Постановка проблеми. Роль інтелектуальної еліти в суспільстві пов’язана з виробництвом та поширенням знань та ідей, формуванням інтелектуального потенціалу суспільства, інформуванням його про події та допомогою в їх оцінці. Також слід відзначити особливості інтелектуальної еліти. По суті, вона позбавлена влади і не має ніяких реальних ресурсів її здійснення. На відміну від бізне- селіти, вона позбавлена грошей, не має знарядь примусу, як правляча політична еліта та бюрократія. Але її вплив ґрунтується на інформації, ідеях та оцінках, які вона продукує та поширює. Інтелекту- альна еліта живить своїми продуктами і владу, і бізнес, і саме суспільство, формуючи його настрої та свідомість, ідеологію, соціальні інститути та цінності. Тому, враховуючи особливу роль інтелектуаль- ної еліти, слід звернути увагу на можливість створення альянсу еліт, який би включав політичну, еко- номічну та інтелектуальну еліти. УДК 323.2:323.39(477) DOI https://doi.org/10.20535/2308-5053.2022.1(53).261108 УДК 323.2:323.39(477) DOI https://doi.org/10.20535/2308-5053.2022.1(53).261108 УДК 323.2:323.39(477) DOI https://doi.org/10.20535/2308-5053.2022.1(53).261108 © Білозерова Я. В. Стаття поширюється на умовах ліцензії CC BY 4.0 ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Також важливо звертати увагу на те, чи сприймається та чи інша людина саме як інтелектуал, як авторитет саме у розумовому аспекті. Варто відзначити, що важливим фактором, який робить людину представником інтелектуальної еліти, є її причетність до вищої освіти відповідно до історичних вимог, поза якими інтелектуал прак- тично не міг сформуватися. Згідно із трактуванням Енциклопедії сучасної України, інтелектуалізм (від лат. intellectualis – розумовий) – це «напрям у філософії та психології, який розглядає пізнання насамперед через інте- лект, абсолютизує мислення, відмежовуючи їх від емпірики; пріоритет розуму над сферою чуттєвості; насиченість думками та ідеями, концептуальна змістовність, вияв розумового й духовного начал» (Мойсеїв, 2011). Слід зазначити, що основи інтелектуалізму, а також з’ясування сутності інтелектуальної еліти можна побачити в античній філософії. Зокрема, Сократ, Платон та Аристотель, визначаючи структуру суспільства та систему управління ним, говорили про еліту аристократії, вважаючи що управляння суспільством мають здійснювати філософи-мудреці. Звідси з’явився термін «аристократія духу», запропонований Платоном (Платон, 2000). Отже, автори доби найбільш продуктивним вважали саме інтелект, за допомогою якого можливо організувати ефективне управління суспільством та державою. Новим етапом розвитку уявлень про інтелектуалізм можна вважати період Нового часу з поя- вою ідеї епохи Просвітництва про необмежені можливості людського розуму та його високий сус- пільний авторитет. Таким чином, інтелектуалізм як явище виникає в добу Просвітництва в Європі. Саме завдяки інтелекту відбувається розвиток філософії, науки та техніки, поява нових технологій. На думку І. Мойсеїва, «інтелектуалізм, втілений в інформаційних технологіях та комп’ютерних мере- жах, реалізується у глобальному штучному інтелекті, який фактично керує техносферою і визначає режим життєдіяльності сучасного суспільства» (Мойсеїв, 2011). Проте інтелектуалізм має бути врів- новажений моральними та духовними принципами для того, щоб приносити користь суспільству та не шкодити йому. Інтелектуальному життю суспільства притаманна як інтелектуальна традиція (форма накопи- чення і передачі знань), так і інтенсивна інтелектуальна творчість. Представники інтелектуальної еліти крім освіти, ерудиції та начитаності, повинні мати також здатність до творчості, вміння вико- ристовувати свої знання для вироблення оригінального та нового у сфері розумової діяльності. Для справжнього інтелектуала розумова праця та інтелектуальна творчість є засобом життя, екзистенцій- ним потягом до пошуку істини. Так, А. Тойнбі у своїх роботах дає найвищу оцінку інтелектуальній еліті. В рамках своєї теорії «виклику та відповіді» дослідник зазначає, що відповіді на виклики оточуючого середовища дає творча меншість – «творці-одинаки» та «соціальні групи творців» (Toynbee, 1988), тобто інтелектуали. Автор вважає, що «інтелектуальна еліта – хоча і є невеликою за розміром, але найціннішою». Основа його концепції в тому, що «єдиним справжнім творцем всіх досягнень є інтелект. ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Цей альянс (влада – бізнес – інтелект) надав би реальну допомогу у вирішенні проблем, пов’язаних із територіальним та державним розвитком. Варто відзначити, що до проблеми елітарності суспільства часто звертаються науковці, аналі- зуючи національну еліту України (наприклад, З. Атаманюк, К. Баранцева, Ю. Войтенко, О. Коваль). Питання ролі політичної еліти також ґрунтовно проаналізовано вітчизняними вченими. Так, теоре- тичні аспекти розвитку ідеї політичної еліти досліджені Н. Дубовик, Т. Костюченко, Л. Мельник. Історія 47 розвитку політико-управлінської еліти України була розкрита Л. Харченко, Л. Козловською, Л. Ман- дзій, С. Щербиною. Питання легітимації політичної еліти та її роль в державотворенні стали пред- метом досліджень В. Горобця, Л. Дащаківської, Ю. Конотопцевої, Є. Остренко. Особливості сучас- ної політико-управлінської еліти визначені в роботах О. Крюкова, О. Науменко, О. Постоловської, Л. Хорішко та інших. розвитку політико-управлінської еліти України була розкрита Л. Харченко, Л. Козловською, Л. Ман- дзій, С. Щербиною. Питання легітимації політичної еліти та її роль в державотворенні стали пред- метом досліджень В. Горобця, Л. Дащаківської, Ю. Конотопцевої, Є. Остренко. Особливості сучас- ної політико-управлінської еліти визначені в роботах О. Крюкова, О. Науменко, О. Постоловської, Л. Хорішко та інших. розвитку політико-управлінської еліти України була розкрита Л. Харченко, Л. Козловською, Л. Ман- дзій, С. Щербиною. Питання легітимації політичної еліти та її роль в державотворенні стали пред- метом досліджень В. Горобця, Л. Дащаківської, Ю. Конотопцевої, Є. Остренко. Особливості сучас- ної політико-управлінської еліти визначені в роботах О. Крюкова, О. Науменко, О. Постоловської, Л. Хорішко та інших. р Однак інтелектуальна еліта не стала предметом політологічних досліджень, залишаючись в полі зору переважно соціологів, істориків та філософів, серед яких варто відзначити роботи К. Діденко, В. Кононенко, К. Михайльової, В. Омельченко, О. Фоміна. Отже, актуальним є вивчення функцій та ролі інтелектуальної еліти в політичному житті суспільства. Мета статті – визначити функції інтелектуальної еліти, які вона виконує у політичному житті сучасного суспільства. Виклад основного матеріалу. Досліджуючи інтелектуальну еліту з позиції політології, слід визначити та окреслити критерії приналежності до неї. У даному випадку важливим є розуміння сут- ності інтелектуалізму, а також саме поняття еліти. Поняття «еліта» є соціальним феноменом та включає в себе провідну роль відповідної соці- альної групи у функціонуванні та розвитку суспільства. Тому приналежність до інтелектуальної еліти визначається участю індивіда у прокладанні ключових векторів культурно-інтелектуального розвитку суспільства, у створенні чи оформленні нових етико-поведінкових моделей, наукових поглядів, філософських систем тощо. Цей критерій допомагає відрізнити представників інтелекту- альної еліти від інтелектуалів-одинаків, які не внесли ніякого істотного вкладу у створення духов- но-інтелектуального вигляду своєї або наступної епохи. ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Інтелект – найбільше багатство країни» (Тойнбі, 1995). Тому слід враховувати принципи збереження та поповнення інтелектуальної еліти у суспільстві. Навіть за варваризації суспільство насамперед зберігає свою еліту, якщо саме не розпадається. Звідси випливає, що формування еліт є консервативною тенденцією суспільства. ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 48 А. Тойнбі вважає, що в ідеалі інтелектуали мають увійти до політичної еліти, саме так вони забезпечать стабільний розвиток держави та ефективну взаємодію влади та суспільства. Водночас інші дослідники розглядають інтелектуалів як проміжну ланку між суспільством та політичною елі- тою. На мою думку, така точка зору є вірною. Саме незалежне становище дає інтелектуальній еліті серйозний інструментарій для взаємодії з політичною елітою та соціальними інститутами. Влада на Заході із середини ХХ століття активно залучає інтелектуалів у політичний процес через їхній сут- тєвий вплив на суспільство та здатність змінити ситуацію. Таке залучення носить форму дорадчих інститутів при органах влади, залучення незалежних експертів та експертних організацій, провідне місце серед яких мають так звані «фабрики думок». р ф р у У контексті вищенаведеного варто віднести до рис інтелектуальної еліти не лише інтелектуалізм та високий рівень освіти, а й високі морально-етичні якості. Стратегічним завданням інтелектуальної еліти є генерування цінностей та норм, вироблення інноваційних технології поширення та збереження знань, традицій та етичних норм. Основу такої еліти складають інтелектуали та інтелігенція. На думку А. Грамші, кожна соціальна група створює прошарки інтелігенції, які «надають цій групі однорідність та розуміння її власної специфічної ролі» в економіці, соціальній структурі та політиці (Gramsci, 1991). Провідним суб’єктом організації інтелектуального життя в демократичному індустріальному та постіндустріальному суспільстві є інтелектуал. За влучним висловом вітчизняного дослідника Я. Пасько, інтелектуали є «новим класом», символом постіндустріальної реальності, рушійною силою мережевого суспільства, однією з підвалин цивілізаційного розвитку кінця ХХ і початку ХХІ століття (Пасько, 2014: 28). Інтелектуала визначають як особу, що володіє певними соціально-професійними якостями, в той час як інтелігент розглядається як ціннісне соціокультурне поняття. Так, П. Голо- буцький проводить розмежування даних понять: «Інтелектуали – це люди з видатними природними розумовими здібностями, які здатні генерувати нові знання; інтелігенти – це люди, які є виразниками народної совісті та моралі, які формують соціальні й історичні цілі народу, а також зберігають його духовні цінності» (Голобуцький, 2005: 508). Я. Пасько до ключових характеристик інтелігенції відно- сить професійність та освітні ознаки (Пасько, 2014: 36). К. ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА До функцій блоку 49 також можна віднести контролюючу функцію, під якою слід розуміти контроль над ментальною сферою суспільства, розумом та почуттями людей щодо доступу до інформації, інтелектуальних відкриттів та досягнень. З одного боку, інтелектуальна еліта не може допустити інтелектуального та культурного декадансу країни, а з іншого – вивільнити вибуховий зміст культури, а також усіх найновіших досягнень науки на загальний огляд, що неминуче призведе до кардинальних культурних трансформацій з непе- редбачуваними результатами. Тобто дана функція передбачає певне регулювання обсягів інформації з метою надання широкій громадськості того її об’єму який остання зможе сприйняти також можна віднести контролюючу функцію, під якою слід розуміти контроль над ментальною сферою суспільства, розумом та почуттями людей щодо доступу до інформації, інтелектуальних відкриттів та досягнень. З одного боку, інтелектуальна еліта не може допустити інтелектуального та культурного декадансу країни, а з іншого – вивільнити вибуховий зміст культури, а також усіх найновіших досягнень науки на загальний огляд, що неминуче призведе до кардинальних культурних трансформацій з непе- редбачуваними результатами. Тобто дана функція передбачає певне регулювання обсягів інформації з метою надання широкій громадськості того її об’єму, який остання зможе сприйняти. р д у р у д фу ц р д р у з метою надання широкій громадськості того її об’єму, який остання зможе сприйняти Формування політичної свідомості та політичної культури складають зміст другого блоку завдань інтелектуальної еліти. Провідною функцією тут є аксіологічна (ціннісно-нормативна), яка передбачає вироблення засобів захисту культурного ядра соціуму від негативних потрясінь, а також продукування стійкої шкали цінностей відповідно до ідей перманентного розвитку та суспільного про- гресу, чим забезпечується ціннісно-нормативна динамічна стійкість адекватно викликам сучасності. Серед інших важливим є формування політичних цінностей та ідей, які в подальшому покладаються в основу суспільно-політичного розвитку на сформованої системи управління суспільством. Ще одна функція в рамках блоку – збагачення та розвиток інтелектуальної культури як одного з ключових її суб’єктів у протиставленні культурі масовій. Дана функція сприяє зростанню рівня загальної та політичної культури громадян, а в результаті – розвитку інтелектуального капіталу. Третій блок завдань можна представити у вигляді цілепокладання у розвитку суспільства в цілому та політичної системи зокрема. Так, інтелектуальна еліта формує стратегічні вектори роз- витку суспільства, координує процес управління культурною і опосередковано іншими сферами. Перша функція блоку – стратегічна, тобто формування стратегії та тактики розвитку суспільства у вигляді вироблення ідеології, концепцій, доктрин, програм, зразків поведінки. Інтелектуальна еліта формує стратегічний вектор соціокультурного розвитку, визначає національні цілі та ідеали, задля досягнення яких можлива мобілізація ресурсів усього суспільства. ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Михайльова дає таке визначення інтелектуальної еліти: «Група, що функціонує у сфері розумової праці, володіє значним інтелектуальним капіталом, відрізняється вищим порівняно із загальною масою населення в тому чи іншому суспільстві рівнем освіти, що поєднується з критич- ним мисленням, творчими здібностями і високим рівнем мотивації своєї інтелектуальної діяльності; здатна до активної соціально-інноваційної діяльності, втіленої в конкретних результатах, заснованих на високому рівні професійної компетентності і таких, що мають особливе значення для розвитку суспільства» (Михайльова, 2008: 15). На мою думку, інтелектуальна еліта, як головний виробник інте- лектуальних знань, включає наукову еліту, творчу еліту, інтелектуалів, що пояснюють соціальні, еко- номічні, політичні та культурні процеси та явища (експертів, журналістів та ін.); діячів системи освіти (Білозерова, 2021: 7–8). Дж. Бренхен (Burnham, 1941) та Г. Лассуел (Lasswell, 1952) відзначають, що еліта здатна виконати таке завдання в суспільстві, як актуалізація вищих цінностей, що гарантують суспільний добробут, та підготовка психіки мас до прийняття нової соціальної структури. Л. Гудков та Б. Дубін визначають три провідні функції інтелектуальної еліти: прагнення іннова- ційної діяльності; критика та селекція соціокультурних та соціально-політичних досягнень суспільства; зберігання та ретрансляція цінностей та знань, які складають інтелектуальний ресурс суспільства (Гудков, Дубінін, 1995: 113). Проте в умовах демократичного розвитку суспільства на неї поклада- ється значно більше функцій. На мою думку, інтелектуальна еліта відіграє важливу роль в суспільстві в цілому, а також виконує низку функцій, що пов’язані з політичним життям суспільства. Зокрема, фактична реалізація її інтелек- туального капіталу відбувається через виконання кількох блоків завдань, в рамках яких реалізуються її функції. Зокрема, до першого блоку можна віднести інформування громадськості та пояснення полі- тичних процесів та явищ. З реалізацією цього напряму пов’язано виконання таких функцій, як пізна- вальна (гносеологічна), що полягає в перманентному дослідженні соціокультурної дійсності у найбільш значущих сферах щодо їх вдосконалення і поліпшення, з чого виявляються закономірності, розши- рюється діапазон знання про суспільство, його культуру та політику. Наступна функція блоку – кри- тична рефлексія життєво важливих сфер суспільної системи, що зачіпають широкі верстви населення (в першу чергу, політичної). Вона передбачає те, що критичний дискурс інтелектуальної еліти є рефлек- сивним процесом перманентної корекції соціокультурного розвитку відповідно до побудованого аргу- ментованого ідеалу та зразка в умовах політичних трансформацій. До даного блоку примикає також консультаційна (або експертна) функція, що включає дієві експертні оцінки подій, що відбуваються, при вирішенні проблемних ситуацій, а також при прийнятті рішень у значущих сферах суспільного життя, внаслідок чого інтелектуальна еліта опосередковано впливає на прийняття таких рішень. Спо- живачем продукту від виконання такої функції виступає політико-управлінська еліта. ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА Саме навколо таких цілей та іде- алів відбувається побудова векторів соціально-політичного розвитку політичною елітою. Друга функ- ція – управлінська, що суттєво відрізняється від аналогічної функції політичної еліти та передбачає особливе культурне та інтелектуальне керівництво суспільством, що визначає логіку історичного роз- витку. Авторитет членів інтелектуальної еліти дозволяє розробляти і впроваджувати в масову свідо- мість моделі поведінки та осмислення соціальної дійсності або ж генерувати в політичну еліту канали впливу на масову свідомість і поведінку людей, визначаючи, зрештою, культурно-історичну динаміку всього суспільства. Остання функція блоку – інноваційна, в основі якої лежить створення принципово нових інтелектуальних продуктів, ідей та інноваційних технологій, що сприяють ефективному роз- витку всього суспільства та його успішному позиціонуванню на світовій арені (в першу чергу, це дося- гається завдяки розробкам в галузі політичного та публічного управління, іміджевої політики тощо). До четвертого блоку завдань інтелектуальної еліти можна віднести інтеграцію та раціоналізацію суспільно-політичного життя. До функцій блоку слід віднести комунікативну, тобто переклад та інте- грацію різних культурних кодів у єдину комунікативну площину для ефективних загальносоціальних, професійних, міжкультурних та політичних комунікацій як усередині країни, так і за її межами з метою збагачення та розвитку суспільства. Сюди також належить інтегративна функція, що передбачає досягнення загального об’єднавчого консенсусу різних соціальних сил щодо культурних, моральних, політичних та інших проблем, що породжуються викликами часу, шляхом подолання міжгрупових протиріч та розбіжностей з-поміж них з урахуванням сформульованих елітою переконань, поглядів, цінностей і ідеалів. Проте слід зазначити, що одночасно інтелектуальна еліта виконує і таку функцію, як структурування нової ієрархії та нерівності, що ґрунтується на положеннях природного інтелекту- ального елітаризму в культурі і пропонує стратифікаційний поділ суспільств на підставі нематеріаліс- тичних критеріїв, інтелектуальної переваги та багатого культурного капіталу, внаслідок чого відбува- ється природне відокремлення інтелектуальної еліти. Наступна функція четвертого блоку – стабілізаційна, тобто забезпечення балансу між культур- ною спадщиною минулого та інноваційними відкриттями, між традиціями та інноваціями з метою їх гармонізації та впорядкування у єдиний дієвий вектор інтелектуального розвитку. Крім того, до даного блоку варто віднести прогностичну функцію, яка передбачає вироблення наукових прогнозів розвитку у ключових сферах (і перш за все у сфері політичного розвитку та публічного управління), визначення тенденцій у розвитку у середньостроковій і довгостроковій перспективі, у найбільш кризових і невизна- чених ситуаціях. Раціоналізація наявного порядку – це ще одна функція блоку, її мета – знизити рівень нестабільності та невизначеності в нових умовах, створити наново передбачуваний світ шляхом інте- лектуалізації та раціоналізації все більш ускладненого по мірі свого розвитку інституційного порядку. ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 ФУНКЦІЇ ІНТЕЛЕКТУАЛЬНОЇ ЕЛІТИ В ПОЛІТИЧНОМУ ЖИТТІ СУСПІЛЬСТВА З урахуванням досвіду минулого та враховуючи функціональне призначення інтелектуальної еліти в політичному життя суспільства, сьогодні стає особливо важливим не допустити елітоциду, тобто відбраковування людей вольових, самостійних, талановитих, сумлінних, які мають високий 50 інтелектуальний рівень. Інтелектуальна еліта повинна регулярно поповнюватися найкращими пред- ставниками суспільства, їй потрібне оновлення, попередження накопичення баласту. А для цього має справно працювати механізм соціального ліфту, головна функція якого – підвищення якості інтелекту- альної еліти, введення до неї найкращих громадян, найобдарованіших, наділених суспільним темпе- раментом. Якщо ця функція виконується погано, еліта і суспільство рано чи пізно почне деградувати. інтелектуальний рівень. Інтелектуальна еліта повинна регулярно поповнюватися найкращими пред- ставниками суспільства, їй потрібне оновлення, попередження накопичення баласту. А для цього має справно працювати механізм соціального ліфту, головна функція якого – підвищення якості інтелекту- альної еліти, введення до неї найкращих громадян, найобдарованіших, наділених суспільним темпе- раментом. Якщо ця функція виконується погано, еліта і суспільство рано чи пізно почне деградувати. Висновки. В умовах демократичного суспільства інтелектуальна еліта відіграє значну роль у житті суспільства, впливаючи на сферу освіти, науки, творчості, політичне життя, зокрема на фор- мування політичної еліти, політичну свідомість та політичну культуру членів суспільства, визначення пріоритетів його розвитку та прогнозування суспільно-політичних трансформацій. До основних блоків функцій інтелектуальної еліти в політичному життя необхідно віднести: інформування громадськості та пояснення політичних процесів та явищ (пізнавальна, консультаційна, контролююча функція, а також критична рефлексія життєво важливих сфер суспільної системи), формування політичної сві- домості та політичної культури (аксіологічна функція та збагачення та розвиток інтелектуальної куль- тури), цілепокладання в розвитку суспільства в цілому та політичній системі зокрема (стратегічна, управлінська, інноваційна функції), інтеграцію суспільства та раціоналізацію суспільно-політичного порядку (комунікативна, інтегративна, стабілізаційна, прогностична функція, структурування нової ієрархії та нерівності та раціоналізація наявного порядку). Через війну, яка відбувається в Україні, інтелектуальна еліта наділяється частиною специфіч- них функцій, притаманних українському соціуму, його культурних і ментальних характеристик. Напри- клад, такі функції, як збереження духовних цінностей, трансляція знань та ідей, вміння служити сус- пільству, його інформування, допомога в оцінці подій, що відбуваються, їх «правильного» прочитання та багато інших – практично повсюдно проектуються на інтелектуальну еліту. Саме завдяки їх вико- нанню відбувається консолідація нації, що є запорукою гармонізації соціально-політичних процесів та політичного життя в цілому. Тому доцільним є подальше дослідження функцій інтелектуальної еліти в сучасному політичному процесі України. Література: 1. Білозерова Я. В. Інтелектуальна еліта в культурній та науковій дипломатії. European Political and Law Discourse. 2021. Volume 8. Issue 6. Р. 6–13. 2 Г б й П В І і і і Е і і ії У ї Т 3 Е Й / Р р ур 1. Білозерова Я. В. Інтелектуальна еліта в культурній та науковій дипломатії. European Political and Law Discourse. 2021. Volume 8. Issue 6. Р. 6–13. 2. Голобуцький П. В. Інтелігенція та інтелектуали. Енциклопедія історії України: Т. 3: Е–Й / Редкол.: В А Смолій (голова) та ін ; НАН України Інститут історії України Київ : В во «Наукова думка» р ур 1. Білозерова Я. В. Інтелектуальна еліта в культурній та науковій дипломатії. European Political and Law Discourse. 2021. Volume 8. Issue 6. Р. 6–13. 2. Голобуцький П. В. Інтелігенція та інтелектуали. Енциклопедія історії України: Т. 3: Е–Й / Редкол.: В. А. Смолій (голова) та ін.; НАН України. Інститут історії України. Київ : В-во «Наукова думка», 2005. С. 507–511. URL: http://history.org.ua/LiberUA/ehu/3.pdf ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 2. Голобуцький П. В. Інтелігенція та інтелектуали. Енциклопедія історії України: Т. 3: Е–Й / Редкол.: В. А. Смолій (голова) та ін.; НАН України. Інститут історії України. Київ : В-во «Наукова думка», 2005. С. 507–511. URL: http://history.org.ua/LiberUA/ehu/3.pdf Bilozerova Ya. Functions of the intellectual elite in the political life of society p y The article analyzes the functions and role of the intellectual elite in political life. It is established that in a democratic society the intellectual elite plays a significant role, influencing all spheres of public life, including political life, including the formation of political elite, political consciousness and political culture of society, prioritizing its development and forecasting socio-political transformations. The division of the functions of the intellectual elite into four blocks is proposed. The main blocks of the functions of the intellectual elite in political life include: informing the public and explaining political processes and phenomena, forming political consciousness and political culture, goal setting in the development of society in general and the political system in particular, integration of society and rationalization of socio-political order. It is determined that the first block includes cognitive, consultative, controlling function, as well as critical reflection on vital areas of the social system. It was found that the second block of tasks of the intellectual elite is represented by the axiological function and the function of enrichment and development of intellectual culture (as a resource for the political development of society). It is established that the third functional unit consists of strategic, managerial and innovation functions. It is shown that the fourth block is represented by communicative, integrative, stabilizing, prognostic functions, as well as the function of structuring a new hierarchy and inequality and the function of rationalizing the existing order. It is substantiated that the main specific functions of the intellectual elite in Ukraine are related to the current political situation. In particular, it is determined that such functions as the preservation of spiritual values, transmission of knowledge and ideas, the ability to serve society, informing it, assistance in assessing events that are happening almost everywhere are projected on the intellectual elite. It is determined that due to their implementation the nation is consolidating, which is the key to harmonization of political life. Key words: elite intellectual elite intellectualism functions of intellectual elite political life p g y p Key words: elite, intellectual elite, intellectualism, functions of intellectual elite, political l 51 3. Гудков Л. Д., Дубин Б. В. Интеллигенция: Заметки о литературно-политических иллюзиях. Харь- ков : Эпицентр, Фолио, 1995. 187 с. 4. Михайльова К. Г. Інтелектуальна еліта сучасного суспільства: особливості формування та діяль- нісний потенціал : автореф. дис. ... д-ра соціал. наук: 22.00.01; Харк. нац. ун-т ім. В. Н. Каразіна. Bilozerova Ya. Functions of the intellectual elite in the political life of society Харків, 2008. 32 с. р , 5. Мойсеїв І. К. Інтелектуалізм. Енциклопедія Сучасної України. 2011. URL: https://esu.com.ua/ search_articles.php?id=12389 6. Пасько Я. Пострадянська інтеліґенція: ціннісна марґіналізація чи інтеріоризація європейського культурно-історичного спадку? Філософська думка. 2014. № 2. С. 27–42. URL: http://nbuv.gov.ua/ UJRN/Philos_2014_2_5 7. Платон. Держава / Пер. з давньогр. Д. Коваль. Київ : Основи, 2000. 355 с. URL: http://litopys.org.ua/ plato/plat01.htm 8. Тойнбі А. Дослідження історії. Том 1 / Пер. з англ. В. Шовкуна. Київ : Основи, 1995. 614 Д д р р у , 9. Toynbee A. A Study of History: Volume I: Abridgement of Volumes I–VI. New York : Oxford University Press, 1988. 630 р.f , р 10. Gramsci A. Selections from Cultural Writings. Ed. by David Forgacs and Geoffrey Nowell-Smith. Cambridge : Harvard University Press, 1991. 448 р. 11. Burnham J. The Managerial Revolution: What is Happening in the World. New York : John Day Co, 1941. 342 р. 12. Lasswell H. The comparative study of elites an introduction and bibliography. Stanford : Stanford University Press, 1952. URL: https://find.library. spbu.ru /vufind/Record/apa07618070 References: 1. Bilozerova, Ya. V. Intelektual’na elita v kul’turnij ta naukovij dy’plomatiyi [Intellectual elite in cultural and scientific diplomacy. European Political and Law Discourse]. European Political and Law Discourse. 2021. Volume 8. Issue 6. Р. 6–13. [in Ukrainian] 2. Burnham, J. The Managerial Revolution: What is Happening in the World. New York : John Day Co, 1941. 342 р. [in English] 3. Golobucz’ky’j, P. V. Inteligenciya ta intelektualy’ [Intellectuals and intellectuals]. Ency’klopediya istoriyi Ukrayiny’: T. 3: E–J / Redkol.: V. A. Smolij (golova) ta in. NAN Ukrayiny’. Insty’tut istoriyi Ukrayiny’ [Encyclopedia of the History of Ukraine: Vol. 3: EY / Editor: VA Smoliy (chairman) and others. NAS of Ukraine. Institute of History of Ukraine]. Ky’yiv [Kyiv] : V-vo “Naukova dumka”, 2005. S. 507–511. URL: http://history.org.ua/LiberUA/ehu/3.pdf [in Ukrainian]f 4. Gramsci, A. Selections from Cultural Writings. Ed. by David Forgacs and Geoffrey Nowell-Smith. Cambridge : Harvard University Press, 1991. 448 р. [in English] 5. Gudkov, L. D., Dubin, B. V. Intelligentsiya: Zametki o literaturno-politicheskikh illyuziyakh [Intelligentsia: Notes on Literary and Political Illusions]. Khar’kov [Kharkov] : Epitsentr, Folio, 1995. 187 s. [in Russian] 6 L ll H Th ti t d f lit i t d ti d bibli h St f d St f d 5. Gudkov, L. D., Dubin, B. V. Intelligentsiya: Zametki o literaturno-politicheskikh illyuziyakh [Intelligentsia: Notes on Literary and Political Illusions]. Khar’kov [Kharkov] : Epitsentr, Folio, 1995. 187 s. [in Russian] 6. Lasswell, H. The comparative study of elites an introduction and bibliography. Stanford : Stanford University Press, 1952. URL: https://find.library. spbu.ru /vufind/Record/apa07618070 [in English] y ] [ ] p [ 6. Lasswell, H. The comparative study of elites an introduction and bibliography. Stanford University Press, 1952. URL: https://find.library. spbu.ru /vufind/Record/apa07618070 [in E 7. Mojseyiv, I. K. Intelektualizm [Intellectualism]. Ency’klopediya Suchasnoyi Ukrayiny’ [Ency Modern Ukraine]. 2011. URL: https://esu.com.ua/search_articles.php?id=12389 [in Ukrainia 8. My’xajl’ova, K. G. Intelektual’na elita suchasnogo suspil’stva: osobly’vosti formuvannya ta diyal’nisny’j potencial : avtoref. dy’s. ... d-ra social. nauk: 22.00.01; Xark. nacz. un-t im. V. N. Karazina [Intellectual elite of modern society: features of formation and activity potential : author’s ref. dis. ... Dr. Social. Science: 22.00.01; Hark. nat. Univ. VNKarazin]. Xarkiv [Kharkiv], 2008. 32 s. [in Ukrainian] 9. Pas’ko, Ya. Postradyans’ka intelig’enciya: cinnisna marg’inalizaciya chy’ interiory’zaciya yevropejs’kogo kul’turno-istory’chnogo spadku? [Post-Soviet intelligentsia: value marginalization or internalization of European cultural and historical heritage?]. Filosofs’ka dumka [Philosophical thought]. 2014. # 2. S. 27–42. URL: http://nbuv.gov.ua/UJRN/Philos_2014_2_5 [in Ukrainian] 10. Platon. ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 Стаття надійшла до редакції 22.02.2022 Стаття рекомендована до друку 29.04.2022 References: Derzhava / Per. z davn’ogr. D. Koval’ [State / Per. from ancient Greek D. Koval]. Ky’yiv [Kyiv] : Osnovy’, 2000. 355 s. URL: http://litopys.org.ua/plato/plat01.htm [in Ukrainian] 11. Tojnbi, A. Doslidzhennya istoriyi. Tom 1 / Per. z angl. V. Shovkuna [The study of history. Volu from English V. Shovkun]. Ky’yiv [Kyiv] : Osnovy’, 1995. 614 s. [in Ukrainian] 12. Toynbee, A. A Study of History: Volume I: Abridgement of Volumes I–VI. New York : Oxford University Press, 1988. 630 р. [in English] 12. Toynbee, A. A Study of History: Volume I: Abridgement of Volumes I–VI. New York : Oxford University Press, 1988. 630 р. [in English] ВІСНИК НТУУ «КПІ». Політологія. Соціологія. Право. Випуск 1(53) 2022 52
https://openalex.org/W4313545950	https://escholarship.org/content/qt2pr9z624/qt2pr9z624.pdf?t=rt03j3	English	null	Impact of the Las Vegas Mass Shooting Event on the Graduate Medical Education Mission: Can There Be Growth from Tragedy?	The western journal of emergency medicine/Western journal of emergency medicine	2,022	cc-by	8,766	Title Copyright Information Copyright 2023 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Irvine Western Journal of Emergency Medicine: Integrating Emergen Care with Population Health Title Impact of the Las Vegas Mass Shooting Event on the Graduate Medical Education Mission Can There Be Growth from Tragedy? Permalink https://escholarship.org/uc/item/2pr9z624 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 24(2) ISSN 1936-900X Authors Guldner, Gregory T. Roozendaal, Suzanne M. Berkeley, Ross P. et al. Publication Date 2023 DOI 10.5811/westjem.2022.9.56221 Copyright Information Copyright 2023 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Western Journal of Emergency Medicine: Integrating Emergen Care with Population Health Title Impact of the Las Vegas Mass Shooting Event on the Graduate Medical Education Mission Can There Be Growth from Tragedy? Permalink https://escholarship.org/uc/item/2pr9z624 Journal Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health, 24(2) ISSN 1936-900X Authors Guldner, Gregory T. Roozendaal, Suzanne M. Berkeley, Ross P. et al. Publication Date 2023 DOI 10.5811/westjem.2022.9.56221 Copyright Information Copyright 2023 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC e Western Journal of Emergency Medicine: Integrating Emergency Care with Population Health Impact of the Las Vegas Mass Shooting Event on the Graduate Medical Education Mission: Can There Be Growth from Tragedy? Gregory T. Guldner, MD† Suzanne M. Roozendaal, DO‡ Ross P. Berkeley, MD§ Michael P. Allswede, DO‡ Kristina H. Domanski, MD‡ Obadha M. Sairafe, MD‡ Dylan F. Davey, MD, PhD\|\| Hoda Abou-Ziab, PsyD# Jason T. Siegel, PhD¶ Gregory T. Guldner, MD† Suzanne M. Roozendaal, DO‡ Ross P. Berkeley, MD§ Michael P. Allswede, DO‡ Kristina H. Domanski, MD‡ Obadha M. Sairafe, MD‡ Dylan F. Davey, MD, PhD\|\| Hoda Abou-Ziab, PsyD# Jason T. Siegel, PhD¶ University of California Riverside School of Medicine, Department of Emergency Medicine, Riverside, California HCA Healthcare GME, Brentwood, Tennessee Sunrise Health GME Consortium, Department of Emergency Medicine, Las Vegas, Nevada Kirk Kerkorian School of Medicine at UNLV, Department of Emergency Medicine, Las Vegas, Nevada Sunrise Health GME Consortium, Department of Surgery, Las Vegas, Nevada University of California Riverside School of Medicine, Riverside Community Hospital, Department of Health Sciences, Riverside, California Claremont Graduate University, Division of Behavioral & Organizational Sciences, Claremont, California * † ‡ § \|\| # ¶ University of California Riverside School of Medicine, Department of Emergency Medicine, Riverside, California HCA Healthcare GME, Brentwood, Tennessee Sunrise Health GME Consortium, Department of Emergency Medicine, Las Vegas, Nevada Kirk Kerkorian School of Medicine at UNLV, Department of Emergency Medicine, Las Vegas, Nevada Sunrise Health GME Consortium, Department of Surgery, Las Vegas, Nevada University of California Riverside School of Medicine, Riverside Community Hospital, Department of Health Sciences, Riverside, California Claremont Graduate University, Division of Behavioral & Organizational Sciences, Claremont, California * † ‡ § \|\| # ¶ Section Editor: Christopher Kang, MD Submission history: Submitted January 22, 2021; Revision received August 15, 2022; Accepted September 21, 2022 Electronically published December 9, 2022 Full text available through open access at http://escholarship.org/uc/uciem_westjem DOI: 10.5811/westjem.2022.9.56221 Introduction: Our aim was to determine the psychological and educational impact of the 2017 Las Vegas mass shooting on the graduate medical education (GME) mission within two cohorts of resident physicians and attending faculty at two nearby academic trauma centers. Methods: A cross-sectional survey assessed 55 resident physicians and attending faculty involved in the acute care of the patients from the mass shooting. We measured the psychological impact of the event, post-traumatic growth, team cohesion, social support, and known risk factors for post-traumatic stress disorder (PTSD). Additionally, we assessed the impact of the event on GME-specific tasks. Powered by the California Digital Library University of California eScholarship.org Original Research Original Research INTRODUCTION Two nearby trauma centers, Sunrise Hospital and Medical Center (SHMC) and University Medical Center of Southern Nevada (UMC), both academic training facilities, received most of the injured patients and cared for hundreds of gunshot patients over the span of a few hours. The attending and resident physicians from these centers were exposed to an extraordinary number of emotionally intense traumatic injuries, far beyond the typical experience of clinicians. What was the major finding of the study? The Impact on academics was negative for residents, positive for attendings (2.5 vs 4.9 on 1 to 7 scale; P < 0.01). How does this improve population health? Academic trauma centers can better understand the complex impact of mass shooting events on their physicians, potentially resulting in improved care for their community. the development of PTSD.10,11 Individual factors linked to the development of PTSD include age (older individuals are at higher risk), the amount of professional training and education (more years are protective), and the presence of pre-existing mental health disorders (those impacted are at increased risk).5 While traumatic events may cause substantial negative impact, for some individuals trauma may lead to growth. Post-traumatic growth is a well-researched process in which an individual exposed to a psychologically traumatic event undergoes productive personal development leading to higher functioning.12 Post-traumatic growth occurs through changes in one or more of several psychological domains: appreciation of life, relationship with others, new possibilities or purpose in life, personal strength, or spiritual change.13 Psychologically traumatic exposures such as this can lead to significant post-traumatic stress for some healthcare workers.5 The emergency department (ED) setting, where attending and resident physicians stabilize victims of trauma, has historically reported high rates of post-traumatic stress.6 Rates of post-traumatic stress disorder (PTSD) range from 2.2-24%, depending on definition and measurement technique.5 Affected physicians may show typical signs of post-traumatic stress including involuntary upsetting memories, flashbacks, involuntary responses to external cues, and physical symptoms such as palpitations upon exposure to a reminder of the event.7 However, most physicians exposed to psychologically traumatic events do not develop PTSD. INTRODUCTION Population Health Research Capsule What do we already know about this issue? Caring for patients from mass shootings and catastrophes can impact clinicians’ personal and professional lives, with a minority developing symptoms of post-traumatic stress disorder. What was the research question? How did the Las Vegas mass shooting impact the academic activities of the residents and faculty involved? What was the major finding of the study? The Impact on academics was negative for residents, positive for attendings (2.5 vs 4.9 on 1 to 7 scale; P < 0.01). How does this improve population health? Academic trauma centers can better understand the complex impact of mass shooting events on their physicians, potentially resulting in improved care for their community. Population Health Research Capsule Population Health Research Capsule Teaching hospitals serve a dual role, providing for both graduate medical education (GME) and patient care. This necessarily intertwines teaching and learning activities with unpredictable and traumatic patient care events such as mass casualty incidents (MCI). Mass shootings, which occur in the United States at a rate of approximately one every 12.5 days,1 represent a subset of MCIs with a potential to inflict profound psychological distress on physicians. Exposure to such events may lead to disruption of personal and professional activities and lead to acute stress disorders, sleep disturbances, anxiety, depression, complicated grief, and psychological distress.2 What do we already know about this issue? Caring for patients from mass shootings and catastrophes can impact clinicians’ personal and professional lives, with a minority developing symptoms of post-traumatic stress disorder. What was the research question? How did the Las Vegas mass shooting impact the academic activities of the residents and faculty involved? One such event occurred on October 1, 2017, when an individual armed with multiple weapons opened fire on spectators at the Route 91 Harvest Music Festival in Las Vegas, Nevada. This event was the deadliest mass shooting in US history3: 58 people died at the scene or soon thereafter, with two additional deaths from subsequent complications of injuries. Over 400 additional patients sustained penetrating injuries from gunshots and/or shrapnel, with hundreds of other injuries sustained in the subsequent panic of the crowd.4 The overwhelming volume and acuity of critically injured patients exceeded the capacity of emergency medical services at the concert venue, and large numbers of patients were transported to hospitals by private vehicles. Impact of the Las Vegas Mass Shooting Event on the Graduate Medical Education Mission: Can There Be Growth from Tragedy? Results: Attending faculty and physicians in training in GME residencies evaluated over 300 penetrating trauma patients in less than 24 hours, and approximately 1 in 3 physicians had a patient die under their care. Despite this potential for psychological trauma, the majority of clinicians reported minimal distress and minimal impact on GME activities. However, 1 in 10 physicians screened positive for possible PTSD. Paradoxically, the minority of physicians who sought psychological counseling after the event (20%) were not those who reported the highest levels of distress. Residents generally assessed the event as having an overall negative impact on their educational goals, while attendings reported a positive impact. Psychological impact correlated inversely with social support and the amount of prior education relating to mass casualty incidents (MCI) but correlated directly with the degree of stress prior to the event. Conclusion: Despite the substantial level of exposure, most resident physicians did not report significant psychological trauma or an impact on their GME mission. Some reported post-traumatic growth. However, a minority reported a significant negative impact; institutions should consider broad screening efforts to detect and assist these individuals after a MCI. Social support, stress reduction, and education on MCIs may buffer the effects of future psychologically traumatic events on physicians in training. [West J Emerg Med. 2023;24(2)249–258.] Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 249 Guldner et al. Impact of the Las Vegas Mass Shooting Event on the GME Mission Western Journal of Emergency Medicine METHODS Study Design and Subjects to a very great degree”).15 This scale captures the degree of positive changes in each of the five domains of growth that may occur following a traumatic event. to a very great degree”).15 This scale captures the degree of positive changes in each of the five domains of growth that may occur following a traumatic event. Approximately six months following the shooting, we performed a post-exposure, cross-sectional survey involving resident and attending physicians who were present during the event at the two teaching hospitals impacted: SHMC is a Level II trauma center that treated over 200 mass casualties that evening, and UMC is a Level I trauma center that treated 104 patients. The timing of the survey represented the earliest point that the researchers were able to develop a protocol and obtain institutional review board (IRB) approval at both institutions following the event. No contemporaneously recorded logs of physicians present during the event exist due to the chaos that evening and the addition of unscheduled clinicians from multiple disciplines who arrived on scene spontaneously. Thus, we obtained a list from both institutions of all credentialed physicians who had potentially assisted during the mass shooting event. All physicians were contacted by email with an introduction to the study, a link to the survey, and a request for participants who were involved in caring for the patients from this event either the evening of October 1, 2017, or the morning after. Those who confirmed involvement were included in the study. The study was approved by the IRBs of both SHMC and UMC. We assessed the impact of environmental factors using two scales: the Multidimensional Scale of Perceived Social Support (MSPSS) and four items measuring team cohesion from the Team Development Measure. The MSPSS is a 12- item scale with ranges between 1 (“very strongly disagree”) and 7 (“very strongly agree”) that captures perceived social support from family, friends, and significant others.16 The team cohesion factor (TCF) consists of a four-item scale that measures the degree to which the respondent feels the team they were on was united and that they personally contributed to the overall mission of the team, using a five-point Likert scale from “strongly agree” to “strongly disagree.”17 In addition, several questions were asked to assess the perceived personal impact of the event, scored on a seven-point Likert scale. INTRODUCTION Thus, systemic or personal factors, beyond mere exposure, contribute to the psychological impact of a traumatic event on an individual physician.8 p g p g While several studies have looked at the impact of stressors such as pandemics and MCIs on physicians’ personal lives, and to a lesser extent their professional lives, there is a paucity of evidence on the impact of traumatic events specifically on the GME mission. The lack of understanding of this potential impact is especially concerning given that academic hospitals, which are frequently larger urban trauma centers, often provide a disproportionate amount of patient care during these MCIs. Thus, in our study we sought to evaluate the self-reported impact of the deadliest mass shooting event in US history on peri-traumatic stress, post- traumatic growth, and GME-related activities among residents and attending faculty at two teaching hospitals. The best researched systemic factor demonstrating a consistent inverse relationship with the risk of development of PTSD is the degree of social support.9 Prior training in stressful events, and methods of coping with the psychological aftermath, have also correlated with a protective effect against Volume 24, NO.2: March 2023 250 Impact of the Las Vegas Mass Shooting Event on the GME Mission Guldner et al. METHODS Study Design and Subjects METHODS Study Design and Subjects These questions were developed by a review of the literature, item development, and then content validation by group discussion among authors with expertise in clinical psychology. The question structure was developed by one author who is an academic psychologist with expertise in survey design methodology. These questions included the following: • How frequently have you found yourself avoiding a particular type of patient? For example, avoiding treating patients with penetrating trauma. [Anchors of “Never” to “All the Time”] Assessment Tools To determine the range of exposure to potentially psychologically traumatic events participants were asked a series of “yes” or “no” questions regarding their overall involvement in the events following the shooting. While many participants assumed direct patient care roles some may have provided non-clinical activities such as assisting with supplies, providing information, or offering psycho-social support. These questions included the following: • How frequently have you found yourself having difficulty taking care of a particular type of patient? For example, having strong emotions while treating patients with penetrating trauma. [Anchors of “Never” to “All the Time”] These questions included the following: • Did you personally provide direct care to a shooting victim? • In general, how would you say the Las Vegas shooting experience impacted your academic clinical practice? (Ability to teach, model, and perform in GME)? [Anchors of “Strong Negative Impact” to “Strong Positive Impact”] • Did you personally have a patient from the Las Vegas shooting, who you were treating, die during your care? • Did you personally have to inform relatives or loved ones of a patient’s death? • I have considered changing my specialty because of the event. [Anchored “Strongly agree” to “Strongly disagree”] • Did you personally witness images resulting from violence that were out of the ordinary for you as a physician? • I have considered leaving the field of medicine because of the event. [Anchored “Strongly agree” to “Strongly disagree”] • Did you feel personally at risk of injury or death during the event? The survey included four previously psychometrically validated scales as outcome measures. The Impact of Events Scale – Revised (IES-R) is a 22-item self-report of the degree of subjective distress following a traumatic event. Respondents assess the degree to which they experience each item on a five- point scale ranging from “not at all” to “very much.”14 Post- traumatic growth was assessed with the Post Traumatic Growth Inventory - Short Form (PTGI), a 10-item scale with ranges between zero (“did not experience this”) and five (“experienced Other known risk factors for peri-traumatic stress were assessed by the following questions: • Prior to the shooting, did you ever seek treatment for any of the following conditions? Anxiety, depression, PTSD, obsessive/compulsive disorder, personality disorder, Any other mental health condition. Prompt: What impact if any has the event had on your ability to complete these GME-related tasks? • Prior to the shooting, approximately how much prior formal training had you had regarding mass casualty events? [Coded as “none,” “1-2 hours,” “2-3 hours,” “3-4 hours,” or “more than 4 hours”] • Reading or studying CME articles or other material relevant to training residents • Participation in teaching rounds, educational half-days, noon conferences, or didactics • Completing the required residency administrative tasks such as resident evaluations and time sheets • Prior to the shooting, approximately how much formal training had you completed regarding the psychological impact of critical incidents such as the shooting? [Coded as “none,” “1-2 hours,” “2-3 hours,” “3-4 hours,” or “more than 4 hours”] • Providing teaching during resident presentation of patients • Performing procedures such as operating, intubating, chest tubes, etc. • Communicating with patients and families • Working on research projects or academic scholarly activities i Participant age was not included in the survey due to concerns about maintaining anonymity. • Recalling specific information when you need it (memory) Providing day to day feedback and guidance to residents • Recalling specific information when you need it (memory) • Providing day-to-day feedback and guidance to residents We determined types of GME activities common to residents and attendings by a review of the Accreditation Council for Graduate Medical Education Program Requirements and discussion between authors. Participants were asked, “What impact, if any, has the event had on your ability to complete these education-related tasks?” Responses for each type of activity fell on a nine-item Likert scale ranging from “Much Easier Now” to “Much Harder Now,” with the mid-range labeled as “No impact.” Table 1 shows the GME activity-related questions for attendings and residents. Resident Physician • Reading or studying the material you need to know • Participation in teaching rounds, educational half-days, noon conferences, or didactics • Completing the required residency administrative tasks such as procedure logs, evaluations, and case logs • Presenting patients to an attending, fellow, or senior resident • Performing procedures such as operating, intubating, chest tubes, etc. • Communicating with patients and their families • Teaching medical students or other learners i Psychological Impact and Comparisons by GME Role Psychological Impact and Comparisons by GME Role Psychological Impact and Comparisons by GME Role Table 2 shows the degree of exposure to psychologically traumatic events reported by study participants, and Table 3 shows the summary results of our outcome variables. The results of the IES-R (psychological impact) and PTGI (post- traumatic growth) did not differ by group when comparing those who endorsed specific exposures (directly provided care, had a patient die in their care, participated in death notification) to those who did not. Six of 15 residents (40%) and 19 of 36 attendings (53%) endorsed a prior Assessment Tools [Coded as Yes/No] • Other than the Las Vegas shooting have you previously had an exposure to an event you considered to be Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 251 Guldner et al. Impact of the Las Vegas Mass Shooting Event on the GME Mission Western Journal of Emergency Medicine psychologically traumatic to you? [Coded Yes/No] Table 1. Graduate medical education (GME)-related survey questions by GME role. Table 1. Graduate medical education (GME)-related survey questions by GME role. • In the seven days leading up to the shooting, how stressed would you say you were? [Coded on a seven- point Likert scale from “Not at all” to “Extremely”] Prompt: What impact if any has the event had on your ability to complete these GME-related tasks? Statistical Analysis • Working on research projects or academic scholarly activities GME, graduate medical education; CME, continuing medical education. • Working on research projects or academic scholarly activities GME, graduate medical education; CME, continuing medical education. We present the survey results with descriptive statistics (mean, standard deviation). Univariate associations between continuous variables were determined by Pearson product moment correlations. The association between binary “yes/no” questions and the IES-R and PTGI were measured by point biserial correlation. We measured the associations between both the IES-R and the PTGI with questions with ordinal answer sets (eg, “Less than 1 hour,” “1-2 hours,” or “2-3 hours”) with Spearman’s rho. We used a two-tailed Student’s t-test for comparisons between residents and attending physicians on continuous variables that were normally distributed according to the Shapiro-Francia test for normality. Non-normally distributed variables were compared with the Mann-Whitney U test. To determine whether the event differentially impacted certain types of GME-related activities, we conducted two separate within-subjects ANOVAs for attendings and residents. There was minimal missing data, but when present in any given statistical analysis it was handled by listwise deletion. We calculated statistics with STATA version 15 (StataCorp., College Station, TX). 17 residents. We cannot determine the response rate as a function of all physicians who actually participated in the care of patients during the shooting (as opposed to all physicians credentialed at both hospitals) because no accurate record exists from the event itself. Of the attending physicians 15 identified as emergency medicine (EM), eight as general surgery, four as anesthesiology, three as surgical subspecialties, and two as radiology, while six did not identify their role. Of the residents, there were 11 general surgery, four EM, and one family medicine resident, and one who did not identify their role. Description of Participants A total of 320 physicians were contacted by email. Of these, 55 (17%) confirmed their involvement in the event and completed the survey: 38 attending faculty and Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 252 Impact of the Las Vegas Mass Shooting Event on the GME Mission Impact of the Las Vegas Mass Shooting Event on the GME Mission Guldner et al. Residents N = 17 Attendings N = 38 Provided direct care for a shooting victim. 14 (82%) 30 (79%) Patient died in the care of the participant. 7 (41%) 12 (32%) Participant informed a relative or loved one about a death. 2 (12%) 2 (5%) Personally witnessed images resulting from violence that were out of the ordinary for them as a physician. 5 (29%) 19 (50%) Did you personally feel at risk of injury or death? 0 (0%) 0 (0%) Table 2. Proportion of respondents with exposure to potentially traumatizing experiences. psychologically traumatizing experience. (Two in each group did not answer.) This ratio does not differ by role (chi square = 0.27, P = .60). Four of 15 residents (27%) and six of 36 attendings (17%) endorsed a prior mental health condition. (Two in each group did not answer.) This ratio does not differ by role (chi square = 0.69, P =.41). Comparing those participants who endorsed prior mental health conditions to those who did not we found no statistically significant differences in social support (MSPSS), psychological impact (IES-R), team cohesion, PTGI or the global impact question. We found a similar lack of significant differences when comparing those participants who endorsed a prior psychologically traumatic experience to those who did not. The large majority of both attendings (89%) and residents (82%) reported no subsequent difficulties involving either avoiding certain types of patients (eg, trauma) or with distress associated with seeing certain types of patients after the event. Few participants reported they would consider either changing specialties (4%) or leaving medicine (7%) specifically as a result of exposure to this event. The mean and standard deviation of the standardized scales and the degree of stress prior to the event are shown in Table 4. All scales were non-normally distributed. Residents and attendings did not differ on the IES-R, PTGI, TCF, or the degree of stress perceived prior to the event. Residents reported slightly higher social support. Description of Participants Four of the 38 attending physicians (11%) and two of the 17 residents (12%) scored above the standard cutoff of 24 to signal concern for PTSD. Two of the four attending physicians scored above 33, the standard cutoff for “probable” PTSD.18 P ti i t h d iti f PTSD f EM Table 3. Summary outcomes of measured variables. Construct Instrument Outcome Psychological impact/risk for PTSD IES-R 4 of 38 attendings and 2 of 17 residents screened positive for possible PTSD. Of these, 2 attendings and 0 residents screened positive for probable PTSD. Post-traumatic growth PTGI 0 of 17 residents and 4 of 38 attendings scored at or above moderate post- traumatic growth cutoffs. Perceived social support MSPSS Residents report slightly higher perceived social support than attendings.* Team cohesion TCF Residents and attendings reported similar team cohesion. Perceived stress prior to the event Stress Residents and attendings reported similar perceived stress prior to the event. Prior mental health condition Yes/no questions 6 of 26 attendings and 4 of 15 residents endorsed prior mental health conditions. Prior psychologically traumatic event Yes/no question 19 of 36 attendings and 6 of 15 residents endorsed a prior psychologically traumatic event. Prior MCI training Ordinal options The modal response for both residents and attendings was >4 hours. Prior training on psychological impact of MCIs Ordinal options The modal response for both residents and attendings was “none.” However, 6 of the 38 attendings and 4 of 17 residents reported > 4 hours. Impact of event on core GME tasks Ordinal options Attendings: 94% “no impact,” 2% “negative impact,” and 4% “positive impact.” Residents: 71% “no impact,” 18% “negative impact,” and 11% “positive impact.” Overall impact on GME Likert Scale Residents reported the event had a negative overall impact and attendings reported the event had a slightly positive overall impact.* Unless noted, rates do not differ statistically between residents and attendings psychologically traumatizing experience. (Two in each group did not answer.) This ratio does not differ by role (chi square = 0.27, P = .60). Four of 15 residents (27%) and six of 36 attendings (17%) endorsed a prior mental health condition. (Two in each group did not answer.) This ratio does not differ by role (chi square = 0.69, P =.41). Unless noted, rates do not differ statistically between residents and attendings. P<0.05. Cutoff for IES-R “possible” PTSD is a score > 23 and “probable” PTSD is a score > 32. Cutoff for PTGI for “moderate” post-traumatic growth is >29. IES-R, Impact of Event Scales – Revised; PTGI, Post Traumatic Growth Inventory; PTSD, post-traumatic stress disorder; MCI, mass casualty incident; GME, graduate medical education. Description of Participants Comparing those participants who endorsed prior mental health conditions to those who did not we found no statistically significant differences in social support (MSPSS), psychological impact (IES-R), team cohesion, PTGI or the global impact question. We found a similar lack of significant differences when comparing those participants who endorsed a prior psychologically traumatic experience to those who did not. The large majority of both attendings (89%) and residents (82%) reported no subsequent difficulties involving either avoiding certain types of patients (eg, trauma) or with distress associated with seeing certain types of patients after the event. Few participants reported they would consider either changing specialties (4%) or leaving medicine (7%) specifically as a result of exposure to this event. The mean and standard deviation of the standardized scales and the degree of stress prior to the event are shown in Table 4. All scales were non-normally distributed. Residents and attendings did not differ on the IES-R, PTGI, TCF, or the degree of stress perceived prior to the event. Residents reported slightly higher social support. Four of the 38 attending physicians (11%) and two of the 17 residents (12%) scored above the standard cutoff of 24 to signal concern for PTSD. Two of the four attending physicians scored above 33, the standard cutoff for “probable” PTSD.18 Participants who screened positive for PTSD came from EM, anesthesiology, and one additional specialty in the “other” category. Only three of the 17 residents (18%) and seven of the 34 attendings (21%) undertook specific formal efforts to mitigate the psychological impact of the event. Notably, of the six participants scoring above the cutoff on the IES-R Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 253 Impact of the Las Vegas Mass Shooting Event on the GME Mission Guldner et al. Table 4. Outcomes of measured variables by graduate medical education role. Mann-Whitney U Test Sample size* Construct Instrument Attending M (SD) Resident M (SD) U Att Res P Psychological impact/post-traumatic stress IES-R 10.0 (12.5) 12.1 (17.1) 312.5 38 17 0.61 Post-traumatic growth PTGI 12.5 (9.5) 10.8 (11.9) 306.5 38 17 0.54 Perceived social support MSPSS 59.2 (17.7) 62.8 (17.0) 220.5 37 17 0.04* Team cohesion TCF 18.8 (2.5) 17.1 (4.0) 241.0 36 17 0.13 Perceived stress prior to event Stress 3.2 (1.6) 3.5 (1.8) 284.0 38 17 0.31 Table 4. Outcomes of measured variables by graduate medical education role. P < 0.05. Description of Participants * Sample size varied due to missing data, Cutoff for IES-R “possible” PTSD is a score > 23 and “probable” PTSD is a score > 32. Cutoff for PTGI for “moderate” post-traumatic growth is > 29. Stress: Degree of perceived stress prior to the event, scored from 1 = “not at all” to 7 = “extremely.” M, median; IES-R, Impact of Events Scale – Revised; PTGI, Post Traumatic Growth Inventory; MSPSS, Multidimensional Scale of Perceived Social Support; TCF, Team Cohesion Factor; Att, attending physician; Res, resident. ordinal or binomial variables (attended to a patient, witnessed a patient’s death, performed death notification, or prior diagnosis of mental health condition) correlated at a statistically significant level with IES-R for residents or attendings. None of the ordinal or binomial variables correlated at a statistically significant level with the PTGI for either residents or attendings. signifying more distress, only one also noted a formal effort to mitigate the impact. The single-item global assessment of impact on GME was normally distributed and differed significantly between residents and attendings by two-tailed t-test (t = 7.03, df = 50, P < 0.01). Residents reported an overall negative impact with a mean score of 2.53 on the seven-point scale (SD = 1.33), and attendings reported an overall positive impact with a mean of 4.83 (SD = 0.98). A score of 4 on the scale is anchored as “no effect.” Univariate Associations with Peri-Traumatic Stress and Post-traumatic Growth Overall, a majority of both residents and attendings reported that the mass shooting had little effect on GME- specific activities. Attendings in particular reported minimal impact, with 94% of responses relating to GME-specific activities reported as “no impact,” 2% indicating “negative impact,” and 4% indicating “positive impact.” Among the residents, 71% of the GME-specific activities were rated as “no impact,” 18% as “negative impact,” and 11% as “positive impact.” Analysis of the distribution of responses revealed that there were outlier participants responsible for the majority of non-neutral responses. Two of the 17 residents (12%) scored, on average, more than two standard deviations above the mean (reflecting that GME activities were much harder). Seven of the 34 attending physicians (21%) who answered all items scored, on average, two standard deviations below the mean (reflecting that GME activities were easier). Table 5 demonstrates the correlation matrix for the standardized scales and the degree of stress prior to the event for all participants (residents and attending physicians combined). Team cohesion correlated positively with the degree of social support. However, this relationship held only among the residents (r = 0.50, P < 0.05), not among the attendings (r = 0.10, P = 0.55). Western Journal of Emergency Medicine DISCUSSION A study using a survey comprised of the full Diagnostic and Statistical Manual of Mental Disorders, 5th ed, criteria found a PTSD rate of 2.2% in physicians.20f Regarding GME-specific tasks, the majority of both attendings and residents reported minimal to no impact from the event. Overall, the impact on educational activities was independent of the psychological impact of the event, as evidenced by the near-zero correlation between the IES-R and the global assessment of educational impact. However, residents differed from attendings in their assessment of the educational impact on GME activities. Residents reported the event as negatively impacting their GME experience, while attendings presumably reframed the event as one in which growth occurred. This bias toward growth was seen despite the fact that attendings, far more than residents, correctly recognized the MCI as “outside the normal experience for a physician.” Residents may have perceived the MCI as creating a substantial increase in workload. This may have created a work and learning environment perceived as too heavily focused on clinical service vs education. Additionally, residents faced with increased work demands may not have recognized the potential educational impact of the MCI.i and 22% of surgery residents screened at risk for PTSD. Surprisingly, not one of the 15 trauma surgeons screened positive. This is consistent with our current study, which also showed no surgery attending or resident endorsing a level of psychological impact that would suggest PTSD. However, other studies of trauma surgeons have demonstrated a 15% rate of probable PTSD.19 Thus, it remains unclear whether surgery selects for or develops individuals with a lower risk of PTSD overall or whether social response or selection bias accounts for the lack of surgeons endorsing mental health symptoms in some studies. Importantly, almost all studies used short screening surveys to screen for PTSD, which may overestimate the true rates of clinical PTSD. A study using a survey comprised of the full Diagnostic and Statistical Manual of Mental Disorders, 5th ed, criteria found a PTSD rate of 2.2% in physicians.20f This suggests that efforts to mitigate the impact of psychologically traumatic events in the GME community should begin with screening to detect those more likely to benefit from additional interventions, rather than comprehensive trauma intervention programs designed for all involved. DISCUSSION We sought to determine the potential psychological and educational impact of the worst mass shooting event in US history on members of the GME community who cared for the patients. Consistent with prior literature, most participants, both attendings and residents, reported relatively low levels of post-traumatic stress symptoms five to six months after the event. The vast majority of participants did not intend to either leave medicine or change specialty as a result of this specific exposure to a MCI. Roughly 1 in 10 participants reported symptoms severe enough to be considered PTSD. A previous prevalence screening study of 190 physicians at trauma centers in Texas found a similar rate, with 13% reporting they had previously sought treatment for PTSD-type symptoms.6 In the Texas study, 16% of ED attendings, 29% of EM residents, and 22% of surgery residents screened at risk for PTSD. We sought to determine the potential psychological and educational impact of the worst mass shooting event in US history on members of the GME community who cared for the patients. Consistent with prior literature, most participants, both attendings and residents, reported relatively low levels of post-traumatic stress symptoms five to six months after the event. The vast majority of participants did not intend to either leave medicine or change specialty as a result of this specific exposure to a MCI. Roughly 1 in 10 participants reported symptoms severe enough to be considered PTSD. A previous prevalence screening study of 190 physicians at trauma centers in Texas found a similar rate, with 13% reporting they had previously sought treatment for PTSD-type symptoms.6 In the Texas study, 16% of ED attendings, 29% of EM residents, and 22% of surgery residents screened at risk for PTSD. Surprisingly, not one of the 15 trauma surgeons screened positive. This is consistent with our current study, which also showed no surgery attending or resident endorsing a level of psychological impact that would suggest PTSD. However, other studies of trauma surgeons have demonstrated a 15% rate of probable PTSD.19 Thus, it remains unclear whether surgery selects for or develops individuals with a lower risk of PTSD overall or whether social response or selection bias accounts for the lack of surgeons endorsing mental health symptoms in some studies. Importantly, almost all studies used short screening surveys to screen for PTSD, which may overestimate the true rates of clinical PTSD. Impact of the Las Vegas Mass Shooting Event on the GME Mission Impact of the Las Vegas Mass Shooting Event on the GME Mission Guldner et al. Table 5. Pearson correlation matrix for scales, stress, and general impression of event on graduate medical education. IES-R MSPSS PTGI TCF Stress General impression IES-R 1.00 -0.28* 0.50* 0.11 0.28* 0.01 MSPSS 1.00 0.20 0.27* 0.06 -0.12 PTGI 1.00 -0.05 0.13 0.03 TCF 1.00 -0.17 -0.23 Stress 1.00 -0.03 *P<0.05 by Pearson product moment correlation. N=55. Stress: Degree of perceived stress prior to the event, scored from 1 = ”not at all” to 7 = “extremely.” General Impression: Scored 1 = “Strongly Negative” to 7 “Strongly Positive” with 4 = “No Effect.” MSPSS, Multidimensional Scale of Perceived Social Support; IES-R, Impact of Events Scale – Revised. PTGI, Post Traumatic Growth Inventory; TCF, team cohesion factor. Despite the profound potential of the Las Vegas mass shooting to create psychological trauma, we found that few physicians chose to mitigate the impact with help-seeking behaviors. Paradoxically, the few participants who did seek help were not those with the highest reported distress. This inverse relationship between help-seeking and degree of distress is also seen in the depression literature, which has shown that the most distressed individuals are often the least likely to seek help.23 This pattern has serious implications regarding the typical institutional practice of suggesting to physicians, “If you need help, ask for help.” Those who most need help often will not ask. i Trauma Of the residents, 11 (65%) reported some prior training on MCIs, with six (35%) reporting some training on the psychological impact of MCIs on clinicians. Attending physicians more frequently reported some exposure to MCI training (31 of 38 [82%]) and its psychological impact (22 of 38 [58%]). Combining both groups, the average amount of training on MCIs was 2-3 hours with an average of 1-2 hours on the psychological impact. Among residents, IES-R correlated inversely with the amount of MCI training (r = -0.67, P < 0.01), and with attendings, IES-R correlated inversely with the amount of training on the psychological effects of MCI (r = -0.39, P = 0.02). None of the other The relationship between the overall impression of the impact of the event on GME tasks and post-traumatic growth differed between residents and attendings. Among attendings, the more positive impact they felt the event had on GME tasks, the more post-traumatic growth they reported (r = 0.33, P = 0.05). However, with residents this relationship was reversed, although not statistically significant, likely due to the smaller sample size (r = -0.31, P = 0.23). Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 254 LIMITATIONS Our research has several limitations. The total number of physicians who actually participated in the care of patients during the Las Vegas MCI is unknown, and thus our survey response rate is unknown. The degree of individual distress may have impacted physicians’ willingness to participate in the survey, thereby biasing our study population to reflect a less generalizable cohort. Given that participants self- selected to complete the survey our cohort may suffer from selection bias, as the overall population of physicians who experienced the MCI may differ from those who agreed to participate. Similarly, given the anonymity of the study and the contemporaneous chaos of the event, we cannot confirm that all participants were actually involved in the event other than through their endorsement of being eligible for the study, nor can we determine the extent or nature of the experience of individual participants. While some participants were directly involved in patient care others may have been involved in providing ancillary services such as transportation, logistics assistance, or psycho-social support. i Consistent with prior literature, we found that social support was inversely associated with distress. Social support plays a substantial role in overall well-being, as it mitigates depression, encourages work engagement, and buffers stressors in the environment.25 Deliberate institutional efforts to develop and sustain high levels of collegiality and perceived social support create positive work environments. This likely mitigates the psychological impact of catastrophic events such as the Las Vegas shooting on the healthcare team. Similarly, the association between perceived baseline stress prior to the event and subsequent psychological impact26 provides a target for institutions hoping to mitigate the impact of a similar event. Broad efforts to improve the workplace environment and lessen perceived stress on the GME community should be supported for many reasons. Our study demonstrates yet another domain in which the high levels of baseline stress can negatively impact GME physicians. The survey was distributed five to six months following the mass shooting; therefore, participant responses reflect their understanding of the event after contemplation. A follow-up survey to assess trends and possible longer term impact of the event is under development. Typically, disasters create predictable psychological phases of various durations.31 Initially, the heroic and honeymoon phases last weeks to months and create a sense of social support and hope. DISCUSSION Most individuals exposed to psychological trauma do not develop PTSD,21 and early interventions such as mandatory debriefing sessions for all clinicians have not demonstrated efficacy.22 Despite reporting similar profiles in overall psychological impact, post-traumatic growth, team cohesion, and perceived stress, attending physicians perceived the impact of this traumatic event on the didactic environment differently than the resident physicians. Some possible explanations may include differences in age, psychological resources, sense of purpose, autonomy, confidence in patient care, or commitment to an organization. Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 255 Guldner et al. Impact of the Las Vegas Mass Shooting Event on the GME Mission The retrospective assessment of the impact of the event may be a function of the demands placed on the individual and the resources they employ to meet those demands. For example, since residents do not possess a complete skillset and work under supervision, it is possible they may have experienced a greater sense of helplessness, which has been linked to the development of peri-traumatic distress.24 It is also possible that an MCI may be perceived by certain experienced physicians as an opportunity to demonstrate competency, while for residents such an event may potentially expose weaknesses or knowledge gaps related to their level of training. Although residents did not report greater psychological distress, they did report a more negative perceived impact on their education. Residents’ primary developmental goal is professional growth toward independent practice, while attendings have achieved this milestone and are focused on various other objectives. The impact of an MCI appears to disrupt educational goals variably, more frequently for physicians in training, and only for a minority of residents. psychological trauma and growth in hopes of promoting positive individual development, rather than maladaptive behaviors, after exposure to trauma. Western Journal of Emergency Medicine LIMITATIONS The disillusionment phase follows when the realities of the impact of the disaster may be unopposed by the more positive support from the earlier phases. This may last between 3-36 months followed by the final restorative phase. Thus, the timing of our survey likely corresponds with the disillusionment phase; surveys conducted in earlier or later phases may have yielded different results. Prior studies have shown a relationship between the risk of PTSD and both a sense of helplessness and the degree of prior training in MCIs.5,27,28 Consistent with this finding we found an inverse relationship between prior training in MCIs, including training on their psychological impact, and the impact of a traumatic exposure on GME physicians. Institutions should prioritize training in MCIs and the psychological impact of these events as a strategy for mitigating clinician distress. These training events do not require inordinate time commitments. In our study incremental differences of 1-2 hours predicted less psychological distress. Age, which is related to psychological distress and thus can be a confounder, was not assessed to avoid identification of any specific individual’s responses. However, the analysis by group (resident vs attending) serves as an imperfect proxy assessment of this variable. Due to the small sample size we did not attempt to compare various specialties to one another in their response to the event. Some specialties, such as the two radiologists who completed the survey, may have had a different level of exposure to trauma than other specialties. However, unlike in routine care, some radiologists came to the bedside during the event to interpret radiographs on portable imaging machines, exposing them to unusual scenes of violence. For both cohorts, the degree of psychological impact positively correlated with post-traumatic growth: a relationship noted in prior research in general29 and specifically among emergency physicians.30 Post-traumatic growth arises out of the psychological struggle to integrate traumatic events with one’s prior understanding of the world. Further research is needed to explore the relationship between Some of the measures, such as the TCF, asked participants about the level of teamwork at the time of the event which could have resulted in recall bias, as residents Volume 24, NO.2: March 2023 256 Impact of the Las Vegas Mass Shooting Event on the GME Mission Guldner et al. CONCLUSION 7. American Psychiatric Association. (2013) Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (271-272). American Psychiatric Association. This study of 55 attending and resident physicians involved in the aftermath of the tragic events of the Las Vegas mass shooting found that, months after the event, most physicians reported low levels of PTSD symptoms and minimal impact on GME-specific activities. However, approximately 10% of both resident and attending physicians screened positive for possible PTSD. Attendings and residents differed in their overall global assessment of the impact of the event on their educational mission, with some attendings viewing it as resulting in growth while residents generally perceived it as either neutral or negative. 8. Sareen J, Erickson J, Medved MI, et al. Risk factors for post-injury mental health problems. Depress Anxiety. 2013;30(4):321-7. 9. Prati G, Pietrantoni L. The relation of perceived and received social support to mental health among first responders: a meta-analytic review. J Community Psychol. 2010;38(3):403-17. 10. Engel CC, Litz B, Magruder KM, et al. Delivery of self training and education for stressful situations (DESTRESS-PC): a randomized trial of nurse assisted online self-management for PTSD in primary care. Gen Hosp Psychiatry. 2015;37(4):323-8. REFERENCES with greater or lesser overall impact may have recalled their team cohesion differently. The unpredictability of mass shootings creates significant barriers to any prospective research on the impact of psychological trauma on the GME mission. 1. Meindl JN, Ivy JW. Mass Shootings: the role of the media in promoting generalized imitation. Am J Public Health. 2017;107(3):368-70. 2. Vance MC, Kovachy B, Dong M, et al. Peritraumatic distress: a review and synthesis of 15 years of research. J Clin Psychol. 2018;74(9):1457-84. Although we asked participants whether they sought psychological assistance following the event, we did not inquire as to the specific type of intervention obtained. Some evidence suggests a differential impact on post-traumatic symptoms depending on the type of psychological approach used; and we could not determine what approaches were employed in our sample population. 3. CNN. Deadliest Mass Shootings in the US Fast Facts. 2021. Available at: https://edition.cnn.com/2019/08/19/us/mass-shootings- fast-facts/index.html. Accessed September 25, 2022. 4. Las Vegas Metropolitan Police Department. LVMPD Criminal Investigative Report of the 1 October Mass Casualty Shooting. 2018. Available at: https://www.lvmpd.com/en-us/Documents/1- October-FIT-Criminal-Investigative-Report-FINAL_080318.pdf. Accessed June 25, 2021. Future research on psychological trauma within the GME population may help better characterize the factors that determine the likelihood of an individual developing post-traumatic growth or PTSD symptoms. An examination of why some groups retrospectively view trauma with growth while others view it as entirely negative could yield valuable insights to assist future development of pre- and peri-event interventions. 5. D’Ettorre G, Pellicani V, Ceccarelli G. Post-traumatic stress disorder symptoms in healthcare workers: a ten-year systematic review. Acta Bio Medica Atenei Parm. 2020;91(Suppl 12):1-10. 6. Luftman K, Aydelotte J, Rix K, et al. PTSD in those who care for the injured. Injury. 2017;48(2):293-6. ACKNOWLEDGMENTS 11. Mealer M, Conrad D, Evans J, et al. Feasibility and acceptability of a resilience training program for intensive care unit nurses. Am J Crit Care. 2014;23(6):e97-105. Erratum in: Am J Crit Care. 2016 Mar;25(2):172. PMID: 25362680. The authors would like to thank Ayutyanont Napatkamon, PhD, for her assistance in preparing the study and Heineke Hyunju for her assistance in logistics and submission. 12. Tedeschi RG, Calhoun LG. (1995). Trauma & Transformation: Growing in the Aftermath of Suffering. Thousand Oaks, CA: Sage Publications, Inc. Address for Correspondence: Gregory Guldner, MD, HCA Healthcare National GME Wellness Committee, 2000 Health Park Drive Brentwood, TN 37027. Email: Gregory.Guldner@hcahealthcare.com. 13. Taku K, Cann A, Calhoun LG, et al. The factor structure of the posttraumatic growth inventory: a comparison of five models using confirmatory factor analysis. J Trauma Stress. 2008;21(2):158-16. Conflicts of Interest: By the WestJEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. No author has professional or financial relationships with any companies that are relevant to this study. There are no conflicts of interest or sources of funding to declare. 14. Weiss DS, Marmar CR. (1996). The Impact of Events Scale - Revised. In: Wilson J, Keane TM, eds. Assessing Psychological Trauma and PTSD (399-411). New York, NY: Guilford. 15. Cann A, Calhoun LG, Tedeschi RG, et al. A short form of the Posttraumatic Growth Inventory. Anxiety Stress Coping. 2010;23(2):127-37. 16. Zimet G, Dahlem NM, Farley GK. Multidimensional Scale of Perceived Social Support. J Pers Assess. 1988;52:30-41. Copyright: © 2023 Guldner et al. This is an open access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) License. See: http://creativecommons.org/ licenses/by/4.0/ 17. Stock R, Mahoney E, Carney PA. Measuring team development in clinical care settings. Fam Med. 2013;45(10):691-700. Western Journal of Emergency Medicine Western Journal of Emergency Medicine Volume 24, NO.2: March 2023 257 Guldner et al. Impact of the Las Vegas Mass Shooting Event on the GME Mission Can J Psychiatry. 2012;57(2):122-9. Can J Psychiatry. 2012;57(2):122-9. 18. Creamer M, Bell R, Failla S. Psychometric properties of the Impact of Event Scale - Revised. Behav Res Ther. 2003;41(12):1489-96. 18. Creamer M, Bell R, Failla S. Psychometric properties of the Impact of Event Scale - Revised. Behav Res Ther. 2003;41(12):1489-96. 25. Law M, Lam M, Wu D, et al. Western Journal of Emergency Medicine ACKNOWLEDGMENTS Changes in personal relationships during residency and their effects on resident wellness: a qualitative study. Acad Med. 2017;92(11):1601-6. 19. Joseph B, Pandit V, Hadeed G, et al. Unveiling posttraumatic stress disorder in trauma surgeons: a national survey. J Trauma Acute Care Surg. 2014;77(1):148-54. 26. Brewin CR, Andrews B, Valentine JD. Meta-analysis of risk factors for posttraumatic stress disorder in trauma-exposed adults. J Consult Clin Psychol. 2000;68(5):748-66. 20. Van Steijn ME, Scheepstra KWF, Yasar G, et al. Occupational well- being in pediatricians—a survey about work-related posttraumatic stress, depression, and anxiety. Eur J Pediatr. 2019;178(5):681-93. 27. Joshi A, Abdelsattar J, Castro-Varela A, et al. Incorporating mass casualty incidents training in surgical education program. Global Surg Educ. 2022;1(1):17 21. Kessler RC, Rose S, Koenen KC, et al. How well can post-traumatic stress disorder be predicted from pre-trauma risk factors? An exploratory study in the WHO World Mental Health Surveys. World Psychiatry. 2014;13(3):265-74. 28. Andreatta PB, Maslowski E, Petty S, et al. Virtual reality triage training provides a viable solution for disaster-preparedness. Acad Emerg Med. 2010;17(8):870-6. 22. Rose S, Bisson J, Churchill R, et al. Psychological debriefing for preventing post traumatic stress disorder (PTSD). Cochrane Database Syst Rev. 2002;(2):CD000560. 29. Shakespeare-Finch J, Lurie-Beck J. A meta-analytic clarification of the relationship between posttraumatic growth and symptoms of posttraumatic distress disorder. J Anxiety Disord. 2014;28(2):223-9. 23. Siegel JT, Lienemann BA, Rosenberg BD. Resistance, reactance, and misinterpretation: highlighting the challenge of persuading people with depression to seek help. Soc Personal Psychol Compass. 2017;11(6): e12322. 30. Yaakubov L, Hoffman Y, Rosenbloom T. Secondary traumatic stress, vicarious posttraumatic growth and their association in emergency room physicians and nurses. Eur J Psychotraumatology. 11(1):1-12. 31. Math SB, Nirmala MC, Moirangthem S, et al. Disaster management: mental health perspective. Indian J Psychol Med. 2015;37(3):261-71. 24. Thomas É, Saumier D, Brunet A. Peritraumatic distress and the course of posttraumatic stress disorder symptoms: a meta-analysis. Volume 24, NO.2: March 2023 Western Journal of Emergency Medicine 258
https://openalex.org/W2966240019	https://journal.iainkudus.ac.id/index.php/Konseling_Edukasi/article/download/5580/pdf	Indonesian	null	Peran Konselor dalam Menghadapi Perilaku Temper Tantrum	Konseling edukasi : journal of guidance and counseling	2,019	cc-by-sa	3,252	Peran Konselor dalam Menghadapi Perilaku Temper Tantrum David Ari Setyawan Institut Agama Islam Negeri (IAIN) Kudus, Indonesia davidarisetyawan@iainkudus.ac.id Kata kunci: Peran Konselor, Temper Tantrum. Abstrak THE ROLE OF COUNSELOR IN DEALING WITH TEMPER TANTRUM BEHAVIOR. Children aged 4-7 years is a time to get acquainted and learn to deal with disappointment when their desire cannot be fulfilled. Disappointment, anger, sadness, and so on is a natural feeling. However, often, parents unwittingly 'clog up' the emotions felt by the children. For example, when a child cries out in disappointment, parents in various ways try to entertain, distract, scold, and so on in order to stop the child's crying. This actually makes the children's emotions do not come out freely. If this continues, the result will be what is called the emotional pile. This pile of emotions can later explode out of control and appear as temper tantrum. Bursting anger with dangerous actions and causing injury is one form of tantrum so that children get what they want. The realization of the children’s tantrum that can pose a risk of injury can be either dropping to the floor, hitting the head, or throwing things. These are considered as the first stage of temper tantrum when the children are able to express their frustration. If temper tantrums have already appeared in the form of harmful behaviors and have the potential to cause damage, then intervention must be done immediately. The bigger the child, the more powerful the energy is and the more difficult it will be for parents to control or prevent uncontrolled behavior. Keywords: The role of counselor, Temper Tantrum. Keywords: The role of counselor, Temper Tantrum. Abstrak Anak usia 4-7 tahun merupakan masa untuk berkenalan dan belajar menghadapi rasa kecewa saat apa yang dikehendaki tidak dapat terpenuhi. Rasa kecewa, marah, sedih dan sebagainya merupakan suatu rasa yang wajar dan natural. Namun seringkali, tanpa disadari orang tua ‘menyumbat’ emosi yang dirasakan oleh anak. Misalnya saat anak menangis karena kecewa, orangtua dengan berbagai cara berusaha menghibur, mengalihkan perhatian, memarahi dan sebagainya demi menghentikan tangisan anak. Hal ini sebenarnya membuat emosi anak tak tersalurkan dengan lepas. Jika hal ini berlangsung terus menerus, akibatnya timbullah yang disebut dengan tumpukan emosi. Tumpukan emosi inilah yang nantinya dapat meledak tak terkendali dan muncul sebagai temper tantrum. Meluapkan kemarahan dengan tindakan - tindakan yang berbahaya dan menimbulkan cedera adalah salah satu bentuk tantrum agar anak mendapatkan apa yang ia inginkan. Perwujudan tantrum pada anak yang dapat menimbulkan resiko cedera tersebut dapat berupa menjatuhkan badan ke lantai, memukul kepala, atau melempar barang, hal ini diduga merupakan bentuk awal dari temper tantrum pada saat anak sudah mampu mengekspresikan rasa frustasinya. Jika temper tantrum telah terlanjur muncul dalam bentuk perilaku yang membahayakan dan berpotensi menimbulkan kerusakan, maka tindakan intervensi harus segera dilakukan. Semakin besar anak, tenaga juga semakin kuat dan akan semakin sulit bagi orang tua untuk mengendalikan atau mencegah tingkahlakunya yang tidak terkendali. Kata kunci: Peran Konselor, Temper Tantrum. Konseling Edukasi: Journal of Guidance and Counseling 123 David Ari Setyawan A. Pendahuluan Temper tantrum adalah salah satu dari sekian banyak kelainan pada kebiasaan-kebiasaan anak, sebagai suatu usaha untuk memaksakan kehendaknya pada orang tua, yang biasanya tampak dalam bentuk menjerit-jerit, berteriak dan menangis sekeras-kerasnya, berguling-guling di lantai dan sebagainya (Kartono, 1991: 13). Temper tantrum merupakan luapan emosi yang meledak-ledak dan tidak terkontrol. Kejadian ini seringkali muncul pada anak usia 15 bulan sampai 5 tahun. Tantrum terjadi pada anak yang aktif dengan energi yang melimpah (Hasan, 2011: 185). Temper tantrum merupakan ledakan amarah, ketakutan yang hebat dan iri hati yang tidak masuk akal Hurlock (1998: 115). Hal ini tampak mencolok pada anak-anak usia 2,5 sampai 3,5 dan 5,5 sampai 6,5 tahun. Ledakan amarah mencapai puncaknya antara usia dua dan empat tahun, setelah itu amarah berlangsung tidak terlampau lama. Temper tantrum merupakan gangguan tingkah Vol. 3, No. 1, Jan-Jun 2019 124 laku yang terjadi pada anak usia tiga sampai tujuh tahun, gangguan ini ditandai dengan adanya suatu pola tingkah laku dissosial, agresif atau menentang yang berulang dan menetap (Maslim, 2003: 137). Temper tantrum merupakan ledakan emosi yang cukup kuat, disertai rasa marah, serangan agresif, ,menangis, menjerit- jerit, menghentak-hentakkan kedua tangan dan kaki di lantai atau tanah (Chaplin, 2009:502). Temper tantrum merupakan perilaku destruktif dalam bentuk luapan yang bisa bersifat fisik (memukul, menggigit, mendorong), maupun verbal (menangis, berteriak, merengek) atau terus menerus merajuk Salkind (2002: 408). Dari pengertian di atas dapat disimpulkan bahwa temper tantrum adalah suatu ledakan amarah yang sering terjadi pada anak usia tiga sampai enam tahun yang ditandai dengan tindakan menangis, menjerit-jerit, melempar benda, berguling-guling, memukul dan aktivitas destruktif lainnya. tantrum terjadi pada anak yang aktif dengan energi yang berlimpah. Tantrum dapat terjadi pada anak- anak yang dianggap lebih sulit, dengan ciri-ciri sebagai berikut: (1) Memiliki kebiasaan makan, tidur, dan buang air besar tidak teratur, (2) Sulit menerima orang baru, (3) Lambat beradaptasi terhadap perubahan, (4) Suasana hati tidak stabil, (5) Mudah kesal, marah dan mudah terprovokasi, (6) Sulit dialihkan perhatiannya. 1. Pola Asuh Otoriter Pengasuhan yang otoriter (authorian parenting) ialah suatu gaya membatasi dan menghukum yang menuntut anak untuk mengikuti perintah- perintah orang tua dan menghormati pekerjaan dan usaha. Orangtua menuntut anak mengikuti perintah-perintahnya, sering memukul anak, memaksakan aturan tanpa penjelasan, dan menunjukkan amarah. Orang tua yang otoriter menetapkan batas-batas yang tegas dan tidak memberi peluang yang besar kepada anak-anak untuk berbicara atau bermusyawarah. Peraturan yang keras untuk memaksa perilaku yang diinginkan menandai semua jenis pola asuh yang otoriter Hurlock (2010: 93). Tekniknya mencakup hukuman yang berat bila terjadi kegagalan memenuhi standar dan sedikit, atau sama sekali tidak adanya persetujuan, pujian atau tanda-tanda penghargaan lainnya bila anak memenuhi standar yang diharapkan.Orang tua tidak mendorong anak untuk dengan mandiri mengambil keputusan-keputusan yang berhubungan dengan tindakan mereka. Sebaliknya, mereka hanya mengatakan apa yang harus dilakukan. Jadi anak-anak kehilangan kesempatan untuk belajar bagaimana mengendalikan perilaku mereka sendiri. Dengan cara otoriter, ditambah dengan sikap keras, menghukum dan mengancam akan menjadikan anak “patuh” di hadapan orang tua, tetapi di belakangnya ia akan menentang atau melawan karena anak merasa “dipaksa”. Reaksi menentang bisa ditampilkan dalam tingkahlaku-tingkahlaku yang melanggar norma-norma lingkungan rumah, sekolah, dan pergaulan (Gunarsa, 2008:82). Efek pengasuhan ini, antara lain anak mengalami inkompetensi sosial, sering merasa tidak bahagia, kemampuan komunikasi lemah, tidak memiliki inisiatif melakukan sesuatu, dan kemungkinan berperilaku agresif (Soetjiningsih, 2012: 216). Anak dari orang tua yang otoriter sering kali tidak bahagia, ketakutan, minder ketika membandingkan diri dengan orang lain, tidak mampu memulai aktivitas, dan memliki kemampuan komunikasi yang lemah, serta sering berperilaku agresif (Santrock, 2002: 167). B. Pembahasan Pola asuh orang tua adalah suatu metode disiplin yang diterapkan orang tua terhadap anaknya. Metode disiplin ini mempunyai dua konsep yaitu konsep negatif dan konsep positif, Hurlock (1998:82). Konsep positif, disiplin berarti pendidikan dan bimbingan yang lebih menekankan pada disiplin dan pengendalian diri. Konsep negatif, disiplin diartikan pengendalian dengan kekuasaan. Ini merupakan suatu bentuk pengekangan melalui cara yang tidak disukai dan menyakitkan. Dalam kasus ini memotret terjadi kesalahan konsep dalam pendidikan pola asuh orangtua kepada anak, sehingga mengakibatkan anak mengalami temper tantrum. Lebih jauh Hurlock menyebutkan bahwa fungsi pokok dari pola asuh orang tua adalah untuk mengajarkan anak menerima pengekangan- pengekangan yang diperlukan dan membantu mengarahkan emosi anak ke dalam jalur yang berguna dan diterima secara sosial. Pola asuh orang tua akan mempengaruhi kepribadian dan perilaku anak (Dariyo, 2004: 97). Ada beberapa pola asuh orang tua yang dikenal dengan pola asuh otoriter, pola asuh demokratis dan pola asuh permisif Hurlock (1998:94). Dapat disimpulkan bahwa pola asuh adalah bentuk pengasuhan orang tua untuk menanamkan disiplin pada anaknya Konseling Edukasi: Journal of Guidance and Counseling 125 David Ari Setyawan yang pada akhirnya akan membentuk kepribadian dan perilaku anak. Terdapat tiga tipe pola asuh yaitu pola asuh otoriter, pola asuh demokratis dan pola asuh permisif. 2. Pengasuhan Permisif Pengasuhan yang permisif ialah suatu gaya dimana orang tua sangat tidak terlibat dalam kehidupan anak. Anak mengembangkan perasaan bahwa aspek- aspek lain kehidupan orangtua lebih penting daripada diri mereka. Biasanya pola asuh permisif tidak membimbing anak ke pola perilaku yang disetujui secara sosial Vol. 3, No. 1, Jan-Jun 2019 126 dan tidak menggunakan hukuman. Orang tua membiarkan anak-anak meraba-raba dalam situasi yang terlalu sulit untuk ditanggulangi oleh mereka sendiri tanpa bimbingan atau pengendalian. Anak sering tidak diberi batas-batas atau kendali yang mengatur apa saja yang boleh dilakukan. Mereka diijinkan untuk mengambil keputusan sendiri dan berbuat sekehendak mereka sendiri (Hurlock, 2010: 93). Karena harus menentukan sendiri, maka perkembangan kepribadian anak menjadi tidak terarah. Pada anak tumbuh egosentrisme yang terlalu kuat dan kaku, dan mudah menimbulkan kesulitan-kesulitan jika harus menghadapi larangan- larangan yang ada dalam masyarakat. Efek pengasuhan ini anak akan memiliki kendali diri yang buruk, inkopetensi sosial, tidak mandiri, harga diri rendah, tidak dewasa, rasa terasing dari keluarga, serta pada saat remaja akan suka membolos dan nakal (Soetjiningsih, 2012: 218). Anak dari orang tua yang permisif akan memiliki harga diri yang rendah, tidak dewasa, kesulitan belajar menghormati orang lain, kesulitan mengendalikan perilakunya, egosentris, tidak menuruti aturan, dan kesulitan dalam berhubungan dengan teman sebaya (Santrock, 2002: 168). 3. Pengasuhan Demokratis Pola asuh demokratis mendorong anak-anak agar mandiri tetapi masih menetapkan batas-batas dan pengendalian atas tindakan-tindakan mereka. Musyawarah verbal yang ekstensif dimungkinkan dan orang tua memperlihatkan kehangatan serta kasih sayang kepada anak. Pengasuhan yang otoritatif diasosiasikan dengan kompetensi sosial anak. Metode demokratis menggunakan penjelasan, diskusi dan penalaran untuk membantu anak mengerti mengapa perilaku tertentu diharapkan, Hurlock (2010:93). Metode ini lebih menekankan aspek edukatif dari disiplin dari pada aspek hukumannya. Pada pola asuh ini menggunakan hukuman dan penghargaan, dengan penekanan yang lebih besar pada penghargaan. Hukuman tidak pernah keras dan biasanya tidak berbentuk hukuman badan. Hukuman hanya digunakan bila terdapat bukti bahwa anak-anak secara sadar menolak melakukan apa yang diharapkan dari mereka. Bila perilaku anak memenuhi standar yang diharapkan, orang tua yang demokratis akan menghargainya dengan pujian atau persetujuan orang lain. Dengan cara demokratis ini pada anak akan tumbuh rasa tanggungjawab untuk memperlihatkan sesuatu tingkahlaku dan selanjutnya memupuk rasa percaya dirinya. Anak akan mampu bertindak sesuai norma dan menyesuaikan diri dengan lingkungannya (Gunarsa, 2008:84). Konseling Edukasi: Journal of Guidance and Counseling 127 David Ari Setyawan Efek pengasuhan demokratis, yaitu anak mempunyai kompetensi sosial percaya diri, dan bertanggung jawab secara sosial. Juga tampak ceria, bisa mengendalikan diri dan mandiri, berorientasi pada prestasi, mempertahankan hubungan ramah dengan teman sebaya, mampu bekerja sama dengan orang dewasa, dan mampu mengatasi stres dengan baik (Soetjiningsih, 2012: 217). Anak dari orang tua yang demokratis ceria, bisa mengendalikan diri dan mandiri, dan berorientasi pada prestasi, mereka cenderung untuk mempertahankan hubungan yang ramah dengan teman sebaya, bekerja sama dengan orang dewasa, dan bisa mengatasi stres dengan baik (Santrock, 2002: 167). 4. Peran Konselor dalam Proses Konseling Pemberian konseling tidak hanya diberikan pada klien tetapi juga harus dilakukan pada orang tua. Hal ini dikarenakan konsep pola asuh orang tua juga menjadi dasar anak untuk dapat berkembang optimal sesuai dengan tugas perkembangannya. Disini konselor sangat berperan membantu mengarahkan dan mengembangkan potensi yang dimiliki anak. Dalam menangani anak yang mengalami temper tantrum ini konselor harus bekerja sama baik dengan pihak sekolah, keluarga, dan masyarakat. Konselor dapat membantu dalam hal: 1) Memberikan informasi kepada orang tua yang sedang kegundahan memiliki anak yang mengalami temper tantrum. Informasi yang diberikan berhubungan dengan diagnosisnya dan dukungan yang dapat diberikan oleh orang tua dalam membantu anaknya mencapai aktualisasi diri dan melakukan hal-hal sebagaimana petunjuk sekolah yang menanganinya, 2) Membantu mencapai dan mensugestikan respons emosional yang sesuai untuk mengurangi ketegangan yang mungkin ada antara orang tua, konselor maupun anak yang bersangkutan, 3) Membangun kelompok orang tua yang dapat memberi dukungan dan yang mengalami hal-hal yang kurang lebih sama, 4) Menjalankan sesi dan mengenalkan pola asuh dalam pendidikan keluarga, 5) Konselor sangat mampu mengatasi pikiran-pikiran negatif melalui konseling kelompok maupun individual. Demikian pula penjelasan bahwa seseorang dapat saja hebat dalam bidang tertentu, tetapi tidak demikian dalam hal lain. Disini seorang konselor memberikan bantuan bukan hanya kepada orang tua anak yang mengalami temper tantrum tetapi juga terhadap anak yang bersangkutan. Jika diperlukan untuk melakukan proses konseling, karena mereka berbeda dengan teman-temannya yang lain. Anak tersebut sulit bergaul dengan teman-temannya, disini peran kita sebagai konselor dibutuhkan untuk memberikan anak tersebut dukungan bahwa kelebihan yang dimilikinya itu Vol. 3, No. 1, Jan-Jun 2019 128 merupakan anugerah dari Tuhan, yang tidak semua orang memilikinya. Dan memberikan pemahaman bahwa setiap individu itu unik dan memiliki keunikannya masing-masing. Konselor juga wajib mengarahkan anak tersebut untuk memanfaatkan kelebihan yang dimilikinya dengan hal-hal yang positif dan berguna bagi pengembangan potensi nya. Penelitian ini menggunakan metode penelitian kualitatif dengan desain studi kasus. Subyek penelitian ini terdiri dari subyek penelitian yang menjadi informan kunci yang dapat memberikan data yang dibutuhkan oleh peneliti, yakni dua anak usia dini yang berinisial GG dan berinisial AR, adapun subyek sekunder terdiri dari 3 orang kerabat dari GG dan AR. Peneliti menggunakan pendekatan kualitatif dikarenakan data-data yang didapatkan nantinya adalah data kualitatif berupa kata-kata atau tulisan tidak berbentuk angka dan untuk mengetahui serta memahami fenomena secara terinci mendalam dan menyeluruh. Data penelitian dikumpulkan dengan wawancara. 4. Peran Konselor dalam Proses Konseling Selanjutnya hasil penelitian dianalisis dengan pendekatan kualitatif model interaktif sebagaimana diajukan oleh Miles dan Hubermas, yaitu terdiri dari empat hal utama, yakni pengumpulan data, reduksi data, pemaparan data dan penarikan kesimpulan atau verifikasi (Sugiyono, 2009). Hasil penelitian menunjukkan bahwa dari kedua subyek penelitian sama- sama mengalami temper tantrum. Jika subyek GG menghendaki sesuatu yang diinginkan selalu meluapkan emosinya dengan berteriak sangat kencang dan mengguling-gulingkan badannnya dilantai tanpa kontrol, sehingga mengkhawatirkan dapat melukai dirinya sendiri. Sedangkan subyek AR saat menginginkan sesuatu selalu yang diinginkan meluapkan emosinya dengan memukul apa yang ada didepannya, bahkan melempar apa yang ada disekitarnya. Hal ini tentu dapat berdampak pada tingkah laku yang kurang baik dan nantinya akan menjadi rekaman yang buruk ketika seseorang tumbuh kembang menjadi dewasa. Temuan dilapangan munculnya temper tantrum diawali dengan kesalahan cara orangtua berkomunikasi dan bersikap kepada anak. Sehingga secara umum kesalahan yang dilakukan orangtua kepada anak menjadikan anak kecewa, marah dan menangis. Hal ini juga didukung dengan pola asuh yang belum konsisten yang mengakibatkan anak tidak mampu menyesuaikan tugas perkembangannya dengan optimal. Hampir setiap anak mengalami temper tantrum dan umumnya hal ini terjadi pada hampir seluruh periode awal masa kanak-kanak (Hurlock, 1998: 114). Temper tantrum sering terjadi karena anak merasa frustasi dengan keadaannya, sedangkan ia tidak mampu mengungkapkan perasaannya dengan kata-kata atau ekspresi yang diinginkannya (Hasan, 2011: 187) Konseling Edukasi: Journal of Guidance and Counseling 129 David Ari Setyawan Temper tantrum terjadi pada anak yang pemalu, penakut, dan sering cemas terhadap orang asing, Salkind (2002:408),. Keterlambatan dalam perkembangan bahasa, gangguan pendengaran, gangguan system syaraf pusat dapat menyebabkan temper tantrum. Lingkungan anak akan mempengaruhi intensitas dan frekuensi tantrum. Pada anak usia 2-3 tahun, tantrum terjadi karena anak usia tersebut biasanya sudah mulai mengerti banyak hal dari yang didengar, dilihat maupun dialaminya, tetapi kemampuan bahasa atau berbicaranya masih sangat terbatas (Hasan, 2011: 187). Berikut adalah beberapa faktor yang menyebabkan terjadinya temper tantrum (1) Terhalangnya keinginan untuk mendapatkan sesuatu; (2) Ketidakmampuan anak mengungkapkan diri; (3) Tidak terpenuhinya kebutuhan; (4) Pola asuh orang tua.; (5) Anak merasa lelah, lapar atau dalam keadaan sakit yang dapat menyebabkan anak menjadi rewel; (5) Anak sedang stress dan merasa tidak aman, Setiawani (2000:133). 4. Peran Konselor dalam Proses Konseling Beberapa penyebab temper tantrum adalah; (1) Masalah keluarga, keluarga yang tidak harmonis akan membuat anak kehilangan kehangatan keluarga, yang dapat mengganggu kestabilan jiwa anak; (2) Anak yang dimanja akan membuat anak dapat memanfaatkan orang tuanya; (3) Anak yang kurang tidur, kelelahan, memiliki tubuh dan keadaan fisik yang lemah akan membuatnya cepat marah.; (4) Masalah kesehatan, ketika anak mengalami kurang enak badan, ada masalah kesehatan atau tubuh cacat, semua yang mempengaruhi kekuatan pengendalian dirinya, atau hal yang tidak sesuai dengan dirinya, akan mudah membuat anak marah; (5) Masalah makanan, beberapa makanan dapat membuat anak peka atau alergi yang membuat anak menjadi kehilangan kekuatan untuk mengendalikan diri, seperti makanan yang mengandung zat pewarna atau pengawet, dan coklat; (6) Kekecewaan, saat anak menyadari keterbatasan kemampuan dirinya dalam menyatakan keinginannya dan tidak dapat melakukan sesuatu hal, membuat anak mudah marah; (5) Meniru orang dewasa, ketika melihat ada orang dewasa yang tidak dapat menyelesaikan atau menghadapi kesulitan, lalu marah-marah, ditambah di rumah orang tua dan di sekolah konselor juga mudah marah, akan membuat anak meniru mereka menjadi anak yang mudah marah. Aktivitas yang menimbulkan temper tantrum antara lain; (1) Rintangan terhadap gerak yang diinginkan anak, baik rintangan itu berasal dari orang lain atau dari ketidakmampuan diri sendiri, (2) Rintangan terhadap aktivitas yang sudah mulain berjalan, (3) Rintangan terhadap keinginan, rencana, dan niat yang ingin dilakukan anak, Hurlock (2010: 222). M k d t di i lk f kt b b k l i t t t Maka dapat disimpulkan faktor penyebab anak mengalami temper tantrum antara lain: (1) Faktor fisiologis, yaitu lelah, lapar atau sakit; (2) Faktor psikologis, Vol. 3, No. 1, Jan-Jun 2019 130 antara lain anak mengalami kegagalan, dan orangtua yang terlalu menuntut anak sesuai harapan orangtua; (3) Faktor orangtua, yakni pola asuh; (4) Faktor lingkungan, yaitu lingkungan keluarga dan lingkungan luar rumah. Konseling Edukasi: Journal of Guidance and Counseling C. Simpulan Temper tantrum adalah suatu ledakan amarah yang sering terjadi pada anak usia tiga sampai enam tahun yang ditandai dengan tindakan menangis, menjerit-jerit, melempar benda, berguling-guling, memukul dan aktivitas destruktif lainnya. Faktor penyebab anak mengalami temper tantrum antara lain: (1) Faktor fisiologis, yaitu lelah, lapar atau sakit; (2) Faktor psikologis, antara lain anak mengalami kegagalan, dan orangtua yang terlalu menuntut anak sesuai harapan orangtua; (3) Faktor orangtua, yakni pola asuh; (4) Faktor lingkungan, yaitu lingkungan keluarga dan lingkungan luar rumah. Bentuk-bentuk perilaku temper tantrum adalah sebagai berikut: menangis dengan keras, menendang segala sesuatu yang ada di dekatnya, memukul benda, dirinya sendiri, maupun orang lain, membentur- benturkan kepala, melempar-lempar dan merusak barang, menghentak-hentakkan kaki, berteriak- teriak dan menjerit, membanting pintu, merengek, mengancam dan memaki. Pola asuh orang tua sangat mempengaruhi setiap perilaku anak. Model pengasuhan orang tua akan membentuk suatu perilaku atau pengelolaan emosi yang berbeda-beda sesuai apa yang telah diajarkan oleh orang tua. Orang tua merupakan lingkungan pertama bagi anak yang sangat berperan penting dalam setiap perkembangan anak khususnya perkembangan kepribadian dan emosi anak. Sehingga peran konselor dalam membantu orangtua untuk mengatasi permasalahan temper tantrum sangat diperlukan melalui proses konseling yang lebih inten. 131 David Ari Setyawan Daftar Pustaka Dariyo, Agoes. (2007). Psikologi Perkembangan Anak Tiga Tahun Pertama. Bandung: Refika Aditama. Gunarsa, Singgih D. (2008). Psikologi Anak dan Remaja. Jakarta: PT BPK Gunung Mulia. Hayes, Eileen. (2003). Tantrum. Jakarta: Erlangga Hayes, Eileen. (2003). Tantrum. Jakarta: Erlangga Hurlock, E.B. (1998). Psikologi Perkembangan Suatu Pendekatan Sepanjang Rentang Kehidupan. Jakarta: Erlangga. Hagan, Jessica S. (2006). Mendidik Anak Memasuki Usia Prasekolah. Jakarta: PT. Prestasi Pustakaraya. Hasan, Maimunah. (2011). Pendidikan Anak Usia Dini. Jogjakarta: Diva Press. Indraswari, Ayunita. (2012). Perilaku Sosial Pada Kanak-Kanak Awal yang Mengalami Temper Tantrum (Studi Kasus di KB Permata Hati Desa Kebon Agung Kecamatan Kajen, Kabupaten Pekalongan). Skripsi Universitas Negeri Semarang Ismaya, Y. (2010). Pengaruh Penggunaan Timeout Terhadap Penurunan Temper Tantrum Pada Usia Balita. Jurnal. Pekanbaru: PSIK UR. Junita.T, Inneke. (2013). Gambaran Strategi yang Dilakukan Orang Tua dalam Menghadapi Tantrum pada Anak dengan Autism Spektrum Disorder. Jurnal.Pekanbaru. Kartono, Kartini. (1991). Bimbingan Bagi Abak dan Remaja yang Bermasalah. Jakarta: CV. Rajawali. Latipun. (2004). Psikologi Eksperimen Edisi II. Malang: UMM Press. Pratisti, Wiwien Dinar. (2008). Psikologi Anak Usia Dini. Jakarta: PT Indeks. Salkind, Neil J. (2002). Child Development. USA: Macmillan Reference. Santrock, J.W. (2002). Life-Span Development: Perkembangan Masa Hidup (Edisi Kelima). Jakarta: Erlangga. Setiawani, Mary Go. (2000). Menerobos Dunia Anak. Bandung: Yayasan Kalam Hidup. Soetjiningsih, Christiana Hari. (2012). Perkembangan Anak. Jakarta: Prenada Media Group. Sugiyono. (2009). Metodologi Penelitian Kuantitatif, Kualitatif dan R&D. Bandung: Alfabeta. Yusuf, H. Syamsu. 2011. Psikologi Perkembangan Anak dan Remaja. Bandung: Remaja Rosdakarya. Vol. 3, No. 1, Jan-Jun 2019 132
https://openalex.org/W4236590831	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/266A3251341CAA6466DE159B32282E24/S0955603600095209a.pdf/div-class-title-mind-the-vietnam-bike-ride-div.pdf	English	null	Mind – the Vietnam bike ride	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	2,001	cc-by	1,746	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https://doi.org/10.1192/pb.25.1.39-b Published online by Cambridge University Press
https://openalex.org/W3012128794	https://link.springer.com/content/pdf/10.1057/s41260-020-00156-3.pdf	English	null	Factor-based investing in government bond markets: a survey of the current state of research	Journal of asset management	2,020	cc-by	9,618	1 The terms “sovereign” and “government” bonds are used inter- changeably throughout this article. Journal of Asset Management https://doi.org/10.1057/s41260-020-00156-3 Journal of Asset Management https://doi.org/10.1057/s41260-020-00156-3 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Factor investing has become very popular during the last decades, especially with respect to equity markets. After extending Fama–French factors to corporate bond markets, recent research more often concentrates on the government bond space and reveals that there is indeed clear empirical evidence for the existence of significant government bond factors. Voices that state the opposite refer to outdated data samples. By the documentation of rather homogeneous recent empirical evidence, this review underlines the attractiveness of more sophisticated investment approaches, which are well established in equity and even in corporate bond markets, to the segment of government bonds. Keywords Government bonds · Sovereign bonds · Factors · Anomalies · Risk premia · Style investing JEL Classification G11 · G12 · G14 · G15 JEL Classification G11 · G12 · G14 · G15 Demir Bektić1,3 · Britta Hachenberg2 · Dirk Schiereck3 Demir Bektić1,3 · Britta Hachenberg2 · Dirk Schiereck3 © The Author(s) 2021 Introduction (2018, 2019) as well as Israel et al. (2018) show that further well-known factors from equity markets could be translated to corporate bonds. These findings are consistent to early results by Harvey et al. (1994) who show that factors driving bond returns are the same as those driving equity returns. The still fast-growing body of the literature which concen- trates on the predictability of variations in international asset returns has a clear focus on equity markets where more than 300 papers on cross-sectional returns are counted (Crawford et al. 2019; Harvey et al. 2016). Barr and Priestley (2004) argue that it is reasonable to expect equity markets to be less closely integrated than bond markets, largely because differences between bonds in different countries are small. However, Bektić et al. (2019) and Dekker et al. (2019) provide convincing evidence for the explanatory power of the Fama–French factors also in corporate bond markets. Additionally, Houweling and van Zundert (2017), Bektić Compared to these two asset classes, research on govern- ment bond1 return factors was perceived as less attractive because excess returns of long-term government bonds are subject only to interest rate risk in the absence of default risk and cash flow uncertainty (Ilmanen 1995). Besides approaches to explain predictable variation in government bond returns with measures of time-varying risk (Lauterbach 1989; Boudoukh 1993), more technical factors were tested. In this context, Litterman and Scheinkman (1991) support the idea of identifying common factors that affect the returns on US government bonds and related factors. They provide evidence that three attributes of the yield curve—level, steepness and curvature—can explain returns on all fixed- income securities. However, in recent years a number of papers have extended the idea of factor investing from the corporate bond market to the government bond market, and by doing so, they also address the increasing demand for insights in this area as shown in Figs. 1 and 2. 1 International University of Monaco, Deka Investment GmbH and IQ-KAP, Mainzer Landstraße 16, 60325 Frankfurt am Main, Germany * Demir Bektić demir.bektic@gmail.com Introduction 1 International University of Monaco, Deka Investment GmbH and IQ-KAP, Mainzer Landstraße 16, 60325 Frankfurt am Main, Germany 1 International University of Monaco, Deka Investment GmbH and IQ-KAP, Mainzer Landstraße 16, 60325 Frankfurt am Main, Germany 2 Department of Business, Economics and Law, TH Köln University of Applied Sciences, Claudiusstrasse 1, 50678 Cologne, Germany 3 Department of Law and Economics, Technical University of Darmstadt, Hochschulstrasse 1, 64289 Darmstadt, Germany 3 Department of Law and Economics, Technical University of Darmstadt, Hochschulstrasse 1, 64289 Darmstadt, Germany 6789) D. Bektić et al. Fig. 1 Number of articles found via Google Scholar for the period from December 2000 until December 2017 Number of Arcles Cumulative Number of Articles search term: "smart beta" "government bonds" 50 140 120 40 100 30 80 20 60 40 10 20 0 0 Fig. 2 Number of articles found via Google Scholar for the period from December 2000 until December 2017 Number of Articles Cumulave Number of Arcles search term: "factor invesng" "government bonds" 60 50 40 30 20 10 0 140 120 100 80 60 40 20 0 es found he 2000 Number of Arcles Cumulative Number of Articles search term: "smart beta" "government bonds" 50 140 120 40 100 30 80 20 60 40 10 20 0 0 search term: "smart beta" "government bonds" Number of Articles Cumulave Number of Arcles search term: "factor invesng" "government bonds" 60 50 40 30 20 10 0 140 120 100 80 60 40 20 0 These impressive demand patterns are interpreted as a perceived lack of structured overall survey information on the state of research on factor-based investing in government bond markets. We address this research gap and intend to give an overview on factors in government bond markets. These factors are often defined as investments that produce long-term returns using risk factors that are significant and unique, in the sense that they are not subsumed by other tra- ditional risk factors. Such investment strategies are usually applied in a long–short or long-only (smart beta) manner. 2 Traditional approaches in the sovereign bond space primarily included affine term structure models as well as macroeconomic and latent risk factors. Momentum Momentum is probably one of the most popular factors in equity markets. In short, momentum is the tendency for an asset’s recent performance to continue in the short term (Brooks et al. 2018). Hamdan et al. (2016) also discuss that a risk factor is not necessarily a risk premium. A momentum strategy follows the market, and in trend-following strate- gies, there is no additional exposure to a risk during “bad” times. Thus, they argue that momentum is a risk factor and a market anomaly instead of a risk premium. This trend- following definition is also used by Brightman and Shepherd (2016). They note that intuitively this might seem to violate the efficient market hypothesis; however, empirical results show that momentum is a robust strategy even across asset classes. p Brooks and Moskowitz (2017) explore momentum in the cross section across countries using level, slope and cur- vature portfolios, which is a novel approach. In the cross section of government-level returns, momentum is an insig- nificant factor (duration-adjusted). Unlike carry and value, it also does not capture any information from principal components. In the cross section of country slope returns, momentum again is not a significant factor and not related to any principal component. It only becomes significant when principal components and carry are added to the regres- sion, but its coefficient is negative. Principal components can explain only 25.5% and 40.7% of cross-sectional and time series variation of momentum in slope strategies. This drops even further for level strategies, but remains impor- tant for curvature strategies in a time series analysis. When curvature is high, recent returns of intermediate bonds are lower compared to the bonds at the longer end of the curve. This can be observed in the correlation structure of momen- tum which is negatively correlated with the third principal component (related to the curvature). For instance, Asness et al. (2013) show that momen- tum exists in every major asset class. They employ a sim- ple measure of momentum, the past 12-month cumulative raw return not including the most recent month. Their momentum portfolios are constructed as long-only as well as long–short portfolios where securities are weighted according to their signal rank. Additionally, Brightman and Shepherd (2016) use each country’s own historical return in a time series approach and report statistically significant Sharpe ratios and mean returns based on momentum in gov- ernment bond markets. Introduction However, while the most popular applications of these fac- tors are dedicated to the equity market, the extension of risk factors to non-equity markets has been an important trigger for empirical research in the modern asset pricing literature.2 As the underlying asset classes of equity and sover implications for investors seeking to track portfolios versus market capitalization-weighted benchmarks for each asset class, as well. On the one hand, while both equity and fixed- income benchmarks contain a large number of securities, constituents of fixed-income indices are usually changing due to the maturing nature of fixed-income securities. On the other hand, while market capitalization-weighted equity benchmarks enable investors to hold mean variance efficient portfolios, market capitalization-weighted indices in the bond market force investors into the most prolific issuers of debt, which intuitively are associated with elevated levels of risk. This counterintuitive dynamic of market capitalization- weighted indices in bond markets is known as the “bums problem” (Siegel 2003) and, in terms of government bonds, leads to assigning the largest weight to those countries with the largest amounts outstanding in the index. These dynam- ics in fixed-income markets motivate why factor-based strat- egies may significantly and sustainably outperform their market capitalization-weighted peers. For instance, Ilmanen et al. (2019) analyze a century of data across six asset classes and find significant time variation in single-factor returns These factors are often defined as investments that produce long-term returns using risk factors that are significant and unique, in the sense that they are not subsumed by other tra- ditional risk factors. Such investment strategies are usually applied in a long–short or long-only (smart beta) manner. However, while the most popular applications of these fac- tors are dedicated to the equity market, the extension of risk factors to non-equity markets has been an important trigger for empirical research in the modern asset pricing literature.2 As the underlying asset classes of equity and sover- eign bond markets are fundamentally different, so are the Factor‑based investing in government bond markets: a survey of the current state of research result of their opposite sign loadings on liquidity risk as value loads negatively, while momentum loads positively on liquidity. This positive loading on liquidity indeed captures to some extent the variation in momentum returns. 3 See Ludvigson and Ng (2009), who provide a summary of macro- factors in bond risk premia. Introduction and variances, providing support for models of dynamic return premia and, at the same time, rejecting models of macroeconomic risks, which were perceived to have predic- tive power in the past.3i Similar to Asness et al. (2013), Brooks et al. (2018) define momentum as each country’s own 12-month average excess return. They show that the long-only tercile portfolios exhibit positive results, while the long–short alpha is not statistically significant. Additionally, these authors construct a multifactor (COMBO) portfolio and show that momentum has a negative correlation with value, carry and quality for government bonds (− 0.32, − 0.42 and − 0.22). Therefore, investors can exploit significant diversification benefits when combining factors rather than employing just one sin- gle factor in a portfolio (COMBO portfolio alpha = 2.8%, t-stat = 3.89). The negative correlation of − 0.18 between momentum and value is also observed in Brightman and Shepherd (2016). The focus of our article is on the five best documented and most cited factors, namely momentum, value, quality (defensive), carry and liquidity. We compare and analyze different factor definitions as well as data sets which are used to conduct the corresponding research. Finally, we show how investors should pursue factor strategies to generate signifi- cantly higher risk-adjusted returns as well as to further diver- sify their portfolios. Momentum Brooks and Moskowitz (2017) also examine the perfor- mance of factors using traded bond data from J.P. Morgan across level, slope and curvature portfolios. In level portfo- lios, momentum delivers 5.02% alpha (t-stat = 2.46) when the principal components are included in the regression. Additionally, momentum is negatively correlated with the first principal component as well as value across level, slope and curvature portfolios (− 0.36, − 0.13 and − 0.51, respec- tively). These results indeed indicate that momentum is a unique factor in level portfolios and is not captured by other known factors.fi Another important finding from Asness et al. (2013) is the negative correlation of value and momentum. This creates a significant diversification effect on their 50/50 portfolio consisting of value and momentum tilts and leads to stronger results. The negative correlation also can be observed through the first principle component and the liquidity risk’s loadings on value and momentum. They argue that the nega- tive relation between value and momentum might be the Since principle components are not sufficient to explain the variation in momentum, further unspanned factors are examined as well. For instance, momentum has a nega- tive coefficient for macroeconomic variables (growth and D. Bektić et al. y g Factor definition Data period Data source # Countries Countries Article 1-year past return on the bond differences in past year returns between 10- and 2-year bonds, duration-adjusted difference in past year returns between 5-year bonds and the average of the 10- and 2-year bonds, duration- adjusted December 1971 to March 2016 Wright (2011)—till May 2009, Reuters DSFI afterward 7 Australia, Germany, Canada, Japan, Sweden, the UK and the USA Brooks and Moskowitz (2017) 12-month futures return January 1989 to June 2016 Datastream, Bloomberg and Global Financial Data 8 Australia, Canada, Germany, France, the UK, Japan, the USA and Italy Brightman and Shepherd (2016) The return over the past 12 months skipping the most recent month January 1982 to July 2011 Bloomberg and Morgan Markets 10 Australia, Canada, Denmark, Ger- many, Japan, Norway, Sweden, Switzerland, the UK and the USA Asness et al. (2013) The prior 12-month excess return January 1996 to June 2017 J.P. Morgan Government Bond Index 13 Australia, Belgium, Canada, Denmark, France, Germany, Italy, Japan, the Netherlands, Spain, Sweden, the UK and the USA Brooks et al. (2018) inflation), although they are not significant. 4 The term spread is also often used as a sorting variable in carry fac- tor definitions. Momentum The relation to the Cochrane and Piazessi (2005) factor is tested only by using level time-varying portfolios where momentum is negative and significant. Brooks et al. (2018) as well study the exposures to macroeconomic factors (growth, inflation, real yield, volatility and illiquidity) but only for long–short portfolios. However, we can conclude that government bond portfolios based on factors have less sensitivity to shocks in macroeconomic variables than common sovereign bond indices. Factor definition Data period Data source # Countries Countries Article 1-year past return on the bond differences in past year returns between 10- and 2-year bonds, duration-adjusted difference in past year returns between 5-year bonds and the average of the 10- and 2-year bonds, duration- adjusted December 1971 to March 2016 Wright (2011)—till May 2009, Reuters DSFI afterward 7 Australia, Germany, Canada, Japan, Sweden, the UK and the USA Brooks and Moskowitz (2017) 12-month futures return January 1989 to June 2016 Datastream, Bloomberg and Global Financial Data 8 Australia, Canada, Germany, France, the UK, Japan, the USA and Italy Brightman and Shepherd (2016) The return over the past 12 months skipping the most recent month January 1982 to July 2011 Bloomberg and Morgan Markets 10 Australia, Canada, Denmark, Ger- many, Japan, Norway, Sweden, Switzerland, the UK and the USA Asness et al. (2013) The prior 12-month excess return January 1996 to June 2017 J.P. Morgan Government Bond Index 13 Australia, Belgium, Canada, Denmark, France, Germany, Italy, Japan, the Netherlands, Spain, Sweden, the UK and the USA Brooks et al. (2018) Finally, Brooks and Moskowitz (2017) compare govern- ment bond factors with factors from other asset classes. For instance, level momentum is positively correlated across asset classes and the correlation is significant with momen- tum in currency and commodity markets.i These findings indicate that momentum can be an impor- tant factor across asset classes as it has a significant positive loading on equity momentum as well which is also con- firmed by the study of Brightman and Shepherd (2016). Fur- thermore, Durham (2015) shows that momentum produces meaningfully alphas in the US Treasury market. Table 1 summarizes the studies discussed above. # Countries Countries Value The results show that nearly half of the return on the term spread portfolio can be captured by carry, and the term spread value is positively correlated with carry.i They also apply these two definitions to global govern- ment bonds and construct long–short portfolios (a long position in the top third and a short position in the bottom third of the universe). The global term spread portfolio and the global real yield portfolio produce 1.1% return (Sharpe ratio = 0.49) and 0.5% return (Sharpe ratio = 0.26), respec- tively. The global term spread and real yield bond value port- folios are negatively correlated (− 0.33). i To better understand the power of value in explaining the returns of government bonds, other macrofactors such as growth in industrial production and inflation for each coun- try are added to the regressions of level, slope and curvature portfolios. While macrofactors do not capture any informa- tion in slope and curvature portfolios, interesting results are observed in level portfolios. Here, value remains significant when the returns are controlled for macrofactors and prin- cipal components, but the previously significant inflation factor is now subsumed by value.5 In order to explain the variation observed in value, Asness et al. (2013) investigate common macroeconomic and liquid- ity risk factors. The macroeconomic factors are regressed on a portfolio consisting of all the non-stock assets; hence, a more reliable variation effect may be captured when testing on global government bonds only. In general, they also find that global funding liquidity shocks have negative impact on value returns. This may be partly due to the price pres- sure the flight-to-liquidity trades create when a shock occurs. However, the exposure of value returns in global government bonds to liquidity risks is weak. Finally, Brooks and Moskowitz (2017) show that the value factor in level portfolios is positively related to and correlated with value strategies in other asset classes like equities and currencies. The co-movement of value strate- gies is also documented by Asness et al. (2013). Addition- ally, Brightman and Shepherd (2016) and Mazzoleni and Kunz (2018) also find supporting evidence for this finding by showing that value in bonds is correlated with value in currencies and equities (0.20 and 0.25). Therefore, value seems to be a significant and important factor across asset classes and as a result should be an integral component of a government bond portfolio as well. Value Value is a widely known equity market factor and one of the most studied ones across asset classes. In short, it is the tendency for relatively cheap assets to outperform relatively expensive assets (Brooks et al. 2018). Asness et al. (2013) test value across different asset classes and employ a sim- ple and standard definition for equity markets (asset’s book value relative to its current market value). Furthermore, a similar approach is extended to government bonds using the 5-year change in the yields of 10-year bonds. This definition is similar to the negative of the past 5-year return which is shown to have a high correlation with the value factor using book-to-market equity ratios (see Fama and French, 1996). They also analyze value using alternative measures, such as real bond yield and term spread.4f Mazzoleni and Kunz (2018) use two different measures of value (real bond yield as defined by Asness et al. (2013) and the term spread) and create two portfolios accordingly. In the period between March 1989 and October 2017, the term spread portfolio produces a 3.4% return with a 0.47 Sharpe ratio, while the real yield portfolio has a mean return of − 0.7%. It should be noted that the returns used to calculate these results are based on the 10-year Treasury bonds and the US cash rate. They further investigate the positive return Factor definition Factor‑based investing in government bond markets: a survey of the current state of research Brooks and Moskowitz (2017) find that the first principal component (PC1), which is highly related to “level” of the yield curve, becomes insignificant in the presence of value (t-stat drops 2.63 to 1.05). Value also loads positively on PC1 when level-neutral long–short portfolios are employed. The regression is repeated for slope and curvature portfo- lios across countries. Finally, a long–short value portfolio consisting of level, slope and curvature portfolios, weighted according to the inverse of volatility, exhibits a statistically significant alpha with a Sharpe ratio of 0.95. in their term spread portfolio by dividing the total return into two components; carry return and spot return using the Koijen et al. (2018) carry definition. 5 The Cochrane and Piazessi (2005) factor loses its explanatory power as well when value and principal components are present in the regression. Value Table 2 summarizes the above-discussed studies. Brooks et al. (2018) employ a value measure in gov- ernment bonds, where the real bond yield is calculated by comparing nominal yields versus maturity-matched infla- tion expectations. The global government bond data and the portfolio construction method (tercile long-only portfolios and long–short duration-neutral portfolios) are the same as previously described in the momentum section above. When their value definition is applied across countries, the return pattern shows that countries with relatively high yields are preferred. Additionally, the Sharpe ratio improves from the low value to the high-value tercile portfolio (0.87 to 1.07). For the long–short portfolio, value produces a significant and positive alpha (2.02%) with a Sharpe ratio of 0.65. Quality (defensive) Table 2 Summary of the value factor literature in government bond markets Factor definition Data period Data source # Countries Countries Article Real bond yield minus the nominal yield on the bond minus a matu- rity-matched CPI inflation forecast from Consensus Economics December 1971 to March 2016 Wright (2011)—till May 2009, Reuters DSFI afterward 7 Australia, Germany, Canada, Japan, Sweden, the UK and the USA Brooks and Moskowitz (2017) Real bond yield January 1989 to June 2016 Datastream, Bloomberg and Global Financial Data 8 Australia, Canada, Germany, France, the UK, Japan, the USA and Italy Brightman and Shepherd (2016) Term spread difference between the yields of the 10-year US Treasury bond and the 3-month US Treas- ury bill March 1989 to October 2017 Datastream and Bloomberg 8 Australia, Canada, Germany, France, the UK, Japan, the USA and Italy Mazzoleni and Kunz (2018) The 5-year change in the yields of 10-year bonds (negative of the past 5-year return) January 1982 to July 2011 Bloomberg and Morgan Markets, inflation forecasts from Consen- sus Economics 10 Australia, Canada, Denmark, Ger- many, Japan, Norway, Sweden, Switzerland, the UK and the USA Asness et al. (2013) Real bond yield (nominal yields vs. maturity-matched inflation expectations) January 1996 to June 2017 J.P. Morgan Government Bond Index 13 Australia, Belgium, Canada, Denmark, France, Germany, Italy, Japan, the Netherlands, Spain, Sweden, the UK and the USA Brooks et al. (2018) As already stated in the introduction, a meaningful risk factor should gain a return that cannot be obtained by expo- sure to other factors. Hence, Brooks et al. (2018) run regres- sions where the long–short defensive portfolio returns are regressed on traditional market risk premia as well as equity factor premia. However, the defensive factor produces sta- tistically significant alphas even when controlled for these additional factor exposures. Another adoption of the defensive strategy is the betting against beta (BAB) approach introduced by Frazzini and Pedersen (2014). This strategy is based on a similar hypoth- esis that high-risk assets offer lower returns compared to low-risk assets when adjusted for risk. The strategy first sorts assets ranked by their beta value and then short-sells the high-beta assets while taking a long position on the low-beta assets. However, these low-risk assets often require lever- age as the portfolio maintains market neutrality (see Asness et al. 2015). Frazzini and Pedersen (2014) also extend their BAB strat- egy to government bonds. Quality (defensive) The economic intuition behind a defensive strategy can be summarized as the tendency of low-risk assets to deliver higher risk-adjusted returns compared to assets with higher risk and consists of buying low-beta assets while selling high-beta assets (Brooks et al. 2018).f Brooks and Moskowitz (2017) analyze the relationship between value and common bond market factors. They also employ the real bond yield as a measure of value and their strategy performs when the level of yields reverts to expected inflation (which is the maturity-matched CPI infla- tion forecast). The excess return of level portfolios across countries is regressed on value and the coefficient is 0.53 (t-stat = 3.56). The value factor even increases its signifi- cance when carry and momentum are added to the regres- sion (t-stat = 3.72). This finding seems to strengthen the hypothesis that value and other known factors are orthogonal to each other, harvesting different sources of return. Addi- tionally, value remains significant when principal compo- nents are included in the regression as well. Brooks et al. (2018) employ effective duration as their low-risk measure and apply the defensive strategy across maturities unlike other style factors which are investigated across countries. Since the defensive strategy uses duration as the fundamental measure of the quality factor, it pre- fers shorter maturities across all countries. They construct D. Bektić et al. equally weighted portfolios containing the short maturity bucket across all countries in the top tercile and long matu- rity buckets in the bottom tercile. The top minus bottom duration-neutral tercile portfolio goes long on short maturi- ties while taking a short position on higher maturity securi- ties. The resulting mean returns of the long-only defensive strategy are positive and decrease monotonically from the top to the bottom tercile portfolio. The long–short defen- sive portfolio produces a significant alpha as well (1.3%, t-stat = 2.18). Furthermore, the factor also exhibits a low positive correlation with value and carry (0.21 and 0.15) and is negatively correlated with momentum (− 0.22). Thus, it amplifies the diversification benefits when constructing multifactor portfolios. Carry bond portfolios are regressed on the corresponding bond market returns, the global value and momentum bond fac- tors, as well as the time series bond momentum as defined by Moskowitz et al. (2012), to understand what is actually behind the variation in carry returns. The resulting alphas are positive and statistically significant. Hence, carry strate- gies deliver excess returns in each country without a signifi- cant exposure to the corresponding bond market. The alpha remains significantly positive when tested against cross- sectional and time series momentum. Further tests analyze the relation between global carry returns and downside risk where the returns are positively exposed to global liquidity shocks and negatively exposed to volatility risk except for the US Treasuries where this relationship is the other way round. However, the alphas of the carry strategy in global government bonds and Treasuries are statistically signifi- cant. Therefore, to explain the cross section of government bond carry strategy returns, risk premia and exposure to the above-mentioned risk factors are not a sufficient explanation. Similar to Koijen et al. (2018), Brooks et al. (2018) define carry as the tendency for higher-yielding assets to outper- form lower-yielding assets and summarize it as the reach- ing for yield. Brooks et al. (2018) use a sample of govern- ment bonds consisting of all the bonds from the J.P. Morgan Government Bond Index (13 markets in total) and divide them into three maturity buckets (1–5-year, 5–10-year and 10–30-year bucket). Then, they test carry by taking an equal duration-weighted average across the three maturity buckets within each country. The sample period for both long-only tercile portfolios and long–short portfolios is from January 1996 to June 2017. Finally, Brooks et al. (2018), Koijen et al. (2018) and Brightman and Shepherd (2016) report similar Sharpe ratios. Koijen et al. (2018) define bond carry as the return on a government bond when the yield curve does not change during the holding period. The main reason for its popular- ity in fixed income is that it is quick and easy to calculate. The predominantly employed definition specifies carry as an asset’s future (or synthetic) return, assuming that interest rates remain unchanged over time. Hamdan et al. (2016) state that “the underlying idea of a carry strategy is to cap- ture a spread or a return by betting that the underlying risk will not occur or that market conditions will stay the same” (p. Carry 21).i Koijen et al. (2018) adapt this definition for government bonds by relating carry to the slope of the yield curve and by adding the “roll down” component that captures the price change which occurs as the bond moves along the yield curve as time passes. Thus, the carry strategy in general is defined as taking a long position on high carry assets and a short position on low carry assets. Furthermore, Koijen et al. (2018) note that the application in global government bond markets is even easier when bond futures are employed to implement the strategy. However, liquid bond futures con- tracts are only available in a small number of countries. Thus, they use synthetic futures prices to test the application of carry in the cross section of global government bonds. Based on their definition, it is easy to see the connection between bond carry and forward rates. Additionally, when modified duration is included, carry can be separated into two effects: the bond’s yield spreads to the risk-free rate and the price appreciation of the bond as it moves on the yield curve. To account for different duration exposures between global government bonds, they use a duration-adjusted posi- tion sizing. fi Similar to Koijen et al. (2018), Brooks et al. (2018) define carry as the tendency for higher-yielding assets to outper- form lower-yielding assets and summarize it as the reach- ing for yield. Brooks et al. (2018) use a sample of govern- ment bonds consisting of all the bonds from the J.P. Morgan Government Bond Index (13 markets in total) and divide them into three maturity buckets (1–5-year, 5–10-year and 10–30-year bucket). Then, they test carry by taking an equal duration-weighted average across the three maturity buckets within each country. The sample period for both long-only tercile portfolios and long–short portfolios is from January 1996 to June 2017. Finally, Brooks et al. (2018), Koijen et al. (2018) and Brightman and Shepherd (2016) report similar Sharpe ratios. Brooks et al. (2018) also test whether the premium cap- tured by a carry strategy is unique to factor investing or whether it can be earned through investing in well-known risk premia such as (1) the credit risk premium, (2) the equity risk premium and (3) the bond term premium as well as dynamic factors such as size, value and momen- tum. Quality (defensive) However, the alphas of the carry strategy in global government bonds and Treasuries are statistically signifi- cant. Therefore, to explain the cross section of government bond carry strategy returns, risk premia and exposure to the above-mentioned risk factors are not a sufficient explanation.i Quality (defensive) The data are obtained from the Center for Research in Security Prices (CRSP) US Treasury Database and ranges from January 1952 to March 2012. To calculate beta, they use equally weighted portfolios consist- ing of all the bonds in the index. Then, to create the BAB bond factor, they go long the shorter maturity bonds (as lev- eraged low-beta bonds) and short the longer term bonds (as de-leveraged high-beta bonds). The obtained BAB portfo- lio delivers a significant Sharpe ratio of 0.81 (t-stat = 6.26), and the results remain almost unchanged when time-varying market exposure is included in the regression analysis. Finally, Frazzini and Pedersen (2014) note that BAB bond portfolios may be subject to funding constraints as investors would need a high level of leverage to implement such a strategy in practice. Hence, this trade depends on the indi- vidual leverage constraints of each investor. However, Durham (2018) shows that investors should fol- low such a strategy cautiously as it seems to be associated with substantial systematic risk. Table 3 summarizes the studies discussed above. Factor‑based investing in government bond markets: a survey of the current state of research Table 3 Summary of the quality factor literature in government bond markets Factor definition Data period Data source # Countries Countries Article Effective duration January 1996 to June 2017 J.P. Morgan Government Bond Index 13 Australia, Belgium, Canada, Denmark, France, Germany, Italy, Japan, the Netherlands, Spain, Sweden, the UK and the USA Brooks and Moskowitz (2017) Beta value to GDP-weighted index January 1952 to March 2012 CRSP US Treasury Data- base and Kenneth French website 1 the USA Frazzini and Pedersen (2014) bond portfolios are regressed on the corresponding bond market returns, the global value and momentum bond fac- tors, as well as the time series bond momentum as defined by Moskowitz et al. (2012), to understand what is actually behind the variation in carry returns. The resulting alphas are positive and statistically significant. Hence, carry strate- gies deliver excess returns in each country without a signifi- cant exposure to the corresponding bond market. The alpha remains significantly positive when tested against cross- sectional and time series momentum. Further tests analyze the relation between global carry returns and downside risk where the returns are positively exposed to global liquidity shocks and negatively exposed to volatility risk except for the US Treasuries where this relationship is the other way round. Liquidity Government bond yields are heavily affected by the indebt- edness of the particular government; thus, a liquidity premia in government bond markets should be observed. Bernoth et al. (2004) argue that the liquidity component of the yield spreads highlights a lack of financial market integration. They disintegrate the equation of bond yield difference between two bonds into three segments where one of them is the liquidity risk premium. Intuitively, the liquidity risk premium increases when the domes- tic bond market decreases in liquidity. Besides the basic model, Bernoth et al. (2004) test the liquidity variable in a reduced form model as well for 13 European Union (EU) government bond markets between 1991 and 2002.7 In this model, they use the positive correlation between liquid- ity and the total supply of debt (see Gravelle 1999) and assume that the liquidity premium should be proportional to the ratio of the debt issued by a government to the total debt of the European Union issued in Deutsche Mark or euro and US dollars. They show that the liquidity risk pre- mium is larger for USD denominated bonds. Finally, they observe that the effect of EU membership on liquidity is positive as the coefficient of liquidity turns from negative to positive in all regressions when a country becomes a member of the EU. This finding indicates that with EU membership, financial market integration across countries y p gy Brooks and Moskowitz (2017) find that carry subsumes the explanatory power of the slope and the curvature of the yield curve. They investigate both the time series and the cross-sectional performance of international government bonds’ returns in the level, slope and curvature of the yield curve. The level and slope portfolios are built similar to Koijen et al. (2018), using 10-year bond yields and the dif- ference of 10- and 2-year bond yields, respectively. They also include the “butterfly” portfolio where they use the dif- ference between the 5- and an average of the 2- and 10-year bonds. The results for level portfolios in the cross section show that carry is mostly related to the second principal component (PC2). Furthermore, the univariate forecasting regression results show that carry captures significant and positive risk premia in the cross section of government bond- level returns (0.25%; t-stat = 2.11). In slope portfolios, carry is adjusted for duration to make the results more reliable. Carry They document only small exposure to both the credit The data set of Koijen et al. (2018) includes 10 countries starting from November 1983 till September 2012, and the returns are calculated by using the 10-year minus 2-year slope returns in each of the 10 markets. Additionally, they also analyze the 10-year global yields based on the level of the yield curve. Then, the carry returns of global government D. Bektić et al. one-year-ahead forecasts of inflation and industrial produc- tion growth are included in the asset pricing tests.6 risk premium and the bond term premium, and a small but negative exposure to size. However, the R2 of the regres- sion is only 8% which indicates that these risk premia can- not explain the return variability in government bonds. The authors also investigate the sensitivities of long–short government bond portfolios to shocks in macroeconomic and financial conditions. They use quarterly data of growth, inflation, real yield, volatility and illiquidity between Janu- ary 1997 and June 2017. However, the patterns for govern- ment bond carry portfolios are significantly more stable compared to the market as they show little sensitivity to macroeconomic or financial market shocks. Brooks and Moskowitz (2017) also show that both the carry returns of the time variation and the cross section of the level portfolios are positively related to carry returns of equity indices, currencies and global bond indices. Addi- tionally, Brightman and Shepherd (2016) also document a positive correlation of bond carry with equity and currency carry, and a negative correlation with commodity carry. Lastly, Hamdan et al. (2016) state that a carry strategy is betting that the forward price is not a true estimator of the spot price and it goes against the financial theory of rational expectations. Therefore, both Brooks and Moskowitz (2017) and Hamdan et al. (2016) state that carry strategies become extremely risky during market distress mostly due to their higher probability of drawdowns. Table 4 summarizes the above-discussed studies. i Beekhuizen et al. (2019) improve the betting against beta (BAB) results of Frazzini and Pedersen (2014) by using a duration-neutral portfolio instead of a beta-neutral portfolio. An interesting result is that curve carry still performs with a significant alpha when BAB is added as an explanatory variable in the regression although it loads heavily on BAB. Carry The authors believe that this is the result of both strategies having a preference for lower-maturity buckets in times of lower funding rates. However, the reverse regression shows that BAB does not add value beyond curve carry, claiming curve carry a superior strategy.i 6 Interestingly, when carry is included in the regression of the time series-level portfolios on the country Cochrane and Piazessi (2005) factor, this factor is not significant anymore. 7 Until 1998, they use bonds issued in Deutsche Mark, afterward Euro or USD. 6 Interestingly, when carry is included in the regression of the time series-level portfolios on the country Cochrane and Piazessi (2005) factor, this factor is not significant anymore. i 7 Until 1998, they use bonds issued in Deutsche Mark, afterward Euro or USD. Liquidity However, carry still produces significantly positive returns and becomes a stronger return predictor for slope portfolios compared to level portfolios. Similar results are obtained for the butterfly returns across countries for carry as it generates a significant and positive alpha. Additionally, the authors also examine the time variation in expected returns of the level, slope and butterfly portfolios of each country and the results confirm the findings from the cross-sectional analy- sis. Finally, the results show that carry continues to exhibit a significant positive alpha even when macrofactors like Factor‑based investing in government bond markets: a survey of the current state of research Table 4 Summary of the carry factor literature in government bond markets Factor definition Data period Data source # Countries Countries Article The forward rate (slope plus the roll down component) slope of the yield curve long the 10-year bond and short the 2-year bond (sizing is adjusted based on duration) November 1983 to September 2012 Wright (2011)—till June 2009, Bloomberg afterward 10 The USA, Australia, Canada, Germany, the UK, Japan, New Zealand, Norway, Sweden and Switzerland Koijen et al. (2018) Term spread (10-year yield minus the local 3-month rate) differ- ence in 10-year yields relative to the short rate between two bonds, adjusted for duration difference between 5-year yields and the average of the 10- and 2-year bond yields relative to the short rate, duration-adjusted December 1971 to March 2016 Wright (2011)—till May 2009, Reuters DSFI afterward 7 Australia, Germany, Canada, Japan, Sweden, the UK and the USA Brooks and Moskowitz (2017) Slope of the yield curve long the 10-year bond and short the 2-year bond (sizing is adjusted based on duration) January 1989 to June 2016 Datastream, Bloomberg and Global Financial Data 8 Australia, Canada, Germany, France, the UK, Japan, the USA and Italy Brightman and Shepherd (2016) Slope of the term structure plus the roll down component long the shorter maturities and short the longer maturities (funding rates: eurocurrency and T-bill rate) 1985 to 2014 Datastream and J.P. Morgan 10 Australia, Canada, Denmark, Ger- many, Japan, Norway, Sweden, Switzerland, the UK and the UK Beekhuizen et al. (2019) The term spread (difference between the bond’s nominal yield and the local short-term yield) January 1996 to June 2017 J.P. Morgan Government Bond Index 13 Australia, Belgium, Canada, Den- mark, France, Germany, Italy, Japan, the Netherlands, Spain, Sweden, the UK and the USA Brooks et al. Conclusion The concept of factor-based investing in the fixed-income space is still relatively less mature compared to equities, despite the obvious importance and relevance of the subject, and can for certain no longer be denied by looking at cur- rent figures. Especially in times of possible rising rates in a low-yield environment, new approaches to fixed-income investing have sparked investors’ interest. In this review, we therefore clearly state that factor investing in sovereign bond markets represents a new paradigm for a holistic portfolio construction process. Boudoukh et al. (2016) show that if a bond’s liquidity increases, the price of that bond decreases. They analyze the liquidity effect in 10 sovereign bond markets (devel- oped countries) over a 17-year period by comparing newly issued bonds to the most recent previously issued bonds. The traditional market capitalization-weighted approach has often been criticized for its lack of diversification, especially in bond markets. It is questionable at best if fixed-income indices, in the way they are constructed, are representing what they are supposed to do as the level of indebtedness is not necessarily representative for the mar- ket as a whole. Therefore, factors like momentum, value, quality, carry and liquidity should not be neglected when constructing fixed-income portfolios as we observe strong support for the existence of significant factors in global gov- ernment bond markets. Moreover, based on a review of the literature, all examined factor strategies generate a signifi- cant alpha. Intuitively, during times of market turmoil, we would expect investors to prefer more liquid assets and the spread between newly issued government bonds and the previ- ously issued ones to widen. However, their results show that in “bad” times, the newly issued spread actually tight- ens and becomes negative in those countries with rela- tively lower credit quality. Ejsing et al. (2012) investigate the liquidity premium as well and how it is connected with the flight-to-quality phenomenon, especially during the 2008 financial crisis and subsequent sovereign debt crisis. In contrast to other studies, they focus solely on the highly rated and liquid France and Germany government bonds and they measure the liquidity premia as the difference in the term structures of government guaranteed agency bonds over government bonds. Liquidity (2018) Table 4 Summary of the carry factor literature in government bond marketsi D. Bektić et al. 2006 to October 2011) to calculate the corresponding yield curves. increases and members therefore exhibit a lower-liquidity- risk premium.f The increase in liquidity of German/French government and agency bonds is very clear during the 2008/09 financial crisis as well as during the euro area debt crisis. In their model-based framework, they take the liquidity factor as a latent factor of their state–space model. The difference between the two models comes from assuming that the pric- ing of liquidity can also affect agency yields. The estima- tion results show that the liquidity premium in agency yields can explain 30% of the variation in the agency–government yield spread. The results also show that in times of crises, both German and French yields suffered due to an increased demand for highly rated assets. Therefore, this paper also presents evidence on the flight-to-quality effect and shows why liquidity plays an important role in investors’ portfolios. Table 5 summarizes the studies discussed above. Favero et al. (2010) investigate the liquidity effect and how it is priced in the EU area as well. The popularity of the EU area in academia for researchers concerning liquid- ity comes from the elimination of exchange rate risk in bonds and the bonds from different countries’ not being a substitute for each other as they still show some variation. This variation indicates that different sovereign countries in the EU have different risk profiles. fi Favero et al. (2010) develop an asset pricing model with an endogenous liquidity premium variable. The model assumes that trading costs and the availability of other investments in the market affect the liquidity premium. They believe that the liquidity premium decreases when the aggregate market risk increases. This assumption con- tradicts with other studies in the literature supporting the notion that an increase in volatility increases the liquid- ity premium as investors usually want to liquidate their asset in times of market turmoil (flight-to-liquidity). They argue that if the illiquidity and the volatility increase at the same time, asset prices would increase, while the positive change in their return would decrease. The authors con- clude that this effect comes from the interaction between liquidity and aggregate market risk. Liquidity Hence, the liquidity risk premium exists in government bond markets and the yield differentials depend on the interaction between the liquidity variable and the exposure to aggregate risk. Conclusion (2010) Newly issued bond spread (the spread between the hypothetical bond, based on the previous issue curve, and the newly issued bond) January 1998 to December 2015 Data Scope Fixed-Income (DSFI) from Thompson Reuters and Bloomberg 10 Belgium, Canada, France, Italy, Germany, Japan, the Netherlands, Spain, the UK and the USA Brooks and Moskowitz (2017) Model-free indicators of liquidity in a state–space model January 2006 to October 2011 The German Kreditanstalt für Wiederaufbau (KfW) and the French Caisse d’Amortissement de la Dette Sociale (CADES), the German and French governments 2 France and Germany Ejsing et al. (2012) The main criticism of factor outperformance versus conventional benchmarks points at a higher risk exposure to duration and carry trade risk. However, as shown in our literature review, the latest studies control for these exposures and document that the results remain almost unchanged. Additionally, when analyzing alphas of gov- ernment bond portfolios (or funds) one should also take into account the possibility of omitted risk factors as well as considering investable portfolios. While a theoretical long–short portfolio usually leads to higher risk-adjusted returns, implementing long–short sovereign bond portfo- lios is complex and non-practical due to operational diffi- culties and high transaction costs associated with shorting cash bonds, especially for illiquid bonds. Finally, as already discussed, our examined factors are also employed in other asset classes as well. There- fore, for future research, the adoption of a unified asset pricing model that can be applied across asset classes may be worth considering. Additionally, future research should also examine further significant factors that might impact the performance of government bond markets as this would probably lead to greater financial literacy in this rather underdeveloped field and hence larger investor attention. Table 5 Summary of the liquidity factor literature in government bond markets Acknowledgements Views expressed in this paper are those of the authors and do not necessarily reflect those of Deka Investment or its employees. We acknowledge the outstanding research assistance of Gurur Akbas, and we are very grateful for the comments made by Michael Wegener. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Conclusion The data set includes daily bond prices of the Ger- man Kreditanstalt für Wiederaufbau (KfW) and the French Caisse d’Amortissement de la Dette Sociale (CADES), as well as the German and French government bonds (obtained from Bloomberg for the period from January While the corresponding positive returns can be cap- tured through investing in each factor individually, employing multifactor portfolios produces more persistent and even higher risk-adjusted returns. This is mainly due to the low correlation between factors as well as due to different market behavior of each factor across the busi- ness cycle. Therefore, multifactor portfolios provide bet- ter and a more efficient diversification. However, not only the implemented strategy and factor definition preferences Factor‑based investing in government bond markets: a survey of the current state of research but also the specific implementation design like invest- ment universe as well as rebalancing frequency, transac- tion costs, weighting scheme and definition of portfolio configuration has a significant impact on performance and explains why two portfolios (or funds) based on seem- ingly identical factors may perform differently. Addition- ally, from our review of the literature, it is not always clear whether currency risk is hedged or left unhedged. As currency moves and hence volatility can be substan- tial, especially in the short term, significant differences in performance between hedged and unhedged government bond portfolios can occur. Table 5 Summary of the liquidity factor literature in government bond markets Factor definition Data period Data source # Countries Countries Article The ratio of the total debt of the issuer country to the total debt of the EU issued in DM/euro or US$ January 1991 to January 2002 Capital DATA Bondware and Ameco database 13 Austria, Belgium, Denmark, Fin- land, France, Germany, Greece, Italy, Ireland, Portugal, Spain, Sweden and the UK Bernoth et al. (2004) 5-day moving average of the bid– ask spread January 2002 to December 2003 Euro MTS Group’s European Benchmark Market trading platform 9 Austria, Belgium, Finland, France, Germany, Italy, the Netherlands, Portugal and Spain Favero et al. References Asness, C., A. Ilmanen, R. Israel, and T. Moskowitz. 2015. Invest- ing with style. Journal of Investment Management 13: 27–63. Harvey, C., B. Solnik, and G. Zhu. 1994. What determines expected international asset returns? 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Account- ing-based anomalies in the bond Market. Financial Analysts Journal 75: 105–124. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Dekker, L., P. Houweling, and F. Muskens. 2019. Factor investing in emerging market credits. Working paper, Tilburg University (November). https://papers.ssrn.com/sol3/papers.cfm?abstract_ id=3457127. Accessed Dec 2019. Demir Bektic graduated in business informatics from the University of Mannheim, Germany, in 2010. He received the Ph.D. degree in finance from the Technical University of Darmstadt in 2018. Since 2018, he has been an Assistant Professor in the same University and since 2019 an Adjunct Professor at the International University of Monaco as well. 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Hamdan, R., F. Pavlowsky, T. Roncalli, and B. Zheng. 2016. A primer on alternative risk premia. Working paper, Lyxor Asset Manage- ment (June). Business School. In 2008 he got an appointment for the Chair of Cor- porate Finance at Technical University of Darmstadt. Dirk Schiereck is member of the supervisory board of BayernInvest and Creditshelf AG. He focuses his research beyond asset management on sustainable financial solutions. References Grothe, and O. Grothe. 2012. Liquidity and credit risk premia in government bond yields. Working Paper, ECB Work- ing Paper 1440 (July). Fama, E., and K. French. 1996. Multifactor explanations of asset pricing anomalies. Journal of Finance 51: 55–84. Favero, C., M. Pagano, and E.-L. von Thadden. 2010. How does liquidity affect government bond yields. Journal of Financial and Quantitative Analysis 45: 107–134. Britta Hachenberg received her German diploma from the University of Applied Sciences, Cologne, Germany, in 2000. In the same year, she joined the investment bank Goldman Sachs and held various roles in Frankfurt and London as an Executive Director until she left the bank in 2016. From 2016 to 2018, she finalized her Ph.D. at the chair of corporate finance at the Technical University of Darmstadt. In 2018, she started as an Assistant Professor at University of Technology, Arts, Sciences, Cologne, being appointed to Professor of Finance in 2019. Hachenberg is a member of the advisory board of Koelner Boersen- verein e.V. and focuses her research on capital markets and banking regulation. Frazzini, A., and L.H. Pedersen. 2014. Betting against beta. Journal of Financial Economics 111: 1–25. Gravelle, T. 1999. Liquidity of the government of Canada securities market: Stylized facts and some market microstructure compari- sons to the United States treasury market. Working Paper, Bank of Canada Working Paper 99–11 (July). Factor‑based investing in government bond markets: a survey of the current state of research Factor‑based investing in government bond markets: a survey of the current state of research Dirk Schiereck studied Economics at Christian-Albrechts-University in Kiel. After his graduation he became a research and teaching assistant at the Chair of Business Administration and Banking at Mannheim University where he also finalized his doctoral thesis and his habilita- tion. After two years at the Institute for Mergers & Acquisitions of the University Witten/Herdecke he spent six years at the European
https://openalex.org/W3108211527	https://gmd.copernicus.org/articles/13/6077/2020/gmd-13-6077-2020.pdf	English	null	Simulating second-generation herbaceous bioenergy crop yield using the global hydrological model H08 (v.bio1)	Geoscientific model development	2,020	cc-by	12,169	1 Introduction Abstract. Large-scale deployment of bioenergy plantations would have adverse effects on water resources. There is an increasing need to ensure the appropriate inclusion of the bioenergy crops in global hydrological models. Here, through parameter calibration and algorithm improvement, we enhanced the global hydrological model H08 to simulate the bioenergy yield from two dedicated herbaceous bioen- ergy crops: Miscanthus and switchgrass. Site-speciﬁc eval- uations showed that the enhanced model had the ability to simulate yield for both Miscanthus and switchgrass, with the calibrated yields being well within the ranges of the observed yield. Independent country-speciﬁc evaluations further con- ﬁrmed the performance of the H08 (v.bio1). Using this im- proved model, we found that unconstrained irrigation more than doubled the yield under rainfed condition, but reduced the water use efﬁciency (WUE) by 32 % globally. With ir- rigation, the yield in dry climate zones can exceed the rain- fed yields in tropical climate zones. Nevertheless, due to the low water consumption in tropical areas, the highest WUE was found in tropical climate zones, regardless of whether the crop was irrigated. Our enhanced model provides a new tool for the future assessment of bioenergy–water tradeoffs. Bioenergy with carbon capture and storage technology en- ables the production of energy without carbon emissions while sequestering carbon dioxide from the atmosphere, pro- ducing negative emissions. Therefore, bioenergy is consid- ered an important technology in the push to achieve the 2◦C climate target (Smith et al., 2015). With ambitious climate policies, the demand for bioenergy in 2100 could reach 200– 400 EJ per year, based on recent predictions (Rose et al., 2013; Bauer et al., 2018). However, large-scale planting of bioenergy crops requires water consumption to be doubled or even tripled, which would exacerbate the future water scarcity (Beringer et al., 2011; Bonsch et al., 2016; Hejazi et al., 2015; Yamagata et al., 2018). Therefore, representation of bioenergy crops in global hydrological models is critical in elucidating the possible side effects of large-scale imple- mentation of bioenergy. Second-generation bioenergy crops, such as Miscanthus and switchgrass, are generally regarded as a dedicated bioen- ergy source due to their high yield potential and lack of di- rect competition with food production (Beringer et al., 2011; Yamagata et al., 2018; Wu et al., 2019). This is because Miscanthus and switchgrass are rhizomatous perennial C4 grasses, which have a high photosynthesis efﬁciency (Try- bula et al., 2015). Correspondence: Zhipin Ai (ai.zhipin@nies.go.jp) Received: 2 June 2020 – Discussion started: 7 July 2020 Received: 2 June 2020 – Discussion started: 7 July 2020 Received: 2 June 2020 – Discussion started: 7 July 2020 Revised: 30 September 2020 – Accepted: 16 October 2020 – Published: 2 December 2020 eceived: 2 June 2020 – Discussion started: 7 July 2020 evised: 30 September 2020 – Accepted: 16 October 2020 – Published: 2 December 2020 Revised: 30 September 2020 – Accepted: 16 October 2020 – Published: 2 December 2020 Zhipin Ai1, Naota Hanasaki1, Vera Heck2, Tomoko Hasegawa3, and Shinichiro Fujimori4 4Department of Environmental Engineering, Kyoto University, Building C1-3, C-cluster, Kyoto-Daigaku-Katsura, Nishikyo ku Kyoto 615 8504 Japan f Environmental Engineering, Kyoto University, Building C1-3, C-cluster, Kyoto-Daigaku-Katsura, Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Simulating second-generation herbaceous bioenergy crop yield using the global hydrological model H08 (v.bio1) Zhipin Ai1, Naota Hanasaki1, Vera Heck2, Tomoko Hasegawa3, and Shinichiro Fujimori4 1Center for Climate Change Adaptation, National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba 305-8506, Japan 2Potsdam Institute for Climate Impact Research, Telegraphenberg A 31, Potsdam 14473, Germany 3Department of Civil and Environmental Engineering, Ritsumeikan University, 56-1, Toji-in Kitamachi, Kita-ku, Kyoto 603-8577, Japan 4Department of Environmental Engineering, Kyoto University, Building C1-3, C-cluster, Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8504, Japan 2.1 H08 and its crop module H08 is a global hydrological model that can simulate the ba- sic natural and anthropogenic hydrological processes as well as crop growth at a spatial resolution of 0.5◦and at a daily interval (Hanasaki et al., 2008a, b). Main variables related to the water cycle, such as river discharge, terrestrial water stor- age, and water withdrawal have been thoroughly validated in a series of previous studies (Hanasaki et al., 2008a, b, 2018a). H08 consists of six submodules. The six submodules (land surface hydrology, river routing, crop growth, reservoir oper- ation, environmental ﬂow requirements, and anthropogenic water withdrawal) are coupled in a unique way (Fig. 1a). The land surface module can simulate the main water cy- cle components, such as evapotranspiration and runoff. The former is used in the crop module, and the latter is used in the river routing and environmental ﬂow modules. The agri- cultural water demand simulated by the crop module and the streamﬂow simulated by the river routing and reservoir op- eration modules ﬁnally enter into the withdrawal module. Note that the crop module is independent, except for the wa- ter stress calculations, which require evapotranspiration and potential evapotranspiration inputs from the land surface hy- drology module. To the best of our knowledge, LPJmL was the ﬁrst global model that included both bioenergy and the water cycle. It has therefore been widely used to quantify the effects on wa- ter of large-scale planting of bioenergy crops in many previ- ous studies (Beringer et al., 2011; Heck et al., 2016, 2018; Bonsch et al., 2016; Jans et al., 2018; Stenzel et al., 2019). However, it should be noted that Miscanthus and switchgrass are not distinguished in LPJmL, which instead uses a C4 grass to parameterize them. A separate parametrization for the two bioenergy crops could enhance the bioenergy simu- lation since they showed totally different plant characteristics and crop yield (Heaton et al., 2008; Trybula et al., 2015; Li et al., 2018b). CLM5 has been improved and validated for sim- ulating Miscanthus and switchgrass separately based on ob- servations at the University of Illinois Energy Farm (Cheng et al., 2020), but a global validation or application has not been reported. H08 is a global hydrological model that con- siders human activities, including reservoir operation, aque- duct water transfer, seawater desalination, and water abstrac- tion for irrigation, industry, and municipal use (Hanasaki et al., 2008a, b, 2010, 2018a, b). Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6078 Land (LPJmL) (Beringer et al., 2011; Bondeau et al., 2007), H08 (Yamagata et al., 2018), ORCHIDEE (Li et al., 2018b), the High-Performance Computing Environmental Policy In- tegrated Climate model (HPC-EPIC) (Kang et al., 2014; Nichols et al., 2011), the Community Land Model (version 5) (CLM5) (Cheng et al., 2020), MISCANMOD (Clifton- Brown et al., 2000, 2004), MISCANFOR (Hastings et al., 2009), Agricultural Production Systems Simulator (APSIM) (Ojeda et al., 2017), and the Soil & Water Assessment Tool (SWAT) (Trybula et al., 2015). However, among these mod- els, only a few, such as LPJmL, H08, and CLM5, include the global implementation of schemes for irrigation, river rout- ing, or water withdrawal. This severely limits the application of the models to address the global bioenergy–water trade- offs or synergies. Land (LPJmL) (Beringer et al., 2011; Bondeau et al., 2007), H08 (Yamagata et al., 2018), ORCHIDEE (Li et al., 2018b), the High-Performance Computing Environmental Policy In- tegrated Climate model (HPC-EPIC) (Kang et al., 2014; Nichols et al., 2011), the Community Land Model (version 5) (CLM5) (Cheng et al., 2020), MISCANMOD (Clifton- Brown et al., 2000, 2004), MISCANFOR (Hastings et al., 2009), Agricultural Production Systems Simulator (APSIM) (Ojeda et al., 2017), and the Soil & Water Assessment Tool (SWAT) (Trybula et al., 2015). However, among these mod- els, only a few, such as LPJmL, H08, and CLM5, include the global implementation of schemes for irrigation, river rout- ing, or water withdrawal. This severely limits the application of the models to address the global bioenergy–water trade- offs or synergies. of Miscanthus and switchgrass, and (5) illustrate the effects of irrigation on the yield, water consumption, and WUE (wa- ter use efﬁciency, deﬁned here as the ratio of yield to water consumption) of Miscanthus and switchgrass. of Miscanthus and switchgrass, and (5) illustrate the effects of irrigation on the yield, water consumption, and WUE (wa- ter use efﬁciency, deﬁned here as the ratio of yield to water consumption) of Miscanthus and switchgrass. 1 Introduction These two crops have been included in a series of models including Lund–Potsdam–Jena managed ed by Copernicus Publications on behalf of the European Geosciences Union. 2.1 H08 and its crop module The ﬁrst use of H08 to simu- late the bioenergy crop yield was reported in an impact as- sessment of the effects of bioenergy production on water, land, and ecosystem services (Yamagata et al., 2018). Us- ing an identical model to that of Yamagata et al. (2018), an- other recent study also used H08 estimates of Miscanthus and switchgrass yield to predict global advanced bioenergy potential (Wu et al., 2019). Based on the work of Yamagata et al. (2018), here we improved the bioenergy crop simula- tion in H08 by performing a systematic parameter calibration for both Miscanthus and switchgrass using the best available data. gy Figure 1b shows the basic biophysical process of the crop module in H08. The biomass accumulation is based on Mon- teith et al. (1977). The crop phenology development is based on daily heat unit accumulation theory. The harvest index is used to partition the grain yield. Regulating factors, includ- ing water and air temperature, are used to constrain the yield variation. The crop module can simulate the potential yield, crop calendar, and irrigation water consumption for 18 crops, including barley, cassava, cotton, peanut, maize, millet, oil palm, potato, pulses, rape, rice, rye, sorghum, soybean, sugar beet, sugarcane, sunﬂower, and wheat. The parameters for these crops were taken from those of the SWAT model. To better reﬂect the agronomy practice, H08 divides each simulation cell into four sub-cells: rainfed, single-irrigated, double-irrigated, and other (i.e., non-agricultural land uses). Irrigation in H08 is deﬁned as the supply of water other than precipitation to maintain soil moisture above 75 % of ﬁeld ca- pacity during the cropping period. To clarify this as regards the function of the parameters we calibrated below, here we describe the algorithms in the crop module of H08. The crop module of H08 accumulates daily heat units (Huna(t)), which are expressed as the daily mean air temperature (Ta) greater than the plant’s speciﬁc base temperature (Tb; given The objective of this study was to enhance and validate the ability of H08 to simulate the second-generation herba- ceous bioenergy crop yield. The following sections of this paper will (1) describe the default biophysical process of the crop module in H08, (2) explain the enhancement of H08 for Miscanthus and switchgrass, (3) evaluate the enhanced per- formance of the model in simulating yields for Miscanthus and switchgrass, (4) map the spatial distributions of the yield Geosci. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6079 Figure 1. Schematic diagram showing the six submodules (a) and basic biophysical processes of the crop module (b) in the H08 model. Figure 1. Schematic diagram showing the six submodules (a) and basic biophysical processes of the crop module (b) in the H08 model. as a crop-speciﬁc parameter): if if ⌊dpl2⌋· 0.01 ≤Ihun < dlai, LAI = {(dpl2 −dpl2) + 1 −(dpl2 −dpl2) · (Ihun −dpl2 · 0.01) dlai −dpl2 · 0.01 ) · blai; (7) as a crop-speciﬁc parameter): Huna(t) = Ta −Tb. (1) (1) Then the heat unit index (Ihun) is calculated as the ratio of ac- cumulated daily heat units PHuna(t) and the potential heat unit (Hun): (7) if dlai < Ihun if dlai < Ihun LAI = 16 · blai(1 −Ihun)2, (8) Ihun = PHuna(t) Hun . (2) (2) LAI = 16 · blai(1 −Ihun)2, (8) (8) When the accumulated daily heat units PHuna(t) reach the potential heat unit (Hun) required for the maturity of the crop, the crop is mature and is harvested. During the growth period, the daily increase in biomass (1B) is calculated us- ing a simple photosynthesis model: where dpl1 and dpl2 are two complex numbers (see the deﬁ- nition in Table 1) and blai is the maximum leaf area index. REGF is calculated as REGF = min(Ts,Ws,Ns,Ps), (9) (9) 1B = be · PAR · REGF, (3) (3) where Ts, Ws, Ns, and Ps are the respective stress factors for temperature, water, nitrogen, and phosphorous. Temperature stress (Ts) is calculated as an asymmetrical function accord- ing to the relationship between air temperature (Ta) and op- timal temperature (To). When air temperature is below (or equal) the optimal temperature (To), Ts is calculated as where be is radiation use efﬁciency, PAR is photosyntheti- cally active radiation, and REGF is the crop regulating fac- tor. PAR is calculated using shortwave radiation (Rs) and leaf area index (LAI) as follows: PAR = 0.02092 · Rs · [1 −exp(−0.65 · LAI)]. 2.1 H08 and its crop module Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield (13) (13) Water stress (Ws) is calculated as the ratio of actual evapo- transpiration (Ea) to potential evapotranspiration (Ep) as Water stress (Ws) is calculated as the ratio of actual evapo- transpiration (Ea) to potential evapotranspiration (Ep) as Ws = Ea Ep . (14) (14) The crop yield (Yld) is ﬁnally estimated from the above- ground biomass (Bag) using the crop-speciﬁc harvest index (Harvest) at the harvesting date as The crop yield (Yld) is ﬁnally estimated from the above- ground biomass (Bag) using the crop-speciﬁc harvest index (Harvest) at the harvesting date as Bag = [1 −(0.4 −0.2 · Ihun)] X 1B, (15) Yld =Harvest · WSF WSF + exp(6.117 −0.086 · WSF) (15) Yld =Harvest · · WSF + exp(6.117 −0.086 · WSF) · Bag , (16) (16) · Bag · Bag where WSF is the ratio of SWU (the accumulated actual plant evapotranspiration in the second half of the growing season) and SWP (the accumulated potential evapotranspiration in the second half of the growing season): WSF = SWU SWP · 100. (17) WSF = SWU SWP · 100. (17) Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield (4) (4) Ts = expln(0.9) · Ctsl(To −Ta) Ta 2 , (10) (10) LAI is calculated according to the growth stage indicated by Ihun; if Ihun < ⌊dpl1 · 0.01⌋, where Ctsl is the temperature stress parameter for tempera- ture below To and is calculated as LAI = (dpl1 −dpl1) · Ihun dpl1 · 0.01 · blai); (5) (5) Ctsl = To + Tb To −Tb . (11) (11) if ⌊dpl1⌋· 0.01 ≤Ihun < dpl2 · 0.01, LAI = {(dpl1 −dpl1) if ⌊dpl1⌋· 0.01 ≤Ihun < dpl2 · 0.01, LAI = {(dpl1 −dpl1) LAI = {(dpl1 −dpl1) When air temperature is above the optimal temperature, Ts is calculated as + (dpl2 −dpl2) −(dpl1 −dpl1) · (Ihun −dpl1 · 0.01) dpl2 · 0.01 −dpl1 · 0.01 ) Ts = expln(0.9) · (To −Ta) Ctsh 2 , (12) (12) ) · blai; (6) · blai; · blai; (6) https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Geosci. Model Dev., 13, 6077–6092, 2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6080 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Table 1. Parameter abbreviations and explanation. Parameter Full name Physical meaning abbreviation Hun Potential heat unit The value of potential heat units required for the maturity of the crop be Radiation use efﬁciency The potential growth rate per unit of intercepted photosynthetically active radi- ation To Optimum temperature The optimal temperature for plant growth Tb Base temperature The base temperature for plant growth blai Maximum leaf area index The maximum potential leaf area index dlai Fraction of growing season when growth declines Same as the full name dpl1 Complex number1 First point on the optimal leaf area development curve. Before decimal: fraction of growing season; after decimal: max corresponding LAI. dpl2 Complex number2 Before decimal: fraction of growing season; after decimal: max corresponding LAI. Second point on the optimal leaf area development curve. rdmx Maximum rooting depth Same as the full name Hunmax Maximum daily accumulation of temperature Same as the full name TSAW Minimum temperature for planting Same as the full name Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6080 Table 1. Parameter abbreviations and explanation. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield where Ctsh is the temperature stress parameter for tempera- ture below To and is calculated as not reﬂect the characteristics for Miscanthus and switchgrass well, which could lead to serious bias based on the result in Trybula et al. (2015). Second, maturity was deﬁned by ei- ther undergoing an autumn freeze (i.e., the air temperature was below the minimum temperature for growth) or the ex- ceedance of the maximum of 300 continuous days of growth. Because both Miscanthus and switchgrass are perennial, the potential heat unit was set as unlimited (see the old values in Table 2). However, this unlimited potential heat unit is far from the observations (see the new values in Table 2) re- ported by Trybula et al. (2015). Here, further enhancements were made as follows. First, we changed the leaf area de- velopment curve by adopting the potential heat unit (Hun) and leaf-area-related parameters (dpl1 and dpl2) proposed by Trybula et al. (2015). The potential heat unit can deter- mine both the total cropping days and the leaf development. Here, we set the values at 1830 and 1400◦for Miscanthus and switchgrass, respectively, as recommended by Trybula et al. (2015) based on their ﬁeld observations. The dpl1 and dpl2 parameters (see Table 1), which were used for deter- mining the leaf development curve, were also changed to the values suggested by Trybula et al. (2015). This modiﬁcation substantially changed the original heat unit index (Ihun) and the development of the leaf area index curve. Second, we modiﬁed the algorithm for water stress that was used to reg- ulate the radiation use efﬁciency. We took the ratio of actual evapotranspiration to potential evapotranspiration as the wa- ter stress factor for any point in the simulation, similar to the description of the soil moisture deﬁcit used in other studies (Anderson et al., 2007; Yao et al., 2010). Third, we added a new output variable for the water consumption of Miscant- hus and switchgrass to analyze the water consumption and WUE in the crop submodule. Fourth, we introduced the Köp- pen climate classiﬁcation (see Fig. 2) into the source code to provide possible climate-speciﬁc analyses. Finally, we con- ducted parameter calibrations with the best available data. Ctsh = 2 · To −Ta −Tb. 2.4 Yield data To independently calibrate and validate the performance of H08 in simulating the bioenergy yield, we collected and compiled up-to-date site-speciﬁc (varied from 1986 to 2011) and country-speciﬁc (varied from 1960 to 2010) yield data from both observations and simulations (Clifton-Brown et al., 2004; Searle and Malins, 2014; Heck et al., 2016; Kang et al., 2014; Li et al., 2018a). For Miscanthus, the yield data used covered 72 sites (64 rainfed and 8 irrigated; observed) and 15 countries (simulated). The simulated country-speciﬁc data are from MISCANMOD and LPJmL. For switchgrass, the yield data used covered 57 sites (55 rainfed and 2 ir- rigated; observed) and 16 countries (simulated). The simu- lated country-speciﬁc data are from HPC-EPIC and LPJmL. A map showing the locations of the majority of sites under the rainfed condition and the corresponding climate zone is presented in Fig. 2. The data sites were predominantly dis- tributed in Europe and the US. It should be noted that the sites are generally located in temperate and continental cli- mate zones, with few located in the tropics and dry climate zones. Detailed lists of the sites from which the yields of Miscanthus and switchgrass were reported are documented in Tables S1 and S2 (for the rainfed condition) and Table S3 (for the irrigated condition) in the Supplement. Figure 2. Map showing the locations of the Miscanthus (red dots) and switchgrass (blue dots) sites under rainfed condition and the Köppen climate zones. The calibration process is presented below, and the ﬁnalized parameter settings are given in Table 2. We conducted a calibration with ﬁve important parame- ters, the radiation use efﬁciency (be), maximum leaf area index (blai), base temperature (Tb), maximum daily accu- mulation of temperature (Hunmax), and minimum temper- ature for planting (TSAW). The speciﬁc parameter ranges and steps set in the calibration process are shown in Ta- ble 3. In total, 1944 simulations were conducted for Mis- canthus and switchgrass to test all combinations of the pa- rameter sets. The simulations were conducted with the av- eraged daily meteorology data from WATCH-Forcing-Data– ERA-Interim (WFDEI) (1979–2016) for two reasons. First, using multi-year averaged metrology input can exclude the effect of extreme climate (low temperatures in early spring and late autumn) on the yield, and this is recommended in the H08 manual (Hanasaki and Yamamoto, 2010). Second, it can largely save the computation storage. 2.3 Model input data The WFDEI global meteorological data (Weedon et al., 2014) from 1979 to 2016 were used in all simulations. The WFDEI data were based on the methodology used for WA- Ter and global CHange (WATCH) forcing data by utiliz- ing ERA-Interim global reanalysis data. The data cover the whole globe at a spatial resolution of 0.5◦. Eight daily meteo- rological variables (air temperature, wind speed, air pressure, speciﬁc humidity, rainfall, snowfall, and downward short- wave and longwave radiation) were used to run H08. Another meteorological dataset for the period 1979–2013 in S14FD (Iizumi et al., 2017) with the same spatial resolution was also used to check the stability of results to input meteorological data. 2.2 Enhancement of H08 for Miscanthus and switchgrass 2.2 Enhancement of H08 for Miscanthus and switchgrass The original bioenergy crop implementation in H08 (Yam- agata et al., 2018) was conducted in two steps. First, crop parameters (see the old values in Table 2) for Miscanthus (refer to Miscanthus giganteus in this study) and switch- grass (refer to Panicum virgatum in this study) were adopted based on the settings from the SWAT model 2012 version (Arnold et al., 2013). However, the default parameters did https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6081 Figure 2. Map showing the locations of the Miscanthus (red dots) and switchgrass (blue dots) sites under rainfed condition and the Köppen climate zones. 2.4 Yield data The best parameter sets were selected using two steps: ﬁrst, the lowest root mean square error (RMSE) and, second, the highest correlation co- efﬁcient (R) of the simulated and observed yields within the lowest RMSE domain. Additional information on how these parameters affect the model can be found in the equations described in Sect. 2.1. A global yield map of Miscanthus and switchgrass that was generated using a random-forest algorithm (Li et al., 2020) was also used to compare the results. This yield map provides a benchmark for evaluating model performance be- cause it is largely constrained by the observed yield ranges, denoting the yields achievable under current technologies (Li et al., 2020). 2.5 Simulation setting After calibration, four different kinds of simulation were run with different purposes. The ﬁrst simulation was conducted using the original model without irrigation to investigate its performance. The second simulation was conducted using https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6082 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Table 2. Parameters set in the enhanced H08 model. Bioenergy crop Parameter Old value New value Source Miscanthus Hun 9999 1830 Trybula et al. (2015) be 39 38 Calibrated To 30 25 Trybula et al. (2015); Hastings et al. (2009) Tb 10 8 Calibrated blai 11.5 11 Calibrated dlai 0.85 1.1 Trybula et al. (2015) dpl1 10.2 10.1 Trybula et al. (2015) dpl2 50.95 45.85 Trybula et al. (2015) rdmx 4 3 Trybula et al. (2015) Hunmax 12.5 11.5 Calibrated TSAW 10.0 8.0 Calibrated Switchgrass Hun 9999 1400 Trybula et al. (2015) be 47 22 Calibrated To 25 25 Trybula et al. (2015) Tb 12 10 Calibrated blai 6 8 Calibrated dlai 0.7 1 Trybula et al. (2015) dpl1 10.2 10.1 Trybula et al. (2015) dpl2 20.95 40.85 Trybula et al. (2015) rdmx 2.2 3 Trybula et al. (2015) Hunmax 12.5 15.5 Calibrated TSAW 10.0 8.0 Calibrated Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield l 6082 Table 2. Parameters set in the enhanced H08 model. Table 2. Parameters set in the enhanced H08 model. Bioenergy crop Parameter Old value New value Source Table 3. Parameter ranges for the calibration simulations. Table 3. Parameter ranges for the calibration simulations. Bioenergy crop Parameter Range Increment Unit Reference Miscanthus be (30, 40) 2 g MJ−1 × 10 Clifton-Brown et al. (2000); van der Werf et al. (1992); Beale and Long (1995); Heaton et al. (2008); Trybula et al. (2015) blai (9, 11) 1 m2 m−2 Heaton et al. (2008); Trybula et al. (2015) Tb (7, 9) 1 ◦ Beale et al. (1996); Trybula et al. (2015) Hunmax (11.5, 16.5) 1 ◦ H08 endogenous variable TSAW (8, 10) 1 ◦ H08 endogenous variable Switchgrass be (12, 22) 2 g MJ−1 × 10 Heaton et al. (2008); Madakadze et al. (1998); Trybula et al. (2015) blai (6, 8) 1 m2 m−2 Trybula et al. (2015); Giannoulis et al. (2016); Madakadze et al. (1998); Heaton et al. (2008) Tb (8, 10) 1 ◦ Trybula et al. 2.5 Simulation setting (2015) Hunmax (11.5, 16.5) 1 ◦ H08 endogenous variable TSAW (8, 10) 1 ◦ H08 endogenous variable the enhanced model without irrigation to investigate its per- formance under rainfed condition. The third simulation was conducted using the enhanced model with irrigation to inves- tigate its performance under irrigated condition. These three simulations were conducted at a daily scale with annual me- teorological data from WFDEI for the period 1979–2016. The last simulation was conducted using identical model set- tings to the third one, except using different meteorological the enhanced model without irrigation to investigate its per- formance under rainfed condition. The third simulation was conducted using the enhanced model with irrigation to inves- tigate its performance under irrigated condition. These three simulations were conducted at a daily scale with annual me- teorological data from WFDEI for the period 1979–2016. The last simulation was conducted using identical model set- tings to the third one, except using different meteorological data from S14FD for the period 1979–2013. Note that irriga- tion in this study means uniform unconstrained irrigation. 3.2 Site-speciﬁc performance of enhanced H08 (19) An overview of the performance of the enhanced H08 is provided in Fig. 4. The simulated yield is the annual aver- age from 1986 to 2011. Points in a scatterplot comparing simulated yields derived from the enhanced H08 with ob- served yields are well distributed along the 1 : 1 line. It can be seen that the performance of the enhanced H08 was im- proved over that of the original H08. For Miscanthus, the bias of the original model ranged from −84 % to 80 % with a mean of −52 %, while the bias of the enhanced model ranged from −59 % to 53 % with a mean of −9 %. For switchgrass, the bias for the original model ranged from −78 % to 338 % with a mean of 25 %, while the bias for the enhanced model ranged from −52 % to 109 % with a mean of −7 %. Note that Fig. 4 also shows a tendency toward underestimation for some sites, especially for Miscanthus. More detailed site- speciﬁc results are shown in Fig. 5a (Miscanthus) and Fig. 5b (switchgrass). To depict the uncertainties in the observed yield, the minimum and maximum observed yields are shown as error bars in Fig. 5. It was found that the simulated yields were within or close to the range of the observed yields. The simulated relative error was randomly distributed, was sub- stantially smaller than the range of the observed yields, and showed no climatic bias. This implies that the combination of the Hun identiﬁed by Tryubula et al (2015) and the cal- ibrated parameters of this study are valid for climate zones other than that of the midwestern US, where the Hun was ob- served. We also investigated the performance under irrigated conditions (shown in Fig. 6). We used the reported observed yields for 10 sites globally (Table S3). We found that the sim- ulated yields were within or close to the observed yields for ﬁve sites located in China, the UK, and France (see Table S3) but were overestimated for the remaining sites. This was due to the assumption of irrigation. H08 assumes that irrigation is fully applied to crops and hence the yield represents the maximum potential yield under irrigation condition. There- fore, if the reported yield is within the range of the simulated yield between rainfed and irrigated conditions, it is consid- ered reasonable. 2.7 Sensitivity analysis Figure 3. Overall comparison of the calibrated (Cal.) and observed (Obs.) yields for Miscanthus and switchgrass. The black line is the 1 : 1 line. To see the sensitivity of the calibrated variables to the yield simulation, we calculated the sensitivity index (S) (Cheng et al., 2020) value for each variable: S = X (Vs −Vref)/Vref (Ps −Pref)/Pref , (19) 3.2 Site-speciﬁc performance of enhanced H08 This was found to be the case, as shown in Fig. 6. To investigate the uncertainty in the meteorological where Vs and Vref are the calculated RMSE of the simulated and observed yields for the corresponding calibration simula- tions and the ﬁnalized simulation (with ﬁnal ﬁxed parameters in Table 2) and Ps and pref are the parameter values for the corresponding calibration simulations and the ﬁnalized sim- ulation. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6083 Figure 3. Overall comparison of the calibrated (Cal.) and observed (Obs.) yields for Miscanthus and switchgrass. The black line is the 1 : 1 line. yield to water consumption: WUE = yield water consumption, (18) (18) where yield and water consumption refer to the bioenergy crop yield (kg ha−1 yr−1) and the corresponding water con- sumption (mm yr−1) of Miscanthus and switchgrass. 2.6 Water use efﬁciency WUE is an important indicator that shows the efﬁciency of crops in using water to produce biomass (Ai et al., 2020), which is useful in evaluating bioenergy crop performance (Zeri et al., 2013). Here, WUE is calculated as the ratio of Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 https://doi.org/10.5194/gmd-13-6077-2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6084 Figure 4. Overall comparison of the simulated (Sim.) and observed (Obs.) yields for Miscanthus and switchgrass. The simulated yields in (a) and (b) are from the original H08 model, whereas those in (c) and (d) are from the enhanced H08 model. The black line is the 1 : 1 line. that of HPC-EPIC. For example, both models produced high yields in Brazil, Colombia, Mozambique, and Madagascar, and low yields were found in Australia and Mongolia. y g Miscanthus and switchgrass are not distinguished in LPJmL, and we therefore compared the mixed (mean of Mis- canthus and switchgrass) yield of Miscanthus and switch- grass simulated by H08 and the C4 grass yield simulated by LPJmL. The correlation coefﬁcient of the yield simulated by H08 and LPJmL in the scatterplot (Fig. 7f) was 0.77. A t test showed that the correlation was signiﬁcant at the 0.01 level. An additional comparison under the irrigated condi- tion is presented in Fig. S3. The correlation coefﬁcient of the yield simulated by H08 and LPJmL, as shown in the scatter- plot (Fig. S3), was 0.95. A t test showed that the correlation was signiﬁcant at the 0.01 level. The difference was mainly due to Colombia, Sudan, Mozambique, and Mexico, which are located in tropical zones. The difference in these coun- tries was generally equal to the range of H08. For example, as shown in Fig. 7c, the yield in Colombia simulated by LPJmL was equal to the Miscanthus yield simulated by H08 (upper error bar). A separate comparison of the ensemble yield sim- ulated by LPJmL, and the yield of Miscanthus and switch- grass simulated by H08 under both rainfed and irrigated con- ditions is presented in Fig. S4. It can be seen that the yield of Miscanthus simulated by H08 was closer to the yield sim- ulated by LPJmL, which indicates that the LPJmL-simulated yield was more likely to represent Miscanthus. This can also be inferred from the validation results (Fig. 1a) in Heck et al. (2016) since the LPJmL-simulated yield is close to the yield of Miscanthus compared to those of switchgrass. It was difﬁcult to determine which model performed better due to the lack of observed data in tropical zones. This also indi- rectly indicated the relatively large uncertainty of the existing simulations in tropical zones (Kang et al., 2014). Figure 4. 3.3 Country-speciﬁc performance of enhanced H08 Figure 7 compares the yield simulated by the enhanced H08 with the collected independent country-speciﬁc yields simu- lated by MISCANMOD (Clifton-Brown et al., 2004), HPC- EPIC (Kang et al., 2014), and LPJmL (Heck et al., 2016). Here, the yield was simulated under rainfed conditions. The periods of climate data used as inputs were 1960–1990, 1980–2010, and 1982–2005 for MISCANMOD, HPC-EPIC, and LPJmL, respectively. Here, the comparisons were con- ducted using exactly the same period as that of HPC-EPIC and LPJmL. For MISCANMOD, however, we used the data from 1979–1990 due to data availability. For Miscanthus, the correlation coefﬁcient of the yield simulated by H08 and MISCANMOD in the scatterplot (Fig. 7d) was 0.40. A t test showed that the correlation was not signiﬁcant at the 0.01 level. For consistency with the yield collected by MISCAN- MOD, any area within a country where the yield was less than 10 Mg ha−1 yr−1 was excluded from the analyses. Also, the land available for calculations was set as 10 % of the pas- tureland and cropland. For switchgrass, the correlation coef- ﬁcient of the yield simulated by H08 and HPC-EPIC in the scatterplot (Fig. 7e) was 0.80. A t test showed that the corre- lation was signiﬁcant at the 0.01 level. This indicates that the spatial pattern of the yield simulated by H08 was similar to The differences in model structure, the use of speciﬁc al- gorithms, and the input climate data (different periods and sources) can induce differences in the yield simulated by MISCANMOD, HPC-EPIC, LPJmL, and H08. With regard to model structure, MISCANMOD uses a Kriging interpola- tion method to derive the spatial yield from the original site yield, whereas H08, LPJmL, and HPC-EPIC use grid-based calculations. H08 considers the single-harvest system in trop- ical areas, whereas LPJmL considers a multiple-harvest sys- tem. With regard to the speciﬁc algorithms used, the water stress used to regulate radiation use efﬁciency varies consid- erably among the models. Note that the differences in meteo- rological data sources and spatial–temporal resolution would also contribute to these differences. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Overall comparison of the simulated (Sim.) and observed (Obs.) yields for Miscanthus and switchgrass. The simulated yields in (a) and (b) are from the original H08 model, whereas those in (c) and (d) are from the enhanced H08 model. The black line is the 1 : 1 line. data, a simulation using other meteorological data from the S14FD dataset (Iizumi et al., 2017) was conducted; the re- sults are compared in Fig. S2. The comparison showed that the WFDEI-driven result was very similar to that obtained with the S14FD data. 3.1 Parameter calibration The variation in RMSE and R for all 1944 simulations is presented in Fig. S1. Both RMSE and R have large ranges. Based on the optimal values of RMSE (4.68 and 3.16 Mg ha−1 yr−1 for Miscanthus and switchgrass, respec- tively) and R (0.67 and 0.53 for Miscanthus and switchgrass, respectively), we ﬁnalized the parameter set as shown in Ta- ble 2. The simulations presented in the table are for rainfed conditions because only a few sites were irrigated. The radia- tion use efﬁciency values were set at 38 and 22 (g MJ−1×10) for Miscanthus and switchgrass, respectively. These values are similar to those of Trybula et al. (2015), who recom- mended values of 41 (g MJ−1 × 10) for Miscanthus and 17 (g MJ−1 × 10) for switchgrass. The base temperatures were calibrated to be 8 and 10 ◦C for Miscanthus and switchgrass, respectively. The base temperature is sensitive to the crop growing days. Ranges from 7 to 10 ◦C for Miscanthus and from 8 to 12 ◦C for upland switchgrass were suggested by Trybula et al. (2015). The calibrated values are within the above ranges. The maximum leaf area indices were cali- brated at 11 and 8 for Miscanthus and switchgrass, respec- tively; these values were identical to those suggested by Try- bula et al. (2015). Of the ﬁve parameters we calibrated, radia- tion use efﬁciency was the most sensitive parameter to the re- sult, followed by the base temperature (see Table S5); this is consistent with the result of Trybula et al. (2015). As shown in Fig. 3, the calibrated parameters performed well, since the scatter points are well distributed along the 1 : 1 line. https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6085 Figure 5. Site-speciﬁc performance (presented with latitude increasing from the bottom of the vertical axis) and relative error of the simulate yield obtained using the enhanced H08 model compared with the observed yields for Miscanthus and switchgrass. The longitude and latitud of each location for Miscanthus and switchgrass are given in Tables S1 and S2, respectively. The “x” indicates the site’s climate, where 2, 3, and 4 refer to tropical, dry, temperate, and continental climate zones, respectively. Obs. means the observed mean yield. The blac error bar represents the range of the observed minimum and maximum yield. The red or blue error bar represents the range of the simulate minimum and maximum yield. Figure 5. Site-speciﬁc performance (presented with latitude increasing from the bottom of the vertical axis) and relative error of the simulated yield obtained using the enhanced H08 model compared with the observed yields for Miscanthus and switchgrass. The longitude and latitude of each location for Miscanthus and switchgrass are given in Tables S1 and S2, respectively. The “x” indicates the site’s climate, where 1, 2, 3, and 4 refer to tropical, dry, temperate, and continental climate zones, respectively. Obs. means the observed mean yield. The black error bar represents the range of the observed minimum and maximum yield. The red or blue error bar represents the range of the simulated minimum and maximum yield. 3.4 Further evaluation of the performance of enhanced H08 As shown in Fig. 8, we compared our simulation with the latest available global bioenergy crop yield map, generated from observations using a random-forest (RF) algorithm (Li Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 3.6 Effects of irrigation on yield, water consumption, and WUE in different climate zones Climate is one of the main physical constraints of crop growth and yield. Figure 10a shows the mean yield for Mis- canthus and switchgrass in four different Köppen climate zones (see Fig. S6 in the Supplement). For Miscanthus, a tropical climate (including the northern part of South Amer- ica, central Africa, Southeast Asia, and southern India) pro- duced the highest average yield of 33.0 Mg ha−1 yr−1. A temperate climate (including the eastern US, Europe, south- ern China, and the southern part of South America) produced the second-highest average yield of 19.7 Mg ha−1 yr−1. Dry and continental climate zones had similar average yields of 8.3 and 6.2 Mg ha−1 yr−1, respectively. For switch- grass, a tropical climate had the highest yield, averaging 11.9 Mg ha−1 yr−1. For the other three climate types, the av- erage yields averaged 9.0, 4.7, and 4.0 Mg ha−1 yr−1 for the temperate, continental, and dry climate zones, respectively. As shown in Fig. 10a, irrigation greatly increased the yield, especially in dry climate zones, which had the largest yield increases of 44.2 and 15.7 Mg ha−1 yr−1 for Miscanthus and switchgrass, respectively. In contrast, irrigation had a rela- tively weak effect on yield in the tropical climate zone. Figure 6. Site-speciﬁc performance (shown with increasing latitude from the bottom of the vertical axis) of the simulated yield (sim.) obtained using the enhanced H08 model compared with observed yields (obs.) for Miscanthus (mis.) and switchgrass (swc.) under ir- rigated condition. The longitude and latitude of each location ID for Miscanthus and switchgrass are given in Table S3. Obs. indi- cates the observed mean yield. The black error bar represents the range of the observed minimum and maximum yield. The red or blue error bar represents the range of the simulated minimum and maximum yield. Figure 6. Site-speciﬁc performance (shown with increasing latitude from the bottom of the vertical axis) of the simulated yield (sim.) obtained using the enhanced H08 model compared with observed yields (obs.) for Miscanthus (mis.) and switchgrass (swc.) under ir- rigated condition. The longitude and latitude of each location ID for Miscanthus and switchgrass are given in Table S3. Obs. indi- cates the observed mean yield. The black error bar represents the range of the observed minimum and maximum yield. The red or blue error bar represents the range of the simulated minimum and maximum yield. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6086 Figure 6. Site-speciﬁc performance (shown with increasing latitude from the bottom of the vertical axis) of the simulated yield (sim.) obtained using the enhanced H08 model compared with observed yields (obs.) for Miscanthus (mis.) and switchgrass (swc.) under ir- rigated condition. The longitude and latitude of each location ID for Miscanthus and switchgrass are given in Table S3. Obs. indi- cates the observed mean yield. The black error bar represents the range of the observed minimum and maximum yield. The red or blue error bar represents the range of the simulated minimum and maximum yield. gation plays a critical role in ensuring optimum bioenergy crop yield in arid and semi-arid regions, especially for Mis- canthus. 3.6 Effects of irrigation on yield, water consumption, and WUE in different climate zones Figure 10b shows the water consumption for both Mis- canthus and switchgrass. The annual mean water consump- tion of Miscanthus was around 613 mm yr−1 for the tropi- cal climate zone (with a high yield of 33.0 Mg ha−1 yr−1), whereas it was 155 mm yr−1 for a dry climate (with a low yield of 8.3 Mg ha−1 yr−1) under rainfed conditions. Un- der irrigated conditions, the largest increases in water con- sumption were 1618 and 1054 mm yr−1 for Miscanthus and switchgrass in dry climate zones, respectively. With such large amounts of irrigation, the yield in a dry climate zone can exceed that in a tropical climate zone under rainfed con- ditions. This highlights the yield–water tradeoff effects. for Miscanthus and switchgrass, respectively, indicating that irrigated yield was more than double the rainfed yield. The spatial distributions of yield increases due to irrigation simu- lated by H08 were very similar to those simulated by LPJmL (Beringer et al., 2011). At the continental scale (e.g., Eu- rope), yield increases were located mainly in southern Eu- rope, consistent with the ﬁndings obtained using MISCAN- MOD (Clifton-Brown et al., 2004). The response of yield to irrigation was weaker for switchgrass than for Miscanthus (see Fig. 9b and d). This might have been due to switch- grass having less dependency on water compared to Miscant- hus (Mclsaac et al., 2010). Miscanthus growth has been re- ported to have a high water requirement due to its high yield, large leaf area index, and long growing season (Mclsaac et al., 2010; Lewandowski et al., 2003). As a result, Miscant- hus yield is strongly inﬂuenced by water availability, and an annual rainfall of 762 mm yr−1 is thought to be suitable for growth (Heaton et al., 2019). However, the precipitation in most locations is below this level, especially in arid and semi- arid regions (see Fig. S5 in the Supplement). Therefore, irri- for Miscanthus and switchgrass, respectively, indicating that irrigated yield was more than double the rainfed yield. The spatial distributions of yield increases due to irrigation simu- lated by H08 were very similar to those simulated by LPJmL (Beringer et al., 2011). At the continental scale (e.g., Eu- rope), yield increases were located mainly in southern Eu- rope, consistent with the ﬁndings obtained using MISCAN- MOD (Clifton-Brown et al., 2004). The response of yield to irrigation was weaker for switchgrass than for Miscanthus (see Fig. 3.5 Spatial distributions of the simulated yield under rainfed and irrigated conditions et al., 2020). As shown in Fig. 8a and b, there were small differences between our estimated yield and RF yield for switchgrass, whereas larger differences were found for Mis- canthus, especially in tropical regions. There is a similar case for ORCHIDEE, as shown in Fig. S21 in Li et al. (2020). We also compared the differences in the mean values for Mis- canthus and switchgrass because they are not distinguished in LPJmL. As shown in Fig. 8c and d, the differences be- tween our estimations and the RF yields were generally lower than those between the LPJmL estimations and RF yields. In summary, our estimations were well within the ranges of those of ORCHIDEE and LPJmL. Figure 9 shows the global yield distributions of Miscanthus and switchgrass. Under rainfed conditions, high yields are distributed in the eastern US, Brazil, southern China, Africa, and Southeast Asia. To evaluate the response of yield to irri- gation, we compared the results under rainfed and irrigated conditions. As shown in Fig. 9c and d, unconstrained irri- gation greatly increased yields, especially of areas in arid regions such as the western US, southern Europe, north- eastern China, India, southern Africa, the Middle East, and coastal Australia. At the global scale, the increases (exclud- ing the area with a polar climate) were 20.7 (from 16.8 to 37.5) Mg ha−1 yr−1 and 7.9 (from 7.4 to 15.3) Mg ha−1 yr−1 https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 3.6 Effects of irrigation on yield, water consumption, and WUE in different climate zones 9b and d). This might have been due to switch- grass having less dependency on water compared to Miscant- hus (Mclsaac et al., 2010). Miscanthus growth has been re- ported to have a high water requirement due to its high yield, large leaf area index, and long growing season (Mclsaac et al., 2010; Lewandowski et al., 2003). As a result, Miscant- hus yield is strongly inﬂuenced by water availability, and an annual rainfall of 762 mm yr−1 is thought to be suitable for growth (Heaton et al., 2019). However, the precipitation in most locations is below this level, especially in arid and semi- arid regions (see Fig. S5 in the Supplement). Therefore, irri- As shown in Fig. 10c, the WUE values of Miscanthus in a tropical climate were 53.8 kg DM (dry matter) ha−1 mm−1 H2O and 53.5, 48.2, and 47.0 kg DM ha−1 mm−1 H2O, respectively, in dry, temperate, and continental climate zones under rainfed conditions. The respective WUE values of switchgrass were 41.2, 37.9, 30.4, and 29.7 kg DM ha−1 mm−1 H2O in continental, dry, tropical, and temperate climate zones under rainfed conditions. The WUE values for Miscanthus were higher than those for switchgrass, which is inconsistent with values in previous re- ports (Van Loocke et al., 2012). With irrigation, the WUE decreased for both Miscanthus and switchgrass in all climate zones. Globally, excluding the area with a polar climate, the decreases were 14.2 (from 50.6 to 36.4) kg DM ha−1 mm−1 H2O and 12.2 (from 34.8 to 22.6) kg DM ha−1 mm−1 H2O Geosci. Model Dev., 13, 6077–6092, 2020 https://doi.org/10.5194/gmd-13-6077-2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 608 Figure 7. An independent country-speciﬁc comparison of the yield simulated by the enhanced H08 model with those of three other mode (MISCANMOD, HPC-EPIC, and LPJmL) for Miscanthus (a, d), switchgrass (b, e), and their combination (c, f). The H08 in (c, f) indicat 6087 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6087 Figure 7. An independent country-speciﬁc comparison of the yield simulated by the enhanced H08 model with those of three other models (MISCANMOD, HPC-EPIC, and LPJmL) for Miscanthus (a, d), switchgrass (b, e), and their combination (c, f). 3.6 Effects of irrigation on yield, water consumption, and WUE in different climate zones For example, more land can be allocated to the areas with greater WUE. In summary, our enhanced model provides a new tool that can simultaneously simulate Miscanthus and switchgrass with a consideration of water management (such as irrigation), although it cur- rently considers only herbaceous bioenergy crops. From this perspective, we ﬁrmly believe that our enhanced model con- tributes to the bioenergy crop modeling community. for Miscanthus and switchgrass, respectively, indicating a re- duction in the mean WUE values for Miscanthus and switch- grass of up to 32 %. This is consistent with the current global WUE trend for crops, which is high for rainfed croplands but low for irrigated croplands. However, the general magnitude of this relationship changes if the site or regional scale is considered based on reports for wheat in Syria (Oweis et al., 2000) or for wheat and maize in the North China Plain (Mo et al., 2005). Note that it might be better to use a speciﬁc crop model to investigate WUE at the site or watershed scale. 3.6 Effects of irrigation on yield, water consumption, and WUE in different climate zones The H08 in (c, f) indicates the average yield of Miscanthus and switchgrass, and the upper and lower error bars in (c) represent the yields for Miscanthus and switchgrass, respectively. Figure 7. An independent country-speciﬁc comparison of the yield simulated by the enhanced H08 model with those of three other models (MISCANMOD, HPC-EPIC, and LPJmL) for Miscanthus (a, d), switchgrass (b, e), and their combination (c, f). The H08 in (c, f) indicates the average yield of Miscanthus and switchgrass, and the upper and lower error bars in (c) represent the yields for Miscanthus and switchgrass, respectively. grass). Moreover, the growing seasons for Miscanthus (145– 165) and switchgrass (101–114) during the period 2009– 2011 at the Water Quality Field Station of the Purdue Univer- sity Agronomy Center are consistent with the values of 140 and 120 reported in Trybula et al. (2015). Second, the hy- drological effects of bioenergy crop production implemented in our model are actually not incorporated in some other models; for example, we considered irrigation and analyzed WUE, which was not implemented in ORCHIDEE-MICT- BIOENERGY (Li et al., 2018b) and HPC-EPIC (Kang et al., 2014). Third, we investigated the differences in yield, water consumption, and WUE of both Miscanthus and switchgrass among different climate zones, which was useful for bioen- ergy land-scenario design. For example, more land can be allocated to the areas with greater WUE. In summary, our enhanced model provides a new tool that can simultaneously simulate Miscanthus and switchgrass with a consideration of water management (such as irrigation), although it cur- rently considers only herbaceous bioenergy crops. From this perspective, we ﬁrmly believe that our enhanced model con- tributes to the bioenergy crop modeling community. grass). Moreover, the growing seasons for Miscanthus (145– 165) and switchgrass (101–114) during the period 2009– 2011 at the Water Quality Field Station of the Purdue Univer- sity Agronomy Center are consistent with the values of 140 and 120 reported in Trybula et al. (2015). Second, the hy- drological effects of bioenergy crop production implemented in our model are actually not incorporated in some other models; for example, we considered irrigation and analyzed WUE, which was not implemented in ORCHIDEE-MICT- BIOENERGY (Li et al., 2018b) and HPC-EPIC (Kang et al., 2014). Third, we investigated the differences in yield, water consumption, and WUE of both Miscanthus and switchgrass among different climate zones, which was useful for bioen- ergy land-scenario design. Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6089 al.: Simulating second-generation herbaceous bioenergy crop yield Figure 10. Variation in the average yield (a), crop water consumption (b), and water use efﬁciency (WUE) (c) of Miscanthus and switchgrass under rainfed and irrigated conditions in four different Köppen climate zones (tropical, dry, temperate, and continental climates) based on meteorology data collected from 1979 to 2016. The abbreviations M. and S. in the legend denote Miscanthus and switchgrass, respectively. Figure 10. Variation in the average yield (a), crop water consumption (b), and water use efﬁciency (WUE) (c) of Miscanthus and switchgrass under rainfed and irrigated conditions in four different Köppen climate zones (tropical, dry, temperate, and continental climates) based on meteorology data collected from 1979 to 2016. The abbreviations M. and S. in the legend denote Miscanthus and switchgrass, respectively. (Hastings et al., 2009). Additional investigations of the water stress formulation for different bioenergy crops are needed. There are still several uncertainties and limitations that need to be addressed in the future. First, the bioenergy crop yield simulated by H08 did not include constraints due to nu- trients, such as nitrogen and phosphorus. Nutrient dynamics are inﬂuenced by complex site-speciﬁc soil conditions (soil type, temperature, wetness, carbon, etc.), which remain quite challenging to represent properly in global models. This is why similar assumptions and limitations occur in the latest bioenergy potential or yield studies (Li et al., 2018b; Ya- magata et al., 2018; Wu et al., 2019). Second, the effects of CO2 fertilizer and technological advancements were not considered in the current simulations. Third, our simulation was conducted with historical meteorological drivers. There- fore, yield variations in future climate scenarios under dif- ferent representative concentration pathways need to be ex- amined. Fourth, the current irrigation levels were input to represent uniform unconstrained irrigation. Further evalua- tions need to consider the availability of renewable water sources and planetary boundaries of land, food, and water (Heck et al., 2018). Finally, as with other models, like MIS- CANMOD (Clifton-Brown et al., 2004), SWAT (Neitsch et al., 2011), and LPJmL (Bondeau et al., 2007), we adopted a crop-uniform water stress formulation. However, an earlier study indicated that the water stress could be crop-speciﬁc 3.7 Improvements and limitations Compared with earlier studies, our study made several im- portant improvements. First, rather than using an approxima- tion for C4 grass to represent Miscanthus and switchgrass in the LPJmL model, our enhanced H08 model simultaneously simulated the yields of Miscanthus and switchgrass at the global scale. Compared with the original H08, our enhanced model markedly decreased the mean bias (from −52 % to −9 % for Miscanthus and from 25 % to −7 % for switch- https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Z. Ai et al.: Simulating second-generation herbaceous bioenergy crop yield 6088 g g gy p y Figure 8. Comparison of the yield difference (simulated yields minus RF yields) between model simulations and the RF map (Li et al., 2020): (a) for Miscanthus with the yield from H08 minus that from RF; (b) for switchgrass with the yield from H08 minus that from RF; (c) for the mean of Miscanthus and switchgrass with the yield from H08 minus that from RF; (d) the ensemble yield of Miscanthus and switchgrass with the yield from LPJmL minus that from RF. The units for the legends are Mg ha−1 yr−1. Figure 8. Comparison of the yield difference (simulated yields minus RF yields) between model simulations and the RF map (Li et al., 2020): (a) for Miscanthus with the yield from H08 minus that from RF; (b) for switchgrass with the yield from H08 minus that from RF; (c) for the mean of Miscanthus and switchgrass with the yield from H08 minus that from RF; (d) the ensemble yield of Miscanthus and switchgrass with the yield from LPJmL minus that from RF. The units for the legends are Mg ha−1 yr−1. Figure 9. Spatial distributions of the simulated yields (exceeds 2 Mg ha−1 yr−1) for Miscanthus (a, c) and switchgrass (b, d) under rainfed (a, b) and irrigated (c, d) conditions. The units for the legends are Mg ha−1 yr−1. Figure 9. Spatial distributions of the simulated yields (exceeds 2 Mg ha−1 yr−1) for Miscanthus (a, c) and switchgrass (b, d) under rainfed (a, b) and irrigated (c, d) conditions. The units for the legends are Mg ha−1 yr−1. https://doi.org/10.5194/gmd-13-6077-2020 https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Geosci. Model Dev., 13, 6077–6092, 2020 4 Conclusions In this study, we enhanced the ability of the H08 global hydrological model to simulate the yield of the dedicated second-generation herbaceous bioenergy crops. The en- hanced H08 model generally performed well in simulating the yield of both Miscanthus and switchgrass, with the esti- mations being well within the range of observations and other model simulations. To the best of our knowledge, this study is the ﬁrst attempt to enable a global hydrological model with a consideration of water management, such as irrigation, to simulate the yield of Miscanthus and switchgrass separately. The enhanced model could be a good tool for the future as- sessments of the bioenergy–water tradeoffs. Using this tool, we quantiﬁed the effects of irrigation on yield, water con- sumption, and WUE for both Miscanthus and switchgrass in different climate zones. We found that irrigation more than doubled the yield in all areas under rainfed conditions and reduced the WUE by 32 %. However, due to the low water consumption in tropical areas, the highest WUE was gener- ally found in tropical climate zones, regardless of whether the crop was irrigated. https://doi.org/10.5194/gmd-13-6077-2020 Geosci. Model Dev., 13, 6077–6092, 2020 Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/gmd-13-6077-2020-supplement. Beale C. 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https://openalex.org/W1513406002	https://perioperativemedicinejournal.biomedcentral.com/counter/pdf/10.1186/s13741-015-0016-x	English	null	Stroke volume variation to guide fluid therapy: is it suitable for high-risk surgical patients? A terminated randomized controlled trial	Perioperative medicine	2,015	cc-by	4,521	RESEARCH Open Access Stroke volume variation to guide fluid therapy: is it suitable for high-risk surgical patients? A terminated randomized controlled trial Perioperative Medicine Jammer et al. Perioperative Medicine (2015) 4:6 DOI 10.1186/s13741-015-0016-x RESEARCH Open Access Stroke volume variation to guide fluid therapy: is it suitable for high-risk surgical patients? A terminated randomized controlled trial Perioperative Medicine Jammer et al. Perioperative Medicine (2015) 4:6 DOI 10.1186/s13741-015-0016-x Perioperative Medicine Jammer et al. Perioperative Medicine (2015) 4:6 DOI 10.1186/s13741-015-0016-x © 2015 Jammer et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stroke volume variation to guide fluid therapy: is it suitable for high-risk surgical patients? A terminated randomized controlled trial Ib Jammer1,2, Mari Tuovila3 and Atle Ulvik2 Ib Jammer1,2, Mari Tuovila3 and Atle Ulvik2 Abstract Background: Perioperative goal-directed fluid therapy (GDFT) may improve outcome after high-risk surgery. Minimal invasive measurement of stroke volume variation (SVV) has been recommended to guide fluid therapy. We intended to study how perioperative GDFT with arterial-based continuous SVV monitoring influences postoperative complications in a high-risk surgical population. Methods: From February 1st 2012, all ASA 3 and 4 patients undergoing abdominal surgery in two university hospitals were assessed for randomization into a control group or GDFT group. An arterial-line cardiac output monitor was used to measure SVV, and fluid was given after an algorithm in the intervention group. Restrictions of the method excluded patients undergoing laparoscopic surgery, patients with atrial fibrillation and patients with severe mitral/aortal stenosis. To detect a decrease in number of complication from 40 % in the control group to 20 % in the GDFT group, n = 164 patients were needed (power 80 %, alpha 0.05, two-sided test). To include the needed amount of patients, the study was estimated to last for 2 years. Results: After 1 year, 30 patients were included and the study was halted due to slow inclusion rate. Of 732 high-risk patients scheduled for abdominal surgery, 391 were screened for randomization. Of those, n = 249 (64 %) were excluded because a laparoscopic technique was preferred and n = 95 (24 %) due to atrial fibrillation. Conclusions: Our study was stopped due to a slow inclusion rate. Methodological restrictions of the arterial-line cardiac output monitor excluded the majority of patients. This leaves the question if this method is appropriate to guide fluid therapy in high-risk surgical patients. Trial registration: ClinicalTrials.gov: NCT01473446. Trial registration: ClinicalTrials.gov: NCT01473446. * Correspondence: ib.jammer@helse-bergen.no 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway 2Department of Anaesthesia and Intensive Care, Haukeland University Hospital, 5021 Bergen, Norway Full list of author information is available at the end of the article Background Recent studies show the development and use of sev- eral minimal invasive methods to estimate cardiac out- put and guide fluid therapy [2]. Despite the unclear evidence in the literature and contradictory findings in clinical trials, the pressure on clinicians to use a goal- directed fluid therapy approach is high. In the UK, there is even a governmental financial incentive for hospitals to use Oesophageal Doppler for its patients [3] because the goal-directed approach may be cost effective [4]. Maintaining adequate oxygen supply to body organs is one of the main goals during anaesthesia, and giving intravenous fluid is one way to achieve this goal. A Cochrane systematic review found that complication rate and length of hospital stay, but not mortality, were re- duced when global blood flow is optimized periopera- tively by means of fluid and or drugs [1]. High-risk patient may have the greatest benefit of a goal-directed fluid approach [5, 6]. Less capability to compensate hypo- and hypervolemia may increase the * Correspondence: ib.jammer@helse-bergen.no 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway 2Department of Anaesthesia and Intensive Care, Haukeland University Hospital, 5021 Bergen, Norway Full list of author information is available at the end of the article Page 2 of 6 Jammer et al. Perioperative Medicine (2015) 4:6 rate of complications in a poorly optimized fluid bal- ance [7, 8]. Benes et al. evaluate the effect of minimal invasive cardiac output-monitored fluid therapy exclu- sively in a high-risk abdominal surgery population [9]. Pearse describes the use of an arterial-line cardiac out- put monitoring in a high-risk surgery population [10]. Both studies where done on high-risk patients, using a strict definition of “high-risk”. However, we could not find a consensus in the literature about the definition of “high-risk surgery”. To simplify our approach to high-risk surgery, we defined therefore ASA 3 and 4 patients as high-risk patients. Then we intended to conduct a multicentre international prospective clinical trial to study what impact goal-directed fluid therapy based on continuous SVV (stroke volume variation) moni- toring has on postoperative complications in this patient group. higher complication rate was expected due to inclusion of a population with a higher morbidity. To detect a de- crease in number of complication from 40 % in the con- trol group to 20 % in the GDFT group, n = 164 patients were needed (power 80 %, alpha 0.05, two-sided test). Trial design The primary endpoint was the proportion of patients suffering of one or more complication within 5 days postoperatively. The primary endpoint was the proportion of patients suffering of one or more complication within 5 days postoperatively. We planned a two-centre, assessor concealed, prospect- ive randomized clinical trial conducted in Norway and Finland. The trial was approved by the institutional board in Norway (2011/947/REK Vest) and Finland (EETTMK:10/2012). After termination of the study, we decided to reject the primary outcome due to heavy underpowered sam- ple size. To analyse reasons for exclusion, we determined the amount of excluded patients. No statistical analysis was performed of the numbers collected. Background It was estimated that with an approximate inclusion rate of 80 patients/year in each study centre, the study could be conducted within 2 years. Participants From 1 February 2012 to 31 January 2013, all high-risk patients defined by ASA score 3 and 4, older than 18 years scheduled for major abdominal surgery in two university hospitals were assessed for eligibility. Patients who were able to give consent when an investigator was available were screened for eligibility. Patient undergoing liver or oesophageal surgery where not screened because they follow a more restrictive fluid regimen. Exclusion criteria after screening were the following: atrial fibrilla- tion, severe aortic or mitral stenosis, and laparoscopic surgery or declined participation. Patients were included consecutively. Informed consent was obtained from each randomized patient. Interventions P i Patients were randomized into two groups: a control group receiving traditional fluid therapy and a group with a goal-directed fluid therapy (GDFT) regimen guided by an arterial pressure-based cardiac output de- vice (LiDCOrapid, LiDCO Ltd, London, UK) to measure SVV [11]. For more details of the study protocol, see Additional file 1. Participant flow During 1 year, n = 732 high-risk patients were scheduled for abdominal surgery. During 1 year, n = 732 high-risk patients were scheduled for abdominal surgery. Of these, n = 341 were not screened for inclusion. Rea- sons were either no investigator present or equipment missing (n = 224), scheduled liver or oesophagus surgery (n = 64) or the patient were unable to give informed con- sent (n = 53). Of all scheduled patients, n = 391 patients were screened for randomisation. Of these, 64 % (n = 249) were excluded because a laparoscopic technique was preferred, and 24 % (n = 95) were excluded due to atrial fibrillation. The patient flow through the study can be seen in Fig. 1. Of all screened patients, only 7.7 % (n = 30) could be included in the study. The outcome data is presented by study group allocation in Table 1. A de-identified database containing all collected data of included patients is available online as an additional file (see Additional file 2). Interim analysis Due to an unexpectedly low inclusion rate, an analysis of the exclusion factors was performed after 1 year. This resulted in termination of the study. A retrospective ana- lysis of all patients undergoing abdominal surgery within the last year and their comorbidities and surgical tech- niques was undertaken after approval of the institutional board. Sample size calculation After 1 year, the number of randomized patients we could include in the study was only 18 % of the esti- mated number we have been expected at that time. We The complication rate for lower gastrointestinal surgery in elective patients in one of the study hospitals was 40 % in a previous study [12]. In the present study, a Page 3 of 6 Jammer et al. Perioperative Medicine (2015) 4:6 Fig. 1 Flow diagram for patients’ progression through the trial calculated that at this inclusion speed, the study would last more than 5 years and therefore decided to termin- ate the study early. 30 patients, and a statistical analysis would be meaning- less. Consequently, we present just the patient flow numbers and not a complete statistical analysis of complications. Discussion The low number of patients that could possibly benefit from GDFT is valid for our hospitals where the surgeons prefer to operate on high-risk patients with minimal invasive surgery. In hospitals that perform a higher amount of open surgery, the use of a minimal invasive GDFT approach may be more feasible. Principal findings The primary outcome, postopera- tive morbidity, cannot be assessed since we just included Page 4 of 6 Jammer et al. Perioperative Medicine (2015) 4:6 Page 4 of 6 Table 1 Complications (definition) within 5 days after surgery has even been officially recommended in the National Institute for Health and Clinical Excellence guidelines of the UK (http://www.nice.org.uk/guidance/MTG3). This decision has been criticized due to the lack of proof [24, 25], and the method may not be superior to a strategy of a neutral balance [26]. Other studies have not found benefices in a goal-directed fluid approach [10, 12, 26–30], have not found benefit using a restrict- ive fluid approach [31] or even have worse outcome in physically fit patients [5]. Table 1 Complications (definition) within 5 days after surgery Intervention group Control group n = 14 n = 16 Pulmonary Pneumonia (x-ray + antibiotics) 4 0 Pleural fluid (supplemental oxygen + x-ray) 0 2 Atelectasis (supplemental oxygen + x-ray) 3 1 Pneumothorax 0 0 Respiratory failure (intensive care treatment) 2 1 Pulmonary emboli (computed tomography + treatment) 0 0 Cardial Arrytmia (electrocardiogram + treatment or cardiologist consultation) 2 1 Coronary ischemia (electrocardiogram + troponin) 0 0 Pulmonary stasis/oedema (x-ray or treatment) 1 1 Neurological Postoperative delirium (treatment) 1 2 Focal neurological deficit 0 0 Infectious Wound infection (phlegmone + antibiotics or drainage) 0 0 Intraabdominal infection (computed tomography + antibiotics) 0 0 Central venous catheter infection 0 0 Wound rupture (operation) 0 0 Gastrointestinal (GI) Mechanical ileus (operation) 0 1 GI bleeding (transfusion or gastroscopy) 0 0 Paralytic ileus (unable to tolerate enteral diet > 5 days) 1 2 Others Renal impairment (creatinine increase > 33 %) 0 1 Impaired spontaneous voiding (catheterization > 2 times) 1 0 Venous thrombosis (treatment) 0 0 Sum of complications 15 12 Patients with at least one complication 7 7 It is biologically plausible that the right amount of fluid given at the right time increase oxygen delivery to the or- gans and thereby benefit patient outcome. There has been a meta-analysis confirming that a GDFT approach may decrease postoperative complications. However, many included studies are small single centre studies with a high risk of bias or methodological limitations [1, 32, 6]. The effect on outcome in these studies is mostly small. Principal findings An even statistical distribution of different studies with a small effect size would consequently result in a number of studies that would show no effect or even harm. The marked overweight of studies with a small positive effect on outcome may indicate a publication bias favouring tri- als with positive results. This may mask limitations of the arterial-line-based GDFT method that we report. Other studies investigating high-risk surgical patients do not report the exclusion rate due to atrial fibrillation when this condition restricted the GDFT method used [5, 9]. The OPTIMIZE trial with a study population of 734 patients is the largest trial on GDFT to date. It could not show a reduction of complications after periopera- tive arterial-line-based GDFT. However, when including the OPTIMIZE trial in an updated Cochrane meta- analysis, it indicates a reduced complication rate [10]. Goal-directed fluid therapy may be more important in a high-risk surgery population than in a relatively healthy population. Limitations of the method with an arterial- line-based monitor may cause exclusion of a patient group who may benefit most of the treatment. In the UK, it is recommended to use an Oesophagus Doppler to guide fluid therapy preoperatively. The same limitations that apply to the arterial-line-based method (exclusion of patient with atrial fibrillation and laparoscopic procedures) would apply to this method too. Principal findings In our study, a majority of high-risk surgical patients de- fined as ASA 3 and ASA 4 were not eligible for an arterial-line-based GDFT approach. The main reasons are methodological limitations of the arterial-line wave- form analysis. The majority of patients had to be ex- cluded because a laparoscopic surgical technique was preferred or due to atrial fibrillation. We define the high-risk surgical patient by the ASA score to make the study pragmatic. However, other au- thors define “high-risk surgery” or the “high-risk patient” in different ways [13–15]. This makes comparison of tri- als dealing with this patient group difficult. This study was meant to be a prospective randomized controlled trial with a pragmatic approach to include pa- tients. This would reflect daily routines, strengthening the study. Because the study was conducted in two ter- tiary hospitals, we had a high amount of high-risk surgi- cal patients. Therefore, we expected to include enough patients in short time to run a well-powered study. Of 732 patients, 224 were not screened for randomisation due to investigator or equipment not being available. If this patient group also could have been screened, we may have had a higher number of patients randomized. However, it is to assume that the same fraction of pa- tients would have to be excluded due to laparoscopic surgery and atrial fibrillation. Therefore, we do not be- lieve that the total amount of patients that could be ran- domized would be much higher. Maguire found in a retrospective electronic chart study of his hospital that n = 12.308 patients underwent surgery in 1 year, but only n = 4.792 (39 %) fulfilled the criteria for an arterial-line-based cardiac output monitor, and of these, only 23.2 % had an arterial-line. There was no report on how many of the patients were ASA III/IV patients [16]. Arterial-line-based waveform analysis measures hemodynamics by calculation of stroke volume vari- ation or pulse pressure variation. However, arterial- line-based output methods are not applicable to large patient groups due to their limitations [16]. One limitation is laparoscopic procedures [17]. The increased intraabdom- inal pressure from the pneumoperitoneum affects dynamic parameters independently in changes of volume status [17–19]. Consequently would SVV during pneumoperito- neum increase while the blood volume do not decrease, it We terminated the study early, resulting in a heavily underpowered study. References 1. Grocott MP, Dushianthan A, Hamilton MA, Mythen MG, Harrison D, Rowan K, et al. Perioperative increase in global blood flow to explicit defined goals and outcomes after surgery: a Cochrane Systematic Review. Br J Anaesth. 2013;111(4):535–48. doi:10.1093/bja/aet155. 1. Grocott MP, Dushianthan A, Hamilton MA, Mythen MG, Harrison D, Rowan K, et al. Perioperative increase in global blood flow to explicit defined goals and outcomes after surgery: a Cochrane Systematic Review. Br J Anaesth. 2013;111(4):535–48. doi:10.1093/bja/aet155. 22. Jacques D, Bendjelid K, Duperret S, Colling J, Piriou V, Viale JP. 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Challand C, Struthers R, Sneyd JR, Erasmus PD, Mellor N, Hosie KB, et al. Randomized controlled trial of intraoperative goal-directed fluid therapy in aerobically fit and unfit patients having major colorectal surgery. Br J Anaesth. 2011;108(1):53–62. doi:10.1093/bja/aer273. 26. Brandstrup B, Svendsen PE, Rasmussen M, Belhage B, Rodt SA, Hansen B, et al. Which goal for fluid therapy during colorectal surgery is followed by the best outcome: near-maximal stroke volume or zero fluid balance? Conclusions Our primary goal was to investigate if high-risk surgical patients benefit from SVV-guided fluid therapy. This question still remains open. A majority of our patients had to be excluded from the trial due to methodo- logical limitations. This leaves the question whether or not an arterial-line-based cardiac output monitor is the best method to guide fluid therapy in high-risk surgical patients. would lead to false positive readings [20]. It is therefore not well validated in humans [21, 22]. Other limitations of waveform analysis measurements are cardiac arrhythmias and patients with severe cardiac valvulopatias [23]. Despite criticism about the evidence of the effect of goal-directed therapy, one single method of minimal in- vasive cardiac output monitoring (Oesophagus Doppler) Page 5 of 6 Page 5 of 6 Jammer et al. Perioperative Medicine (2015) 4:6 Additional file 2: De-identified SPSS database containing collected data of all randomized patients. SPSS Statistics Data Document. 13. Pearse R, Harrison D, James P, Watson D, Hinds C, Rhodes A, et al. Identification and characterisation of the high-risk surgical population in the United Kingdom. Crit Care. 2006;10:R81. 13. Pearse R, Harrison D, James P, Watson D, Hinds C, Rhodes A, et al. Identification and characterisation of the high-risk surgical population in the United Kingdom. Crit Care. 2006;10:R81. Authors’ contribution IJ wrote the protocol and the first draft of the manuscript. IJ, MT and AU participated in the study design and data collection. All authors wrote, read and approved the final manuscript. 16. Maguire S, Rinehart J, Vakharia S, Cannesson M. Technical communication: respiratory variation in pulse pressure and plethysmographic waveforms: intraoperative applicability in a North American academic center. Anesth Analg. 2011;112(1):94–6. doi:10.1213/ANE.0b013e318200366b. 16. Maguire S, Rinehart J, Vakharia S, Cannesson M. Technical communication: respiratory variation in pulse pressure and plethysmographic waveforms: intraoperative applicability in a North American academic center. Anesth Analg. 2011;112(1):94–6. doi:10.1213/ANE.0b013e318200366b. Competing interests The authors declare that they have no competing interests 15. Boyd O, Grounds RM, Bennett ED. A randomized clinical trial of the effect of deliberate perioperative increase of oxygen delivery on mortality in high-risk surgical patients. JAMA. 1993;270(22):2699–707. 15. Boyd O, Grounds RM, Bennett ED. A randomized clinical trial of the effect of deliberate perioperative increase of oxygen delivery on mortality in high-risk surgical patients. JAMA. 1993;270(22):2699–707. Received: 6 February 2015 Accepted: 30 June 2015 Received: 6 February 2015 Accepted: 30 June 2015 20. Michard F, Chemla D, Teboul JL. Applicability of pulse pressure variation: how many shades of grey? Crit Care. 2015;19(1):144. doi:10.1186/s13054-015-0869-x. 21. Hoiseth LO, Hoff IE, Myre K, Landsverk SA, Kirkeboen KA. Dynamic variables of fluid responsiveness during pneumoperitoneum and laparoscopic surgery. Acta Anaesthesiol Scand. 2012;56(6):777–86. doi:10.1111/j.1399-6576.2011.02641.x. Acknowledgements Th d 17. Duperret S, Lhuillier F, Piriou V, Vivier E, Metton O, Branche P, et al. Increased intra-abdominal pressure affects respiratory variations in arterial pressure in normovolaemic and hypovolaemic mechanically ventilated healthy pigs. Intensive Care Med. 2007;33(1):163–71. doi:10.1007/s00134-006-0412-2. This study was supported by an unrestricted grant by The Eckbo Foundations, Norway and departmental funding. We thank Dr. Gro Østgaard for s pporting the st d and for critical appraisal of the man script This study was supported by an unrestricted grant by The Eckbo Foundations, Norway and departmental funding. We thank Dr. Gro Østgaard for supporting the study and for critical appraisal of the manuscript. for supporting the study and for critical appraisal of the manuscript. 18. Tournadre JP, Allaouchiche B, Cayrel V, Mathon L, Chassard D. Estimation of cardiac preload changes by systolic pressure variation in pigs undergoing pneumoperitoneum. Acta Anaesthesiol Scand. 2000;44(3):231–5. Abbreviations ASA: American society of anesthesia score; GDFT: Goal-directed fluid therapy; SVV: Stroke volume variation. 14. Montenij L, de Waal E, Frank M, van Beest P, de Wit A, Kruitwagen C, et al. Influence of early goal-directed therapy using arterial waveform analysis on major complications after high-risk abdominal surgery: study protocol for a multicenter randomized controlled superiority trial. Trials. 2014;15:360. doi:10.1186/1745-6215-15-360. 14. Montenij L, de Waal E, Frank M, van Beest P, de Wit A, Kruitwagen C, et al. Influence of early goal-directed therapy using arterial waveform analysis on major complications after high-risk abdominal surgery: study protocol for a multicenter randomized controlled superiority trial. Trials. 2014;15:360. doi:10.1186/1745-6215-15-360. Additional files 11. Cannesson M, Aboy M, Hofer CK, Rehman M. Pulse pressure variation: where are we today? J Clin Monit Comput. 2011;25(1):45–56. doi:10.1007/ s10877-010-9229-1. Additional file 1: Study protocol. Study protocol, Word 2010 document. Additional file 2: De-identified SPSS database containing collected data of all randomized patients. SPSS Statistics Data Document. Additional file 1: Study protocol. Study protocol, Word 2010 document. 12. Jammer I, Ulvik A, Erichsen C, Lodemel O, Ostgaard G. Does central venous oxygen saturation-directed fluid therapy affect postoperative morbidity after colorectal surgery? A randomized assessor-blinded controlled trial. Anesthesiology. 2010;113(5):1072–80. doi:10.1097/ALN.0b013e3181f79337. Additional file 2: De-identified SPSS database containing collected data of all randomized patients. SPSS Statistics Data Document. Author details 1 f 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway. 2Department of Anaesthesia and Intensive Care, Haukeland University Hospital, 5021 Bergen, Norway. 3Department of Anesthesiology and Intensive Care, Oulu University Hospital, PL 21, 90029 Oulu, Finland. 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway. 2Department of Anaesthesia and Intensive Care, Haukeland University 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway. 2Department of Anaesthesia and Intensive Care, Haukeland University Hospital, 5021 Bergen, Norway. 3Department of Anesthesiology and Intensive Care, Oulu University Hospital, PL 21, 90029 Oulu, Finland. 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway. 2Department of Anaesthesia and Intensive Care, Haukeland University 1Department of Clinical Medicine, University of Bergen, 5020 Bergen, Norway. 2Department of Anaesthesia and Intensive Care, Haukeland University 19. Guenoun T, Aka EJ, Journois D, Philippe H, Chevallier JM, Safran D. Effects of laparoscopic pneumoperitoneum and changes in position on arterial pulse pressure wave-form: comparison between morbidly obese and normal-weight patients. Obes Surg. 2006;16(8):1075–81. doi:10.1381/096089206778026253. Hospital, 5021 Bergen, Norway. 3Department of Anesthesiology and Intensive Care, Oulu University Hospital, PL 21, 90029 Oulu, Finland. Hospital, 5021 Bergen, Norway. 3Department of Anesthesiology and Intensive Care, Oulu University Hospital, PL 21, 90029 Oulu, Finland. 32. Hamilton MA, Cecconi M, Rhodes A. A systematic review and meta-analysis on the use of preemptive hemodynamic intervention to improve postoperative outcomes in moderate and high-risk surgical patients. Anesth Analg. 2011;112(6):1392–402. doi:10.1213/ANE.0b013e3181eeaae5. 31. Phan TD, D’Souza B, Rattray MJ, Johnston MJ, Cowie BS. A randomised controlled trial of fluid restriction compared to oesophageal Doppler- guided goal-directed fluid therapy in elective major colorectal surgery within an Enhanced Recovery After Surgery program. Anaesth Intensive Care. 2014;42(6):752–60. References Br J Anaesth. 2012;109(2):191–9. doi:10.1093/bja/aes163. 6. Cecconi M, Corredor C, Arulkumaran N, Abuella G, Ball J, Grounds RM, et al. Clinical review: goal-directed therapy-what is the evidence in surgical patients? The effect on different risk groups. Crit Care. 2013;17(2):209. doi:10.1186/cc11823. 6. Cecconi M, Corredor C, Arulkumaran N, Abuella G, Ball J, Grounds RM, et al. Clinical review: goal-directed therapy-what is the evidence in surgical patients? The effect on different risk groups. Crit Care. 2013;17(2):209. doi:10.1186/cc11823. 27. Srinivasa S, Taylor MH, Singh PP, Yu TC, Soop M, Hill AG. Randomized clinical trial of goal-directed fluid therapy within an enhanced recovery protocol for elective colectomy. Br J Surg. 2013;100(1):66–74. doi:10.1002/bjs.8940. 7. Banz VM, Jakob SM, Inderbitzin D. Review article: improving outcome after major surgery: pathophysiological considerations. Anesth Analg. 2011;112(5):1147–55. doi:10.1213/ANE.0b013e3181ed114e. 28. Srinivasa S, Taylor MH, Singh PP, Lemanu DP, MacCormick AD, Hill AG. Goal-directed fluid therapy in major elective rectal surgery. Int J Surg. 2014;12(12):1467–72. doi:10.1016/j.ijsu.2014.11.010. 8. Lees N, Hamilton M, Rhodes A. Clinical review: goal-directed therapy in high risk surgical patients. Crit Care. 2009;13:231. 29. Pestana D, Espinosa E, Eden A, Najera D, Collar L, Aldecoa C, et al. Perioperative goal-directed hemodynamic optimization using noninvasive cardiac output monitoring in major abdominal surgery: a prospective, randomized, multicenter, pragmatic trial: POEMAS Study (PeriOperative goal-directed thErapy in Major Abdominal Surgery). Anesth Analg. 2014;119(3):579–87. doi:10.1213/ANE.0000000000000295. 9. Benes J, Chytra I, Altmann P, Hluchy M, Kasal E, Svitak R, et al. Intraoperative fluid optimization using stroke volume variation in high risk surgical patients: results of prospective randomized study. Crit Care. 2010;14(3):R118. doi:10.1186/cc9070. 10. Pearse RM, Harrison DA, MacDonald N, Gillies MA, Blunt M, Ackland G, et al. Effect of a perioperative, cardiac output-guided hemodynamic therapy algorithm on outcomes following major gastrointestinal surgery: a randomized clinical trial and systematic review. JAMA. 2014;311(21):2181–90. doi:10.1001/ jama.2014.5305. 30. Moppett IK, Rowlands M, Mannings A, Moran CG, Wiles MD, Investigators N. LiDCO-based fluid management in patients undergoing hip fracture surgery under spinal anaesthesia: a randomized trial and systematic review. Br J Anaesth. 2015;114(3):444–59. doi:10.1093/bja/aeu386. Page 6 of 6 Jammer et al. Jammer et al. Perioperative Medicine (2015) 4:6 References Perioperative Medicine (2015) 4:6 Page 6 of 6 Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W3013484605	https://bmcpediatr.biomedcentral.com/track/pdf/10.1186/s12887-020-1916-6.pdf	English	null	Gastrointestinal hemorrhage before anticoagulant therapy in Kawasaki disease: a case report	BMC pediatrics	2,020	cc-by	3,707	Background gastrointestinal hemorrhage due to duodenal ulcer before the use of aspirin. Favorable outcomes for these patients may depend upon the keen judgment of pediatricians famil- iar with the disease’s presentation and management. Kawasaki disease (KD) is an acute febrile multisystem vas- culitis of unknown etiology, especially involving coronary arteries, and has been recognized to be the most common cause of acquired heart disease in children [1]. This vascu- litis results in exudative change with infiltration of lympho- cytes and large mononuclear cells into the media and adventitia of the vessels with endothelial necrosis [1]. Al- though inflammation of the coronary arteries results in the most important clinical outcomes, these problems of vessels may also occur in many other organs. Although the preva- lence of gastrointestinal involvement in KD is unknown, gastrointestinal symptoms including vomiting, diarrhea, and abdominal pain are not uncommon [2]. However, gastrointestinal hemorrhage in patients with KD is rather rare. Herein, we report a 4-year-old boy with KD initially presenting with abdominal pain and complicated by © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Gastrointestinal hemorrhage before anticoagulant therapy in Kawasaki disease: a case report Chenmin Hu and Yanping Yu* Abstract Background: Kawasaki disease (KD) is an acute febrile multisystem vasculitis and has been recognized to be the most common cause of acquired heart disease in children. Owing to its propensity to involve vessels throughout the entire body, KD often mimics other disease processes. The diagnosis might be delayed if other prominent symptoms appear before the characteristic clinical features of KD. Although gastrointestinal symptoms including vomiting, diarrhea, and abdominal pain are not uncommon in KD patients, KD with gastrointestinal bleeding is quite rare. Case presentation: A previously healthy 4-year-old boy initially presented with abdominal pain, followed by fever, rash, and gastrointestinal hemorrhage, eventually diagnosed as complete KD. The patient recovered smoothly after appropriate management and no subsequent complications occurred in the following months. Conclusion: The diagnosis of KD should be considered in children presenting with abdominal symptoms and fever without definable cause. Pediatricians should be aware of the risk of gastrointestinal bleeding in patients with KD, especially in those with prominent abdominal symptoms. rds: Kawasaki disease, Gastrointestinal hemorrhage, Duodenal ulcer, Abdominal pain, Case report Hu and Yu BMC Pediatrics (2020) 20:32 https://doi.org/10.1186/s12887-020-1916-6 Hu and Yu BMC Pediatrics (2020) 20:32 https://doi.org/10.1186/s12887-020-1916-6 Open Access Case presentation A previously healthy 4-year-old boy presented with a 5- day history of abdominal pain, 4-day history of fever and rash. Five days ago, the boy had a sudden onset of persistent abdominal pain, accompanied by vomiting. Empirically, initial therapy with intravenous antibiotics and cimetidine was performed in local hospital. The next day, fever occurred (39 °C–39.8 °C), accompanied by a miliary rash scattered throughout the body. On the third day, the skin rash increased, accompanied by conjunc- tival hyperemia, erythema and swelling of the hands and feet. The laboratory examinations revealed white blood cell count of 13.14 × 10^9/L with predominance of neu- trophil 90.6%, hemoglobin level of 120 g/L, platelet count of 204 × 10^9/L, and C-reactive protein (CRP) of 101.9 mg/L. Abdominal computed tomography shows * Correspondence: yyp757605@163.com Department of Pediatrics, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China * Correspondence: yyp757605@163.com Department of Pediatrics, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China Hu and Yu BMC Pediatrics (2020) 20:32 Page 2 of 5 Hu and Yu BMC Pediatrics (2020) 20:32 Page 2 of 5 Hu and Yu BMC Pediatrics (2020) 20:32 the accumulation of gas and fluid in the small intestine. An echocardiography showed no dilatation of coronary arteries. On day 5, He vomited a small amount of coffee-like liquid and discharged a moderate amount of tarry stool. Then, the patient was transferred to our hospital by emergency. (the 5th day of fever), a total dose of intravenous im- munoglobulin (IVIG, 2 g/kg) was administered. Aspirin was not administered because of his upper gastrointes- tinal bleeding. After 24 h of IVIG, his fever began to sub- side and the skin rash diminished dramatically. On the second day after IVIG, laboratory findings showed a nor- mal leukocytes of 6.7 × 10^9/L and a remarkable fall of the CRP levels at 5 mg/L and the D-Dimer levels at 1280 μg/L (Table 1). However, the fecal occult blood continued to be positive although his gastrointestinal symptoms were improved. p y g y On admission, physical examination revealed an acutely ill boy with fever of 38.5 °C, pulse of 124 times per minute, breathing of 30 times per minute, and blood pressure of 94/47 mmHg. Case presentation The boy displayed erythema- tous maculopapular rash over the body, bilateral con- junctival hyperemia, erythema and swelling of the hands and feet, dry and cracked lips, oropharyngeal hyperemia and mild strawberry tongue. One enlarged lymph node approximately 1.5 × 1 cm was palpable in the right anter- ior cervical triangle. His abdomen was slightly tense with mild tenderness. Laboratory tests are summarized in Table 1. The main abnormalities were as follows: hemoglobin concentration 108 g/L; white blood cell count 11.5 × 10^9/L, neutrophils 91.5%; platelet count 65 × 10^9/L; CRP 31 mg/L, serum alanine aminotrans- ferase (ALT) 54 U/L, total protein 37.2 g/L, albumin 21.3 g/L, creatine kinase isoenzyme MB (CK-MB) 56 U/ L, potassium 3.11 mmol/L, D-Dimer 6230 μg/L, fecal oc- cult blood +++. In light of these findings and the clinical features, a diagnosis of KD with upper gastrointestinal bleeding was made. The boy received supportive care and intravenous omeprazole (0.6 mg/kg/day). On day 6 y An endoscopy was performed on Day 8 of illness. We found an active ulcer covered with thick “white moss” in duodenal bulb (Fig. 1). Then, the dose of intravenous omeprazole was increased to 0.8 mg/kg/ day. Repeated echocardiography on Day 9 demon- strated a mild dilatation of the left coronary artery (2.8 mm). Oral clopidogrel (1 mg/kg/day) was added to treatment. On day 10, 13C-urea breath test showed that his helicobacter pylori status was negative. On day 12, most of the laboratory parameters were normal or close to normal except for elevated platelet count of 672 × 10^9/L. The next day, he was discharged and continued with oral omeprazole (0.6 mg/kg/day) for 4 weeks and clopidogrel (1 mg/kg/day) for a further 8 weeks. Follow-up at 1 month after discharge revealed no recurrence of fever or remarkable abdominal pain. Repeated echocardiography at 8 weeks after discharge revealed that the dilated left coronary artery regressed to normal. A 1 year follow-up showed no cardiac in- volvement or other complications. Discussion gastrointestinal symptoms in KD identify patients at higher risk for IVIG-resistance and for the development of coronary aneurysms [7]. Therefore, the diagnosis of KD should be considered in children with gastrointes- tinal symptoms and prolonged fever without clear cause. KD is the most common vasculitis in children [3]. Even though more than 50 years have passed since the first description of this disease, the diagnosis of KD continues to remain a clinical dilemma and there is no confirma- tory laboratory test [4]. The diagnosis of KD is based on the presence of fever (lasting > 5 days) together with 4 of the 5 characteristic diagnostic criteria (rash, oropharyn- geal changes, non-purulent conjunctivitis, cervical lymphadenopathy and change of the extremities) [1, 4]. A prompt diagnosis is crucial because prognosis relies on timely initiation of the optimal treatment. The most common and life-threatening complication of KD, cor- onary artery abnormalities (CAA), will occur in approxi- mately 25% of untreated patients which may lead to death by causing coronary thrombosis or myocardial infarction. The delayed diagnosis of KD could result in life-threatening problems particularly including cardio- vascular sequelae. y p p g Our patient presented with abdominal pain at the on- set of disease. Fortunately, the followed fever, rash and other typical signs of KD led to timely diagnosis and treatment. Although laboratory tests are nonspecific, they can provide support for a diagnosis of KD in pa- tients with suggestive clinical features. For our patients, laboratory tests supporting KD diagnosis included changes of markers at acute phase such as leukocytosis, elevated CRP, hypoalbuminemia, mild elevations of ALT, and mild elevations of CK-MB and thrombocytosis on day 12 of illness. Thrombocytosis is a characteristic feature of KD but generally does not occur until the sec- ond week, peaking in the third week and normalizing by 4 to 6 weeks after onset in most cases [1, 5]. In addition, thrombocytopenia and elevations of D-Dimer in our pa- tient were detected in the first week of illness. Although uncommon, thrombocytopenia in acute phase of KD may be a sign of disseminated intravascular coagulation and is a risk factor for the development of coronary ar- tery abnormalities. Owing to its propensity to involve the medium-sized arteries in multiple organs, KD often mimics other dis- ease processes. Discussion The diagnosis might be delayed if other prominent symptoms appear before the characteristic clinical features of KD, especially in children with in- complete or atypical forms. Incomplete KD occurs in pa- tients presenting a typical fever without a sufficient number of main clinical criteria, with or without CAA. This kind of KD is frequent in children younger than 12–24 months and should be considered in the differen- tial diagnosis of prolonged unexplained fever in child- hood associated with any of the characteristic clinical features of the disease [1]. The diagnosis can be consid- ered confirmed when coronary artery aneurysms are identified in such patients by echocardiography. How- ever, coronary artery dilatation is generally not detected by echocardiography until after the first week of illness, and a normal echocardiogram in the first week of illness does not rule out the diagnosis of KD. The term atypical KD is used when there are one or more symptoms or signs different from the main KD clinical features such as pneumonia, meningoencephalitis, facial paralysis, acute abdomen, arthritis, cholestatic jaundice, and renal injury [5]. The diagnosis of KD is more challenging in the presence of one or more atypical manifestations. The mechanism of gastrointestinal involvement at the onset of KD has not yet been clarified. The possible ex- planation is that gastrointestinal tract is the first organ involved with systemic vasculitis of the medium-sized arteries, leading to abdominal pain and vomiting. How- ever, the etiology and the entire pathophysiology of KD remain elusive, despite a host of data supports the no- tion that the pathogenesis of KD is closely associated with dysregulation of immune responses to infectious agents [8, 9]. The gastrointestinal tract has been pro- posed as a major site of entry of infectious agents that might act as superantigens, even though more recent studies have favored a canonical response to a conven- tional antigen [1]. Recently, an increasing number of studies have focused on the contribution of gut micro- biota to KD. Esposito et al. postulated that an imbalance in the gut microbiota might indirectly interfere with the normal function of immunity, and that variable micro- biota interactions with environmental factors, mainly in- fectious agents, might selectively drive the development of KD in genetically susceptible children [10]. The prevalence of gastrointestinal involvement in KD is unknown as available data can only be derived from single case and case series. Zulian et al. Case presentation Table 1 Laboratory data during acute phase Parameters Day 3 Day 5 Day 8 Day 12 Leukocytes (× 10^9/L) 13.14 11.5 6.7 9.8 Neutrophil (%) 90.6 91.5 40.1 57.0 Hemoglobin (g/L) 120 108 102 103 Platelet (×10^9/L) 204 65 115 672 C-reactive protein (mg/L) 101.9 31 5 3 Total protein (g/L) / 37.2 51 52.3 Albumin (g/L) / 21.3 21.7 25.9 Alanine aminotransferase (U/L) / 54 19 11 Total bilirubin (μmol/L) / 6.3 6.6 5.2 Creatinine (μmol/L) / 59 47 37 Creatine kinase (U/L) / 61 39 33 Creatine kinase isoenzyme MB (U/L) / 56 27 23 Potassium (mmol/L) / 3.11 3.59 4.47 Sodium (mmol/L) / 131 132 135 Chlorine (mmol/L) / 99 101 105 Prothrombin time (sec) / 13.3 12.5 / D-Dimer (μg/L) / 6230.0 1280 700.0 Serum amylase (U/L) / 33 / / Fecal occult blood / +++ + / Fig. 1 Gastroscopy results showing an active ulcer covered with thick “white moss” in duodenal bulb Fig. 1 Gastroscopy results showing an active ulcer covered with thick “white moss” in duodenal bulb Hu and Yu BMC Pediatrics (2020) 20:32 Page 3 of 5 Hu and Yu BMC Pediatrics (2020) 20:32 Page 3 of 5 Discussion in their case series reported an incidence of atypical KD with a clin- ical onset characterized by acute abdomen of 4.6% [6]. Gastrointestinal symptoms at KD onset can complicate clinical recognition, lead to unnecessary invasive inter- ventions and cause therapeutic delay, thus increasing the risk of cardiac complications [2]. A retrospective multi- center report from Italy demonstrated that presenting Compared to the frequently reported gastrointestinal symptoms, KD with gastrointestinal hemorrhage is quite rare. Only 5 previous cases have been documented in the English literature, as showed in Table 2. Two cases of severe gastrointestinal hemorrhage complicating high- dose aspirin therapy were iatrogenic [11]. Zulian et al. reported a 20-month-old KD boy who had sudden onset of hematemesis without aspirin and presented with Hu and Yu BMC Pediatrics (2020) 20:32 Page 4 of 5 Table 2 Clinical characteristics of the reported patients with gastrointestinal hemorrhage in Kawasaki disease Year First author Age/sex Clinical onset Details of gastrointestinal bleeding Blood transfusion Endoscopy Surgery 1996 Matsubara 2 y/M Fever Hematemesis on day 19 of illness (day 13 of aspirin) Yes Revealed a 2 cm duodenal ulcer None 1996 Matsubara 4 y/F Fever Melanotic stools followed by emesis of blood on day 31 of illness (day 26 of aspirin) Yes None None 2003 Zulian 20 m/M Fever Hematemesis on day 7 of illness without aspirin Yes Showed diffuse hemorrhagic duodenitis None 2004 Chang 5 y/M Fever, cervical lymphadenitis Tarry stool on day 6 of illness; Massive gastrointestinal bleeding on day 14 of illness (day 5 of aspirin) Yes NA NA 2007 Singh 4.5 y/M Nausea, vomiting, diarrhea and fever Hemorrhagic shock with hematemesis and hematochezia at 2 months after onset (without aspirin) Yes Failed because of the massive bleeding Revaeled a 1.5 cm duodenal ulcer with active bleeding M male, F female, y year, m month, NA not available with gastrointestinal hemorrhage in Kawasaki disease M male, F female, y year, m month, NA not available confer any benefit with regards to inflammation and it did not appear to improve treatment outcomes [14]. A recent meta-analysis revealed that low-dose aspirin (3–5 mg/kg/d) may be as effective as the use of high-dose as- pirin (> 30 mg/kg/d) for the initial treatment of KD [15]. Accordingly, we believe that high-dose aspirin may be unnecessary in KD with prominent gastrointestinal symptoms. Funding This work did not receive funding. Authors’ contributions YY designed the work and drafted the manuscript. CH collected literatures and was a major contributor in writing the manuscript. Both authors read and approved the final manuscript. IVIG and aspirin were considered as the standard ini- tial treatment of KD for decades. The efficacy of IVIG administered in the acute phase of KD is well established to reduce the prevalence of CAA [1]. However, the role of high-dose aspirin in KD remains controversial. Kuo et al. demonstrated that high-dose aspirin in KD did not Discussion However, further well-designed prospective studies are still required to evaluate the efficacy of low- dose aspirin for KD. diffuse hemorrhagic duodenitis by endoscopy [6]. Singh et al. reported a 4.5-year-old KD boy who developed hemorrhagic shock and revealed a duodenal ulcer with active bleeding by surgery [12]. Chang et al. reported a 5-year-old KD boy who had tarry stool on day 6 of fever. Three days later, he was still treated with high-dose as- pirin and massive gastrointestinal bleeding occurred on day 5 of aspirin treatment [13]. Before the definitive diagnosis of KD, our patient de- veloped upper gastrointestinal bleeding confirmed by the subsequent endoscopy. On day 5 of fever, he received intravenous immunoglobulin and omeprazole but not high-dose aspirin. The iatrogenic second bleeding as in Chang’s case was avoided. Generally, when high-dose as- pirin is discontinued after KD patient has been afebrile for 48 to 72 h, low-dose aspirin (3–5 mg/kg/d) should be started and continued until the patient has no evidence of coronary changes by 6 to 8 weeks after onset of ill- ness. For children who develop coronary abnormalities, aspirin may be continued indefinitely. Clopidogrel (0.2– 1 mg/kg/d) can be used in place of low-dose aspirin for patients who are aspirin resistant or allergic to aspirin [1]. For our patient, aspirin may have a higher risk of ag- gravating duodenal ulcer. As an alternative anticoagulant therapy, clopidogrel was started and continued for 8 weeks after discharge when a mild dilatation of coronary artery was revealed on day 9 of illness. Finally, our pa- tient completely recovered. Conclusions The diagnosis of KD should be considered in children presenting with abdominal symptoms and fever without definable cause. Pediatricians should be aware of the risk of gastrointestinal bleeding in patients with KD, espe- cially in those with prominent abdominal symptoms. Acknowledgements Not applicable. Acknowledgements Not applicable. Not applicable. Abbreviations ALT: Alanine aminotransferase; CAA: Coronary artery abnormalities; CK- MB: Creatine kinase isoenzyme MB; CRP: C-reactive protein; IVIG: Intravenous immunoglobulin; KD: Kawasaki disease Abbreviations Abbreviations ALT: Alanine aminotransferase; CAA: Coronary artery abnormalities; CK- MB: Creatine kinase isoenzyme MB; CRP: C-reactive protein; IVIG: Intravenous immunoglobulin; KD: Kawasaki disease References Chang CH, Chen MH, Yang W. Kawasaki disease presenting with lymphadenopathy and gastrointestinal hemorrhage: report of one case. Acta Paediatr Taiwan. 2004;45:171–3. 14. Kuo HC, Lo MH, Hsieh KS, Guo MM, Huang YH. High-Dose Aspirin is Associated with Anemia and Does Not Confer Benefit to Disease Outcomes inKawasaki Disease. PLoS One. 2015;10:e0144603. 14. Kuo HC, Lo MH, Hsieh KS, Guo MM, Huang YH. High-Dose Aspirin is Associated with Anemia and Does Not Confer Benefit to Disease Outcomes inKawasaki Disease. PLoS One. 2015;10:e0144603. 15. Zheng X, Yue P, Liu L, et al. Efficacy between low and high dose aspirin for the initial treatment of Kawasaki disease: Current evidence based on a meta-analysis. PLoS One. 2019;14:e0217274. 15. Zheng X, Yue P, Liu L, et al. Efficacy between low and high dose aspirin for the initial treatment of Kawasaki disease: Current evidence based on a meta-analysis. PLoS One. 2019;14:e0217274. References 1. McCrindle BW, Rowley AH, Newburger JW, et al. Diagnosis, treatment, and long-term Management of Kawasaki Disease: a scientific statement for health professionals from the American Heart Association. Circulation. 2017; 135(17):e927–99. 2. Colomba C, La Placa S, Saporito L, et al. Intestinal Involvement in Kawasaki Disease. J Pediatr. 2018;202:186–93. 3. Singh S, Vignesh P, Burgner D. The epidemiology of Kawasaki disease: a global update. Arch Dis Child. 2015;100(11):1084–8. global update. Arch Dis Child. 2015;100(11):1084–8. 4. Singh S, Jindal AK, Pilania RK. Diagnosis of Kawasaki disease. Int J Rheum Dis. 2018;21:36–44. 5. Marchesi A, Tarissi de Jacobis I, Rigante D, et al. Kawasaki disease: guidelines of the Italian Society of Pediatrics, part I - definition, epidemiology, etiopathogenesis, clinical expression and management of the acute phase. Ital J Pediatr. 2018;44(1):102. 6. Zulian F, Falcini F, Zancan L, et al. Acute surgical abdomen as presenting manifestation of Kawasaki disease. J Pediatr. 2003;142:731–5. 7. Fabi M, Corinaldesi E, Pierantoni L, et al. Gastrointestinal presentation of Kawasaki disease: a red flag for severe disease? PLoS One. 2018;13(9): e0202658. 8. Principi N, Rigante D, Esposito S. The role of infection in Kawasaki syndrome. J Inf Secur. 2013;67(1):1–10. 8. Principi N, Rigante D, Esposito S. The role of infection in Kawasaki syndrome. J Inf Secur. 2013;67(1):1–10. 9. Nakamura A, Ikeda K, Hamaoka K. Aetiological significance of infectious stimuli in Kawasaki disease. Front Pediatr. 2019;7:244. 9. Nakamura A, Ikeda K, Hamaoka K. Aetiological significance of infectious stimuli in Kawasaki disease. Front Pediatr. 2019;7:244. 10. Esposito S, Polinori I, Rigante D. The gut microbiota-host partnership as a potential driver of Kawasaki syndrome. Front Pediatr. 2019;7:124. 10. Esposito S, Polinori I, Rigante D. The gut microbiota-host partnership as a potential driver of Kawasaki syndrome. Front Pediatr. 2019;7:124. 11. Matsubara T, Mason W, Kashani IA, Kligerman M, Burns JC. Gastrointestinal hemorrhage complicating aspirin therapy in acute Kawasaki disease. J Pediatr. 1996;128:701–3. 11. Matsubara T, Mason W, Kashani IA, Kligerman M, Burns JC. Gastrointestinal hemorrhage complicating aspirin therapy in acute Kawasaki disease. J Pediatr. 1996;128:701–3. 12. Singh R, Ward C, Walton M, et al. Atypical Kawasaki disease and gastrointestinal manifestations. Paediatr Child Health. 2007;12:235–7. 12. Singh R, Ward C, Walton M, et al. Atypical Kawasaki disease and gastrointestinal manifestations. Paediatr Child Health. 2007;12:235–7. 13. Chang CH, Chen MH, Yang W. Kawasaki disease presenting with lymphadenopathy and gastrointestinal hemorrhage: report of one case. Acta Paediatr Taiwan. 2004;45:171–3. 13. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Page 5 of 5 Hu and Yu BMC Pediatrics (2020) 20:32 Hu and Yu BMC Pediatrics Received: 17 October 2019 Accepted: 7 January 2020 Received: 17 October 2019 Accepted: 7 January 2020 Ethics approval and consent to participate The study protocol was approved by the Ethics Commission of Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine. Permission to carry out the study and access patient records was sought from the respective hospital administrators. Consent for publication Written consent for publication of this anonymous information was obtained from the patient’s parents and the proof of consent can be requested at any time. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
W4242536624.txt	http://ejournal.skpm.ipb.ac.id/index.php/jskpm/article/download/593/146	id		Halaman Cover Depan	Jurnal Sains Komunikasi dan Pengembangan Masyarakat (e-journal)	1,970	cc-by-sa	439	JURNAL SAINS KOMUNIKASI DAN PENGEMBANGAN MASYARAKAT Vol. 2 No. 4, Juni 2018 ISSN: 2338-8021; E-ISSN: 2338-8269 PEMIMPIN REDAKSI Ivanovich Agusta, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor REDAKTUR Endriatmo Soetarto, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Dwi Retno Hapsari, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Dwi Sadono, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Mahmudi Siwi, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Muhammad Shohibuddin, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Nur Isyana Wianti, Jurusan Penyuluhan Pertanian, Fakultas Pertanian, Universitas Halu Uleo Ratri Virianita, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Rina Mardiana, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Rokhani, Program Studi Agribisnis, Jurusan Sosial Ekonomi Pertanian, Fakultas Pertanian, Universitas Jember Sarwititi Sarwoprasodjo, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor Septri Widiono, Jurusan Sosial Ekonomi Pertanian, Fakultas Pertanian, Universitas Bengkulu Zessy Ardinal Barlan, Departemen Sains Komunikasi dan Pengembangan Masyarakat, Fakultas Ekologi Manusia, Institut Pertanian Bogor REDAKTUR PELAKSANA Titania Aulia; Rai Sita; Widya Hasian Situmeang TEKNISI DAN ADMINISTRASI Cepi Surharyadi; Anggra Irena Bondar; Khairunnisa; Dhiny Diterbitkan Oleh Departemen Sains Komunikasi dan Pengembangan Masyarakat Fakultas Ekologi Manusia – Institut Pertanian Bogor Wing 1 Level 5, Jl. Kamper, Kampus IPB Darmaga, Bogor, Jawa Barat 16680 Kontak Mahmudi Siwi Telp/Fax. 0251 – 8627793; HP. 0811180230; Email: skpm@apps.ipb.ac.id Website: http://ejournal.skpm.ipb.ac.id/index.php/jskpm DOI: https://doi.org/10.29244/jskpm.2.3.i-iii ii JURNAL SAINS KOMUNIKASI DAN PENGEMBANGAN MASYARAKAT Vol. 2 No. 4, Juni 2018 ISSN: 2338-8021; E-ISSN: 2338-8269 DOI: https://doi.org/10.29244/jskpm.2.3.i-iii DAFTAR ISI Analisis Perilaku Ibu Rumah Tangga Bantaran Sungai Citampian dalam Mengelola Sampah Rumah Tangga Rahmat Darmawan, Anna Fatchiya 431 Hubungan Gaya Kepemimpinan Ketua dan Efektivitas Kelompok Wanita Tani TOGA Iqbal Syahroni, Siti Amanah 441 Gaya Kepemimpinan Tokoh Informal dan Tingkat Partisipasi Masyarakat dalam Program Desa Mandiri Pangan Mochammad Yunus Gerry Fitriadi, Sumardjo, Hamzah 455 Modal Sosial dan Partisipasi Masyarakat dalam Pembangunan Desa Wisata Tamansari di Kabupaten Banyuwangi Eka Puspitaningrum, Djuara P Lubis 465 Peran Organisasi Pembelajar dalam Gerakan Petani Fani Dwi Iswari, Endriatmo Soetarto, Mohammad Shohibuddin 485 Kemampuan Aparat Pemerintah Desa dan Efektivitas Pengelolaan Keuangan Desa Yunianingrum, Lala M Kolopaking 495 Hubungan Partisipasi Masyarakat dengan Keberlanjutan Ekologi, SosialBudaya dan Ekonomi dalam Ekowisata Religi Mohammad Irfan Rachman, Rina Mardiana 509 Pencapaian Program Keluarga Harapan (PKH) Wulandari, Pudji Muljono 525 Hubungan Tingkat Partisipasi Laki-Laki dengan Tingkat Keberdayaan Ekonomi Perempuan Fahmi Taufiqurrahman, Titik Sumarti, Sriwulan Ferindian Falatehan 539 Strategi Nafkah, Karakter Sosio-Budaya dan Relasi Sosial Rumahtangga Penambang Belerang Muhammad Rifky Afqari, Arya Hadi Dharmawan 551 iii
https://openalex.org/W4388848649	https://www.mdpi.com/2218-273X/13/12/1681/pdf?version=1700541288	English	null	A Comparative Analysis of MicroRNA Expression in Mild, Moderate, and Severe COVID-19: Insights from Urine, Serum, and Nasopharyngeal Samples	Biomolecules	2,023	cc-by	10,230	Article A Comparative Analysis of MicroRNA Expression in Mild, Moderate, and Severe COVID-19: Insights from Urine, Serum, and Nasopharyngeal Samples Raya Soltane 1,* , Nuha Almulla 1, Ahlam Alasiri 1 , Nabila F. Elashmawy 2, Alaa T. Qumsani 3 , Fatimah M. Alshehrei 3 , Doaa El-Ghareeb Keshek 3,4, Taha Alqadi 1 , Saleh Bakheet AL-Ghamdi 5 and Abdou Kamal Allayeh 6,* 1 Department of Biology, Adham University College, Umm Al-Qura University, Makkah 21955, Saudi Arabia; ll@ d (N A ) j i i@ d (A A ) t di@ d (T A ) 1 Department of Biology, Adham University College, Umm Al-Qura University, Makkah 21955, Saudi Arabia; namull@uqu.edu.sa (N.A.); ajasiri@uqu.edu.sa (A.A.); taqadi@uqu.edu.sa (T.A.) 2 1 Department of Biology, Adham University College, Umm Al-Qura University, Makkah 21955, Saudi Arabia; namull@uqu.edu.sa (N.A.); ajasiri@uqu.edu.sa (A.A.); taqadi@uqu.edu.sa (T.A.) 2 Biology Department, College of Science, Jazan University, Jazan 82817, Saudi Arabia; nelashmawy@jazanu.edu.sa 3 Department of Biology, Jumum College University, Umm Al-Qura University, P.O. Box 7388, Makkah 21955, Saudi Arabia; atqumsani@uqu.edu.sa (A.T.Q.); fmshehrei@uqu.edu.sa (F.M.A.); dekeshek@uqu.edu.sa (D.E.-G.K.) 4 Agriculture Genetic Engineering Research Institute (AGERI), Agriculture Research Centre, Giza 12512, Egypt 5 Biology Department, Faculty of Science, Al-Baha University, Al Baha 65947, Saudi Arabia; sb.alghamdi@bu.edu.sa 6 Virology Lab 176, Environment and Climate Change Institute, National Research Centre, Giza 12622, Egypt * Correspondence: rasoltan@uqu edu sa (R S ); ak allayeh@nrc sci eg (A K A ) Virology Lab 176, Environment and Climate Change Institute, National Research Centre, Giza 12622, Egypt Correspondence: rasoltan@uqu.edu.sa (R.S.); ak.allayeh@nrc.sci.eg (A.K.A.) 6 Virology Lab 176, Environment and Climate Change Institute, National Research Centre, Giza 12622, Egypt * Correspondence: rasoltan@uqu.edu.sa (R.S.); ak.allayeh@nrc.sci.eg (A.K.A.) gy , g , , , gyp * Correspondence: rasoltan@uqu.edu.sa (R.S.); ak.allayeh@nrc.sci.eg (A.K.A.) Abstract: COVID-19, caused by the SARS-CoV-2 virus, manifests with a wide range of clinical symp- toms that vary from mild respiratory issues to severe respiratory distress. To effectively manage and predict the outcomes of the disease, it is important to understand the molecular mechanisms underlying its severity. This study focuses on analyzing and comparing the expression patterns of microRNAs (miRNAs) in serum, urine, and nasopharyngeal samples from patients with mild, mod- erate, and severe COVID-19. The aim is to identify potential associations with disease progression and discover suitable markers for diagnosis and prognosis. Our ﬁndings indicate the consistent upregulation of miR-21, miR-146a, and miR-155 in urine, serum, and nasopharyngeal samples from patients with mild COVID-19. In moderate cases, there were more signiﬁcant changes in miRNA expression compared to mild cases. Article A Comparative Analysis of MicroRNA Expression in Mild, Moderate, and Severe COVID-19: Insights from Urine, Serum, and Nasopharyngeal Samples Speciﬁcally, miR-let-7 demonstrated upregulation, while miR- 146b exhibited downregulation. The most notable alterations in miRNA expression proﬁles were observed in severe COVID-19 cases, with a signiﬁcant upregulation of miR-223. Moreover, our analysis using Receiver-operating characteristic (ROC) curves demonstrated that miR-155, miR-let-7, and miR-223 exhibited high sensitivity and speciﬁcity, suggesting their potential as biomarkers for distinguishing COVID-19 patients from healthy individuals. Overall, this comparative analysis revealed distinct patterns in miRNA expression. The overlapping expression patterns of miR- NAs in urine, serum, and nasopharyngeal samples suggest their potential utility in discriminating disease status. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Citation: Soltane, R.; Almulla, N.; Alasiri, A.; Elashmawy, N.F.; Qumsani, A.T.; Alshehrei, F.M.; Keshek, D.E.-G.; Alqadi, T.; AL-Ghamdi, S.B.; Allayeh, A.K. A Comparative Analysis of MicroRNA Expression in Mild, Moderate, and Severe COVID-19: Insights from Urine, Serum, and Nasopharyngeal Samples. Biomolecules 2023, 13, 1681. https://doi.org/10.3390/ biom13121681 biomolecules biomolecules biomolecules Citation: Soltane, R.; Almulla, N.; Alasiri, A.; Elashmawy, N.F.; Qumsani, A.T.; Alshehrei, F.M.; Keshek, D.E.-G.; Alqadi, T.; AL-Ghamdi, S.B.; Allayeh, A.K. A Comparative Analysis of MicroRNA Expression in Mild, Moderate, and Severe COVID-19: Insights from Urine, Serum, and Nasopharyngeal Samples. Biomolecules 2023, 13, 1681. https://doi.org/10.3390/ biom13121681 Academic Editors: Y-h. Taguchi and Hsiuying Wang Academic Editors: Y-h. Taguchi and Hsiuying Wang Received: 13 October 2023 Revised: 9 November 2023 Accepted: 17 November 2023 Published: 21 November 2023 Keywords: SARS-CoV-2; COVID-19; nasopharyngeal; plasma; urine and MicroRNA expression 1. Introduction COVID-19, a new respiratory infection caused by the SARS-CoV-2 virus, was ﬁrst detected in Wuhan in December 2019 and has led to a signiﬁcant number of deaths world- wide [1]. The prognosis of SARS-CoV-2 infection varies from mild to severe, including conditions like acute respiratory distress syndrome (ARDS) and mortality. Factors such as age, immune response, and underlying health conditions play a role in determining https://www.mdpi.com/journal/biomolecules Biomolecules 2023, 13, 1681. https://doi.org/10.3390/biom13121681 Biomolecules 2023, 13, 1681 2 of 15 the severity of the illness [2,3]. The host’s response to the infection has a signiﬁcant im- pact on the clinical outcomes observed in individuals. Increased levels of cytokines and chemokines, particularly IL-6, IL-8, and tumor necrosis factor, along with lymphopenia and immune cell inﬁltration in affected organs, are known to contribute to the severity of COVID-19 and the accompanying hyper-inﬂammatory responses [4–7]. Clinical literature classiﬁes COVID-19 patients into mild, moderate, or severe cate- gories, but there is a growing consensus among experts that utilizing blood tests to interpret disease progression may yield more accurate results [8]. Given the limited available data, there is a need to identify new non-invasive biomarkers that can differentiate between dif- ferent disease stages of COVID-19. This requires a deeper understanding of the interactions between the virus, host cells, viral pathogenesis, and cellular damage [9]. In the ongoing global effort to combat the COVID-19 pandemic, scientists are actively engaged in studying the underlying molecular mechanisms of the disease. One area of particular interest is the role of microRNAs (miRNAs). MicroRNAs are RNA molecules that do not code for proteins but instead regulate gene expression after transcription by inhibiting translation. Numerous studies have highlighted the crucial roles of microRNAs in controlling various biological processes such as inﬂammation, apoptosis, cell proliferation, and the immune response to viral infections [10,11]. Changes in the levels of cellular miRNAs have been associated with the immune response to respiratory viruses, playing a role in activating antiviral and inﬂammatory pathways [12]. MiRNAs have emerged as potential indicators to identify various diseases, including viral infections, due to their high sensitivity and speciﬁcity [13]. Additionally, during the severe phase of infection, speciﬁc human miRNAs exhibit different expression patterns compared to the moderate phase, suggesting their potential as prognostic biomarkers [14]. Recently, miRNAs have emerged as potential key contributors to the development and progression of COVID-19. 1. Introduction For example, studies suggest that miR-146 may play a role in regulating immune responses and controlling inﬂammation. Dysregulation of this speciﬁc miRNA has been observed in viral infections, including respiratory viruses. Its potential involvement in COVID-19 is currently being explored, with initial evidence suggesting a potential link to disease severity and immune dysregulation. Similarly, miR-193-5p and miR-323a-3p have been associated with renal function and have been linked to kidney injury. In the context of COVID-19, the virus can cause damage to various organs, including the kidneys. Altered expression of these miRNAs may contribute to the renal complications observed in severe cases of COVID-19 [15,16]. Furthermore, miR-17-5p, miR-142-5p, miR-25-5p, miR-29-3p, miR-19b-3p, miR-23a- 3p, miR-92a-3p, and miR-320a are involved in immune regulation and cardiovascular function [17,18]. Other miRNAs, such as miR-21, miR-let-7, miR-146a, miR-146b, miR-155, miR-223, and miR-342, are involved in immune regulation, inﬂammation, lung injury, and ﬁbrosis. For instance, miR-21 plays a role in immune regulation and inﬂammation and has been associated with lung injury and ﬁbrosis, indicating its potential contribution to disease progression [19,20]. Similarly, miR-let-7 is a group of miRNAs involved in immune regulation and inﬂammation, affecting immune cell function and cytokine production. Dysregulation of miR-let-7 has been linked to lung injury and ﬁbrosis, suggesting its potential involvement in these conditions [20]. MiR-146a acts as a crucial regulator of inﬂammation and immune responses, func- tioning as a negative feedback regulator by targeting genes involved in pro-inﬂammatory signaling pathways. Dysregulation of miR-146a has been observed in various inﬂamma- tory diseases and has been associated with lung injury and ﬁbrosis due to its impact on inﬂammatory processes. Similarly, miR-146b is another member of the miR-146 family, which shares similar regulatory functions with miR-146a, acting as a negative regulator of inﬂammation by targeting key signaling molecules involved in immune responses [21,22]. Moreover, miR-155 is a well-known regulator of immune responses and inﬂamma- tion. It is expressed in different immune cells and can inﬂuence the production of pro- inﬂammatory cytokines. Dysregulation of miR-155 has been implicated in several inﬂam- Biomolecules 2023, 13, 1681 3 of 15 3 of 15 matory diseases, and its role in lung injury and ﬁbrosis has been studied, suggesting its potential as a molecular marker in these conditions [23]. MiR-223 is predominantly ex- pressed in myeloid cells and is involved in regulating innate immune responses, affecting the function of immune cells like neutrophils and monocytes and inﬂuencing inﬂammatory processes. 2.1. Subjects and Study Design During a speciﬁc cross-sectional study conducted from January 2021 to June 2021 at Cairo’s Ahmad Maher Teaching Hospital in Egypt, a total of 272 individuals were enrolled as participants. This study aimed to collect various clinical samples, including nasopharyngeal, serum, and urine samples, from both infected individuals and healthy individuals. Ethical approval for the study was secured from the Ethics Committee of the General Organization of Teaching Hospitals and Institutes in Egypt, with reference number #HAM00125. The study adhered to the principles outlined in the Second Declaration of Helsinki, and all participants provided informed consent prior to their involvement. The individuals were divided into distinct groups: healthy donors (n = 57), individu- als with mild COVID-19 cases (n = 106), individuals with moderate COVID-19 cases (n = 71), and individuals with severe COVID-19 cases (n = 39). The diagnosis of COVID-19 was conﬁrmed through molecular testing of nasopharyngeal swabs. The study’s inclusion criteria mandated that participants be at least 18 years of age, while pregnant women and individuals with other respiratory viral infections were excluded from the study. 1. Introduction Dysregulation of miR-223 has been observed in various inﬂammatory diseases, and its involvement in lung injury and ﬁbrosis has been suggested [24]. Lastly, miR-342 is involved in immune response regulation and has been associated with inﬂammation, mod- ulating the expression of genes involved in immune cell function and cytokine production, although its speciﬁc role in lung injury and ﬁbrosis is not extensively studied [25]. g p g j y y In summary, miRNAs have become a focal point of research in understanding COVID- 19. Based on existing literature, miR-21, miR-let-7, miR-146a, miR-146b, miR-155, miR-223, and miR-342, which are associated with inﬂammation, immune regulation, lung injury, and ﬁbrosis, show promise in unraveling the complex mechanisms of COVID-19 and may serve as potential targets for diagnostics and therapeutics. Given the interplay between COVID- 19 infection and these miRNAs, it is reasonable to expect that the expression patterns of these miRNAs during the acute phase of infection would differ from those observed during the mild or moderate phases. Therefore, analyzing the expression patterns of these miRNAs in samples obtained from individuals with different COVID-19 severity levels, in comparison to samples from healthy individuals, can provide valuable insights into the role of these miRNAs in the disease and its severity. 2.2. Deﬁnitions The severity categorization of COVID-19 cases in Egypt was determined by the Min- istry of Health, following the standards set by the World Health Organization (WHO) [26]. The WHO has established guidelines and criteria for classifying the severity of COVID-19 cases, taking into consideration various factors such as clinical symptoms, respiratory function, oxygen saturation levels, and imaging results. In summary, individuals who tested positive for SARS-CoV-2 but did not exhibit symptoms consistent with COVID-19 were categorized as having an asymptomatic infection. Those who displayed various signs and symptoms of COVID-19 (e.g., fever, cough, sore throat, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste and smell) but did not experience shortness of breath, difﬁculty breathing, or abnormal chest imaging were classiﬁed as having mild COVID-19. Individuals who showed evidence of lower respiratory disease during clinical evaluation and maintained oxygen saturation (SpO2) levels of 94% on room air at sea level were classiﬁed as having moderate COVID-19. Those with an SpO2 level <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <30 breaths/min, or lung inﬁltrates exceeding 50% were categorized Biomolecules 2023, 13, 1681 4 of 15 4 of 15 as having severe COVID-19. Cases of critical COVID-19 were identiﬁed in individuals who experienced respiratory failure, septic shock, and/or multiple organ dysfunctions. It is important to note that this study did not include individuals with critical COVID-19 upon hospital admission. 2.3. Data Collection and Sample Processing Demographic information, clinical history, co-morbidities, and additional laboratory data were collected from medical records. The laboratory data included a comprehensive blood analysis, measurements of kidney functions, and markers of inﬂammation. Clinical samples were collected at the hospital on the same day of testing and placed in an RNA stabilization solution. These samples were then transported to the Virology lab at the Egyptian National Research Centre, Egypt. Urine and nasopharyngeal samples were centrifuged, and the resulting supernatants were stored at −80 ◦C. Whole blood was also centrifuged, and the serum obtained was stored at −80 ◦C. 2.6. MicroRNA Proﬁle Expression Reverse transcription was performed on 100 nanograms of RNA to convert it into ﬁrst-strand cDNA. The miScript II RT Kit was used for this process. The expression levels of speciﬁc microRNAs such as has-miR-21 (477975_mir), has-miR-let-7 (4427975), hsa-miR-146a (4427975), hsa-miR-146b (4427975), hsa-miR-155 (4427975), hsa-miR-223 (4427975), and has-miR-342 (4427975) (ThermoFisher, Waltham, MA, USA) were analyzed in different phases of the disease and in various clinical types of patients with SARS-CoV-2 infection, as well as in healthy individuals. The miScript SYBR Green PCR Kit was used for the real-time PCR assay. The Ct values obtained for each miRNA were normalized using reference miRNA (miR-U6; 001093), and the relative levels in urine, nasopharyngeal samples, and plasma were determined. The ampliﬁcation reactions consisted of an initial incubation at 95 ◦C for 5 min, followed by 40 cycles of denaturation at 95 ◦C for 5 s and annealing/extension at 60 ◦C for 30 s. All experiments were performed in triplicate. The ampliﬁcation reactions were performed on a CFX Real-Time Polymerase Chain Reaction platform, and the fold change in expression was analyzed using the 2ˆ(−∆∆Ct) method. 2.5. SARS-CoV-2 Real-Time RT-PCR A real-time RT-PCR assay for the quantitative detection of SARS-CoV-2 was performed according to the instructions of the Sansure COVID-19 Nucleic Acid Test Kit (Sansure Biotech Inc., Changsha, China). 2.7. Data Analysis Statistical analysis was performed using the student’s t-test, one-way ANOVA tests for continuous and categorical variables. A signiﬁcance level of p-value 0.05 was used. The spearman correlation matrix was constructed to analyze the correlation between microRNA expression and clinical data using R package. Receiver-operating characteristic (ROC) curve analysis was conducted to evaluate the sensitivity and speciﬁcity of each miRNA as potential biomarkers for COVID-19 using IBM SPSS Software v. 20. 2.4. Total RNA Extraction Total RNA extraction from urine, nasopharyngeal, and serum specimens was per- formed using the QIAamp RNA Mini Kit (Qiagen, Hilden, Germany). The quantity and quality of the isolated RNA were determined using a NanoDrop spectrophotometer and stored at −80 ◦C. 3.1. Demographic, Laboratory Markers and Clinical Symptoms 3.1. Demographic, Laboratory Markers and Clinical Symptoms 3.1. Demographic, Laboratory Markers and Clinical Symptoms Tables 1–3 present a comprehensive overview of the demographic, biochemical, and clinical parameters of all the participants who took part in this study. The study included Biomolecules 2023, 13, 1681 5 of 15 a total of 273 participants who were categorized into four distinct groups. Among these groups, the mild group consisted of 106 participants who received outpatient treatment without any speciﬁc complications, the moderate group included 71 participants who required hospitalization, the severe group comprised 39 participants who were admitted to the ICU due to speciﬁc symptoms, and there were 57 healthy participants in the control group. The mean age of the participants in each group was as follows: 36.7 ± 11.4 years (ranging from 25 to 49 years) for the healthy group, 38.5 ± 10.6 years (ranging from 28 to 55 years) for the mild group, 45.2 ± 9.3 years (ranging from 36 to 62 years) for the moderate group, and 61.8 ± 9.7 years (ranging from 48 to 71 years) for the severe group (refer to Table 1). Among the participants, the distribution of females within the healthy, mild, moderate, and severe groups was as follows: 14 (24.6%), 29 (27.4%), 32 (45.1%), and 13 (33.4%) (refer to Table 1). Table 1. Demographic parameters of the participants. Groups Age (Mean & SD) Male n (%) Female n (%) Total n (%) Healthy 36.7 ± 11.4 43 (75.4) 14 (24.6) 57 (100) Mild 38.5 ± 10.6 77 (72.6) 29 (27.4) 106 (100) Moderate 45.2 ± 9.3 39 (54.9) 32 (45.1) 71 (100) Sever 61.8 ± 9.7 26 (66.6) 13 (33.4) 39 (100) SD; Standard Deviation, n; number. Table 1. Demographic parameters of the participants. Table 2. Comparisons of the biochemical data of the participants among different groups. Table 2. Comparisons of the biochemical data of the participants among different groups. Test Healthy Mean (Range) Mild Mean (Range) Moderate Mean (Range) Severe Mean (Range) HB%, g/dL 13.4 (12.2–14.5) 13.2 (11.5–14.2) 12.8 (12–13.9) 11.6 (10.0–13.2) WBCs, ×103/µL 7.8 (5.9–9.5) 8.5 (6.3–10.8) 9.6 (7.1–12.7) 10.0 (8.6–14.9) RBCs, ×103/µL 4.7 (3.6–5.5) 4.2 (3.3–5.3) 4.0 (2.8–5.6) 3.9 (2.4–5.8) PLT, ×103/µL 223 (160–285) 191 (166–273) 168 (110–235) 173.4 (107–240) FBS, mg/dL 85.6 (73–110) 93 (88–120) 139 (92–271) 207 (112–405) B. 3.2. Expression Analysis of Tested miRNAs The ﬁndings of this study revealed that mild COVID-19 cases showed signiﬁcantly higher levels of miR-21, miR-146a, and miR-155 in serum, urine, and nasopharyngeal samples compared to healthy individuals (p < 0.01), except for miR-21 expression in na- sopharyngeal samples (p < 0.07). However, there were no signiﬁcant differences in the expression levels of miR-146b, miR-let7, miR-223, and miR-342 between these groups. In moderate COVID-19 cases, the expression level of miR-let-7 was also upregulated (p < 0.01), in addition to the previously upregulated miR-155, miR-146a, and miR-21 observed in mild COVID-19 cases. Conversely, the expression level of miR-146b was downregulated (p < 0.05) compared to the healthy group. No signiﬁcant differences were found in the expression levels of miR-223 and miR-342 between these groups. The most signiﬁcant changes in miRNA expression proﬁles were observed in severe COVID-19 cases. The levels of miR-155, miR-146a, miR-21, and miR-223 were signiﬁcantly upregulated (p < 0.001), while the expression level of miR-146b was downregulated (p < 0.01). Addi- tionally, the expression level of miR-342 was dysregulated in all groups, as depicted in Figures 1 and 2. Based on the correlation analysis between miRNAs and laboratory markers presented in Figure 3, the severe COVID-19 group, which exhibited signiﬁcantly upregulated ex- pression levels of miR-21, miR-155, miR-let-7, miR-146a, and miR-223, showed signiﬁcant positive correlations with platelets (p < 0.01), CRP (p < 0.001), HB% (p < 0.01), urea (p < 0.02), and D-Dimer (p < 0.05), and signiﬁcant negative correlations with RBCs (p < 0.001) and Fasting Blood Sugar (FBS) (p < 0.05). In the moderate COVID-19 group, which had sig- niﬁcantly upregulated expression levels of miR-21, miR-155, miR-let-7, and miR-146a, there was a signiﬁcant positive correlation with creatinine (p < 0.03) and a signiﬁcant negative correlation with RBCs (p < 0.001). In the mild COVID-19 group, which showed signiﬁcantly upregulated expression levels of miR-21, miR-155, and miR-146a, there was a signiﬁcant positive correlation with creatinine (p < 0.04) and D-Dimer (p < 0.02), and a signiﬁcant negative correlation with CRP (p < 0.02), FBS (p < 0.01), HB% (p < 0.02), and RBCs (p < 0.001). ROC curve analysis was performed for all miRNAs in different sample types, as shown in (Tables 4–6) and Supplementary Figure S4. 3.1. Demographic, Laboratory Markers and Clinical Symptoms urea, mg/dL 29.5 (22–45) 37 (27–45) 49 (30–84) 69 (33–123) Creatinine, mg/dL 0.86 (0.5–1.1) 0.95 (0.5–1.1) 1.0 (0.7–1.4) 3.8 (1.1–7.5) CRP, mg/mL 36 (12–96) 72 (18–108) 290 (60–560) 810 (400–1200) D-dimer, ng/mL 210 (170–400) 225 (180–550) 366.2 (242–693) 939.5 (892–1340) HB. Hemoglobin; WBCs. white blood cells; RBCs. red blood cells; Plt. platelets; CRP. C reactive protein. Based on the ﬁndings presented in Table 2, it was observed that patients in the moderate and severe groups, who required hospitalization or were admitted to the ICU, had significantly higher serum levels of CRP (p < 0.001), D-Dimer (p < 0.001), Cr (p < 0.01), Urea (p < 0.01), and FBS (p < 0.01) compared to both the healthy group and the mild COVID-19 group. Moreover, when comparing the clinical characteristics of patients in the moderate and severe groups with those in the healthy and mild groups, it was found that fever (67.6% and 76.9%, respectively), cough (92.9% and 89.7%, respectively), and fatigue (87.4% and 94.8%, respectively) were the most commonly reported symptoms in both the moderate and severe groups. In addition, dyspnea (84.6%) and myalgia (77%) were more frequently reported by patients in the severe group compared to all the other groups. For a comprehensive overview of the clinical parameters of the subjects under study, including the healthy controls, please refer to Table 3. Biomolecules 2023, 13, 1681 6 of 15 Table 3. Comparisons of the clinical parameters of the participants among different groups. Table 3. Comparisons of the clinical parameters of the participants among different groups. Test Healthy (n = 57) Mild (n = 106) Moderate (n = 71) Severe (n = 39) Fever; n, % 0 (0) 24 (22.6) 48 (67.6) 30 (76.9) Cough 5 (8.7) 81 (76.4) 66 (92.9) 35 (89.7) Dyspnea 0 (0) 15 (14.2) 40 (56.3) 33 (84.6) Sore throat 3 (5.2) 19 (17.9) 65 (91.5) 27 (69.2) Diarrhea 6 (10.5) 22 (20.7) 39 (54.9) 23 (58.9) Fatigue 10 (17.5) 69 (65) 62 (87.4) 37 (94.8) Abdominal pain 5 (8.7) 29 (27.3) 34 (47.8) 17 (44) Myalgia 0 (0) 12 (11.3) 36 (50.7) 30 (77) Headache 17 (29.8) 92 (86.8) 51 (71.8) 21 (54) 3.2. Expression Analysis of Tested miRNAs The results of ROC analysis indicated that, in serum samples, miR-155 (Area under the Curve (AUC) = 0.927, p = 0.001) and miR-let-7 (AUC = 0.903, p = 0.017) can be used as biomarkers to distinguish severe COVID-19 patients from healthy individuals. In urine samples, miR-21 (AUC = 0.981, p = 0.001), miR-155 (AUC = 0.936, p = 0.001), and miR-let-7 (AUC = 0.893, p = 0.007) can serve as biomarkers for discrimination. However, no suitable candidate was found in nasopharyngeal samples to discriminate severe COVID-19 patients from healthy individuals. In serum samples, miR- 155 (AUC = 0.942, p = 0.001), miR-let-7 (AUC = 0.903, p = 0.014), and miR-223 (AUC = 0.906, 7 of 15 7 of 15 Biomolecules 2023, 13, 1681 p = 0.004) can serve as biomarkers for moderate COVID-19 patients. However, only miR-let- 7 (AUC = 0.873, p = 0.049) is effective as a biomarker in urine samples for the same group. In nasopharyngeal samples, miR-155 (AUC = 0.879, p = 0.002) demonstrates signiﬁcant p = 0.004) can serve as biomarkers for moderate COVID-19 patients. However, only miR-let- 7 (AUC = 0.873, p = 0.049) is effective as a biomarker in urine samples for the same group. In nasopharyngeal samples, miR-155 (AUC = 0.879, p = 0.002) demonstrates signiﬁcant discriminatory ability between moderate COVID-19 patients and healthy controls. For mild COVID-19 patients, miR-155 (AUC = 0.929, p = 0.001), miR-let-7 (AUC = 0.972, p = 0.011), and miR-223 (AUC = 0.984, p = 0.001) can be utilized as biomarkers in serum samples. In urine samples, miR-146a (AUC = 0.929, p = 0.044), miR-155 (AUC = 0.919, p = 0.001), and miR-223 (AUC = 0.913, p = 0.001) are effective in distinguishing mild COVID- 19 patients from healthy individuals. Lastly, in nasopharyngeal samples, only miR-146b (AUC = 0.962, p = 0.033) and miR-let-7 (AUC = 0.950, p = 0.035) can be employed as markers to differentiate between mild COVID-19 and healthy individuals. EVIEW 7 of 15 Figure 1. Comparison of miR‐let‐7, miR‐21, and miR‐146a expression levels in serum, urine, and nasopharyngeal samples of COVID ‐19 patients admitted to the ICU (Severe), hospitalized COVID‐19 (Moderate), non‐hospitalized COVID ‐19 (Mild), and control subjects (Healthy). (a) MiR‐let‐7 in serum, urine and nasopharyngeal samples, respectively; (b) MiR‐21 in serum, urine and nasopharyngeal samples, respectively; (c) MiR‐146a in serum, urine and nasopharyngeal samples, respectively. Figure 1. 3.2. Expression Analysis of Tested miRNAs Comparison of miR-let-7, miR-21, and miR-146a expression levels in serum, urine, and nasopharyngeal samples of COVID -19 patients admitted to the ICU (Severe), hospitalized COVID-19 (Moderate), non-hospitalized COVID -19 (Mild), and control subjects (Healthy). (a) MiR-let-7 in serum, urine and nasopharyngeal samples, respectively; (b) MiR-21 in serum, urine and nasopharyngeal samples, respectively; (c) MiR-146a in serum, urine and nasopharyngeal samples, respectively. Figure 1. Comparison of miR‐let‐7, miR‐21, and miR‐146a expression levels in serum, urine, and nasopharyngeal samples of COVID ‐19 patients admitted to the ICU (Severe), hospitalized COVID‐19 (Moderate), non‐hospitalized COVID ‐19 (Mild), and control subjects (Healthy). (a) MiR‐let‐7 in serum, urine and nasopharyngeal samples, respectively; (b) MiR‐21 in serum, urine and nasopharyngeal samples, respectively; (c) MiR‐146a in serum, urine and nasopharyngeal samples, respectively. Figure 1. Comparison of miR-let-7, miR-21, and miR-146a expression levels in serum, urine, and nasopharyngeal samples of COVID -19 patients admitted to the ICU (Severe), hospitalized COVID-19 (Moderate), non-hospitalized COVID -19 (Mild), and control subjects (Healthy). (a) MiR-let-7 in serum, urine and nasopharyngeal samples, respectively; (b) MiR-21 in serum, urine and nasopharyngeal samples, respectively; (c) MiR-146a in serum, urine and nasopharyngeal samples, respectively. 8 of 15 8 of 15 8 of 15 8 of 15 Biomolecules 2023, 13, 1681 Biomolecules 2023, 13, x FOR Figure 2. Comparison of miR‐146b, miR‐155, miR‐223 and miR‐342 expression levels in serum, urine, and nasopharyngeal samples of COVID‐19 patients admitted to the ICU (Severe), hospital‐ ized COVID‐19 (Moderate), non‐hospitalized COVID‐19 (Mild), and control subjects (Healthy). (d) MiR‐146b in serum, urine and nasopharyngeal samples, respectively; (e) MiR‐155 in serum, urine and nasopharyngeal samples, respectively; (f) MiR‐223 in serum, urine and nasopharyngeal sam‐ ples, respectively; (g) MiR‐342 in serum, urine and nasopharyngeal samples, respectively. Figure 2. Comparison of miR-146b, miR-155, miR-223 and miR-342 expression levels in serum, urine, and nasopharyngeal samples of COVID-19 patients admitted to the ICU (Severe), hospitalized COVID-19 (Moderate), non-hospitalized COVID-19 (Mild), and control subjects (Healthy). (d) MiR- 146b in serum, urine and nasopharyngeal samples, respectively; (e) MiR-155 in serum, urine and nasopharyngeal samples, respectively; (f) MiR-223 in serum, urine and nasopharyngeal samples, respectively; (g) MiR-342 in serum, urine and nasopharyngeal samples, respectively. REVIEW 9 of 15 2. Comparison of miR‐146b, miR‐155, miR‐223 and miR‐342 expression levels in serum, 2. Comparison of miR-146b, miR-155, miR-223 and miR-342 expression levels in serum, Figure 2. 3.2. Expression Analysis of Tested miRNAs Comparison of miR‐146b, miR‐155, miR‐223 and miR‐342 expression levels in serum, urine, and nasopharyngeal samples of COVID‐19 patients admitted to the ICU (Severe), hospital‐ ized COVID‐19 (Moderate), non‐hospitalized COVID‐19 (Mild), and control subjects (Healthy). (d) MiR‐146b in serum, urine and nasopharyngeal samples, respectively; (e) MiR‐155 in serum, urine and nasopharyngeal samples, respectively; (f) MiR‐223 in serum, urine and nasopharyngeal sam‐ ples, respectively; (g) MiR‐342 in serum, urine and nasopharyngeal samples, respectively. Figure 2. Comparison of miR-146b, miR-155, miR-223 and miR-342 expression levels in serum, urine, and nasopharyngeal samples of COVID-19 patients admitted to the ICU (Severe), hospitalized COVID-19 (Moderate), non-hospitalized COVID-19 (Mild), and control subjects (Healthy). (d) MiR- 146b in serum, urine and nasopharyngeal samples, respectively; (e) MiR-155 in serum, urine and nasopharyngeal samples, respectively; (f) MiR-223 in serum, urine and nasopharyngeal samples, respectively; (g) MiR-342 in serum, urine and nasopharyngeal samples, respectively. REVIEW 9 of 15 y y p sented in Figure 3, the severe COVID‐19 group, which exhibited significantly upreg lated expression levels of miR‐21, miR‐155, miR‐let‐7, miR‐146a, and miR‐223, showe significant positive correlations with platelets (p < 0.01), CRP (p < 0.001), HB% (p < 0.01 urea (p < 0.02), and D‐Dimer (p < 0.05), and significant negative correlations with RBCs < 0.001) and Fasting Blood Sugar (FBS) (p < 0.05). In the moderate COVID‐19 grou which had significantly upregulated expression levels of miR‐21, miR‐155, miR‐let‐ and miR‐146a, there was a significant positive correlation with creatinine (p < 0.03) and significant negative correlation with RBCs (p < 0.001). In the mild COVID‐19 grou which showed significantly upregulated expression levels of miR‐21, miR‐155, an miR‐146a, there was a significant positive correlation with creatinine (p < 0.04) an Figure 3. Cont. sented in Figure lated expression significant posit urea (p < 0.02), a < 0.001) and Fa which had sign and miR‐146a, t significant nega which showed miR‐146a, there Figure 3. Cont. sented in Figure lated expression significant posit urea (p < 0.02), a < 0.001) and Fa which had sign and miR‐146a, t significant nega which showed miR‐146a, there Figure 3. Cont. which exhibited significantly upr let‐7, miR‐146a, and miR‐223, sho < 0.01), CRP (p < 0.001), HB% (p < 0 cant negative correlations with RB 5). In the moderate COVID‐19 gr levels of miR‐21, miR‐155, miR‐ relation with creatinine (p < 0.03) a 0.001). 3.2. Expression Analysis of Tested miRNAs The intensity of the color and the size of the circle are proportional to the correlation coefficients. Figure 3. Spearman correlation matrix comparisons among the expression of miRNAs in serum, urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID-19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Nasopharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Nasopharyngeal samples of mild group. Positive connections are shown in blue, while negative correlations are highlighted in red. The intensity of the color and the size of the circle are proportional to the correlation coefﬁcients. urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID‐19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Naso‐ pharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Na‐ sopharyngeal samples of mild group. Positive connections are shown in blue, while negative cor‐ relations are highlighted in red. The intensity of the color and the size of the circle are proportional to the correlation coefficients. g p p g p , urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID-19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Nasopharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Nasopharyngeal samples of mild group. Positive connections are shown in blue, while negative correlations are highlighted in red. The intensity of the color and the size of the circle are proportional to the correlation coefﬁcients. ROC curve analysis was performed for all miRNAs in different sample types, as shown in (Tables 4–6) and Supplementary Figure S4. The results of ROC analysis indi‐ cated that, in serum samples, miR‐155 (Area under the Curve (AUC) = 0 927, p = 0 001) Table 4. ROC curve analysis of the expression of miRNAs in serum samples of various COVID-19 groups vs. control group. shown in (Tables 4–6) and Supplementary Figure S4. 3.2. Expression Analysis of Tested miRNAs In the mild COVID‐19 gr ure 3, the severe COVID‐19 group ion levels of miR‐21, miR‐155, mi sitive correlations with platelets (p , and D‐Dimer (p < 0.05), and signi Fasting Blood Sugar (FBS) (p < 0 gnificantly upregulated expressio a, there was a significant positive c egative correlation with RBCs (p which showed miR‐146a ther Figure 3. Cont. 9 of 15 Biomolecules 2023, 13, 1681 Figure 3. Spearman correlation matrix comparisons among the expression of miRNAs in serum, urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID‐19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Naso‐ pharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Na‐ sopharyngeal samples of mild group. Positive connections are shown in blue, while negative cor‐ relations are highlighted in red. The intensity of the color and the size of the circle are proportional to the correlation coefficients. ROC curve analysis was performed for all miRNAs in different sample types as Figure 3. Spearman correlation matrix comparisons among the expression of miRNAs in serum, urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID-19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Nasopharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Nasopharyngeal samples of mild group. Positive connections are shown in blue, while negative correlations are highlighted in red. The intensity of the color and the size of the circle are proportional to the correlation coefﬁcients. Figure 3. Spearman correlation matrix comparisons among the expression of miRNAs in serum, urine, and nasopharyngeal samples with the laboratory data of severe, moderate, and mild COVID‐19 patients. (a) Serum of severe group; (b) Urine of severe group; (c) Nasopharyngeal samples of severe group; (d) Serum of moderate group; (e) Urine of moderate group; (f) Naso‐ pharyngeal samples of moderate group; (g) Serum of mild group; (h) Urine of mild group; (i) Na‐ sopharyngeal samples of mild group. Positive connections are shown in blue, while negative cor‐ relations are highlighted in red. 3.2. Expression Analysis of Tested miRNAs The results of ROC analysis indi‐ cated that, in serum samples, miR‐155 (Area under the Curve (AUC) = 0.927, p = 0.001) and miR‐let‐7 (AUC = 0.903, p = 0.017) can be used as biomarkers to distinguish severe COVID‐19 patients from healthy individuals. In urine samples, miR‐21 (AUC = 0.981, p = 0.001), miR‐155 (AUC = 0.936, p = 0.001), and miR‐let‐7 (AUC = 0.893, p = 0.007) can serve as biomarkers for discrimination. However, no suitable candidate was found in naso‐ pharyngeal samples to discriminate severe COVID‐19 patients from healthy individuals. In serum samples, miR‐155 (AUC = 0.942, p = 0.001), miR‐let‐7 (AUC = 0.903, p = 0.014), and miR‐223 (AUC = 0.906, p = 0.004) can serve as biomarkers for moderate COVID‐19 patients. However, only miR‐let‐7 (AUC = 0.873, p = 0.049) is effective as a biomarker in urine samples for the same group. In nasopharyngeal samples, miR‐155 (AUC = 0.879, p = 0.002) demonstrates significant discriminatory ability between moderate COVID‐19 patients and healthy controls. For mild COVID‐19 patients, miR‐155 (AUC = 0.929, p = 0.001), miR‐let‐7 (AUC = 0.972, p = 0.011), and miR‐223 (AUC = 0.984, p = 0.001) can be utilized as biomarkers in serum samples. In urine samples, miR‐146a (AUC = 0.929, p = 0.044), miR‐155 (AUC = 0.919, p = 0.001), and miR‐223 (AUC = 0.913, p = 0.001) are effec‐ tive in distinguishing mild COVID‐19 patients from healthy individuals. Lastly, in na‐ Table 4. ROC curve analysis of the expression of miRNAs in serum samples of various COVID-19 groups vs. control group. Group Target AUC p Value Sensitivity Speciﬁcity Severe Group MiR-21 0.838 0.006 100 85.7 MiR-155 0.927 0.001 96.6 87.3 MiR-146a 0.853 0.045 100 86.7 MiR-146b 0.857 0.093 100 94.3 MiR-let-7 0.903 0.017 100 94.4 MiR-223 0.800 0.033 100 90.6 MiR-342 0.731 0.028 91 100 Moderate Group MiR-21 0.851 0.004 86.7 71.4 MiR-155 0.942 0.001 100 75 MiR-146a 0.843 0.051 97.1 86.7 MiR-146b 0.614 0.591 100 97.2 MiR-let-7 0.903 0.014 100 90.6 MiR-223 0.906 0.004 100 87.5 MiR-342 0.764 0.012 90.9 100 10 of 15 10 of 15 Biomolecules 2023, 13, 1681 Table 4. Cont. Group Target AUC p Value Sensitivity Speciﬁcity Mild Group MiR-21 0.836 0.006 93.3 85.7 MiR-155 0.929 0.001 86.6 75 MiR-146a 0.775 0.119 97.1 100 MiR-146b 0.757 0.227 100 97.3 MiR-let-7 0.972 0.011 100 96.9 MiR-223 0.984 0.001 100 87.5 MiR-342 0.863 0.001 91 100 Table 5. 3.2. Expression Analysis of Tested miRNAs ROC curve analysis of the expression of miRNAs in urine samples of various COVID-19 groups vs. control group. Table 5. ROC curve analysis of the expression of miRNAs in urine samples of various COVID-19 groups vs. control group. Group Target AUC p Value Sensitivity Speciﬁcity Severe Group MiR-21 0.981 0.001 100 81.2 MiR-155 0.936 0.001 100 90.9 MiR-146a 0.829 0.122 100 80 MiR-146b 0.866 0.006 95 82.5 MiR-let-7 0.893 0.007 100 88.9 MiR-223 0.884 0.011 100 81.5 MiR-342 0.861 0.001 95.5 100 Moderate Group MiR-21 0.810 0.001 100 87.5 MiR-155 0.800 0.002 100 95.5 MiR-146a 0.821 0.131 100 97 MiR-146b 0.678 0.065 100 94.2 MiR-let-7 0.873 0.049 100 96.3 MiR-223 0.825 0.004 100 92.9 MiR-342 0.727 0.020 94.8 100 Mild Group MiR-21 0.866 0.001 100 75 MiR-155 0.855 0.009 100 90.9 MiR-146a 0.929 0.044 100 91 MiR-146b 0.819 0.001 100 94.1 MiR-let-7 0.919 0.001 100 96.3 MiR-223 0.913 0.001 100 96.4 MiR-342 0.876 0.001 100 93.2 Biomolecules 2023, 13, 1681 11 of 15 11 of 15 Table 6. ROC curve analysis of the expression of miRNAs in nasopharyngeal samples of various COVID-19 groups vs. control group. Group Target AUC p Value Sensitivity Speciﬁcity Severe Group MiR-21 0.779 0.19 100 94.3 MiR-155 0862 0.003 100 71.5 MiR-146a 0.869 0.002 100 82.4 MiR-146b 0.766 0.100 100 80.6 MiR-let-7 0.806 0.074 100 87.8 MiR-223 0.569 0.574 100 96.7 MiR-342 0.784 0.017 87.5 100 Moderate Group MiR-21 0.886 0.07 100 91.4 MiR-155 0.879 0.002 90 78.6 MiR-146a 0.767 0.022 100 87.3 MiR-146b 0.839 0.116 100 98 MiR-let-7 0.800 0.095 100 88.9 MiR-223 0.788 0.019 100 90 MiR-342 0.897 0.001 100 96.4 Mild Group MiR-21 0.921 0.08 100 97.1 MiR-155 0.769 0.028 100 71.4 MiR-146a 0.677 0.130 100 87.5 MiR-146b 0.962 0.033 100 96.8 MiR-let-7 0.950 0.035 100 96.3 MiR-223 0.667 0.175 100 93.3 MiR-342 0.784 0.015 87.5 100 Table 6. ROC curve analysis of the expression of miRNAs in nasopharyngeal samples of various COVID-19 groups vs. control group. Table 6. ROC curve analysis of the expression of miRNAs in nasopharyngeal samples of various COVID-19 groups vs. control group. 4. Discussion [35] demonstrated an eight-fold upregulation of miR-21-3p during lung infection caused by SARS-CoV-2. This upregulation facilitated the survival and replication of the virus. Another study investigating both miR-146 and miR-21 revealed that the levels of these miRNAs in the serum decreased in COVID-19 patients after treatment with the drug Tocilizumab [16]. Our ﬁndings are consistent with these previous studies and demonstrate that miR-146a, miR-155, and miR-21 were consistently upregulated in the serum, urine, and nasopharyngeal samples of COVID-19 patients with different disease severity groups (mild, moderate, and severe) compared to the healthy control group. y g p p y g p Regarding miR-let-7, a study by Pessôa et al. [36] demonstrated that its over expression led to a reduction in the expression of inﬂammatory cytokines and chemokines (IL-6, IL-8, and TNF-α). As for miR-223, alveolar epithelial type II cells are one of the main targets of SARS-CoV-2. In the context of lung injury, the migration of neutrophils to the site of inﬂammation worsens the inﬂammatory response and causes damage to the tissues. MiR-223 is released in microvesicles derived from neutrophils and is involved in lung damage. These microvesicles then transfer miR-223 to alveolar epithelial type II cells to contribute to lung protection [37]. Another study proposed that miR-223 plays a role in the post-transcriptional regulation of the NLRs-inﬂammasome pathway, and its excessive activation has been suggested to contribute to the progression of inﬂammation and the development of a cytokine storm in cases of acute COVID-19 [38]. Our research aligns with these previous studies and demonstrates that miR-let-7 consistently exhibits upregulation in the serum, urine, and nasopharyngeal samples of moderate and severe COVID-19 patients. On the other hand, miR-223 consistently shows upregulation, speciﬁcally in the serum, urine, and nasopharyngeal samples of severe COVID-19 patients. p y g p p The role of miR-146b in SARS-CoV-2 infection remains controversial, as contradictory results have been published. Some authors reported that it acts as a dominant negative regulator of the innate immune response by decreasing NK cell degranulation and the expression of downstream factors in the Toll-like receptor signaling pathway [28]. In this study, the downregulation of miR-146b was associated with an increased expression of pro-inﬂammatory cytokines, such as IL-6 and TNF-α, in severe COVID-19 patients [39]. However, other studies have reported conﬂicting results, showing upregulation of miR- 146b in COVID-19 patients compared to healthy controls [40]. 4. Discussion The primary objective of this study was to identify unique expression patterns of speciﬁc miRNAs (miR-21, miR-let-7, miR-146a, miR-146b, miR-155, miR-223, and miR-342) in COVID-19 patients with different disease states using various clinical samples, such as serum, urine, and nasopharyngeal samples, in comparison to healthy individuals. In recent years, circulating miRNAs have emerged as potential biomarkers for viral disorders and valuable tools for therapeutic applications, which could improve diagnostic accuracy [27–31]. A previous study suggested that measuring circulating miRNA-320a/b and D-dimer together enhances the diagnostic power for deep venous thrombosis. Differential expression of miRNAs has also been associated with the categorization of COVID-19 patients based on D-dimer levels. For example, another study found signiﬁcant downregulation of miR-155, miR-146a, and miR-21, as well as a substantial over-expression of miR-342, in the circulating exosomes of COVID-19 patients with high D-Dimer levels [29,30]. Therefore, the selection of the panel of seven miRNAs in this study was based on their previous associations with inﬂammation, immune control, cell signaling, and viral infections [27–30]. Prior to the COVID-19 pandemic, miR-155 and miR-146a were identiﬁed as the ﬁrst miRNAs stimulated by immune activation. They modulate the Toll-Like Receptor signaling pathway, which triggers the production of various inﬂammatory cytokines, type I interferon, and antiviral proteins [31]. A study by Soni et al. [32], reported higher levels of miR-155-5p in COVID-19 patients compared to healthy individuals. The study also investigated the effect of inhibiting miR-155 in the lungs of SARS-CoV-2-infected transgenic mice and found that the inhibition of miR-155 promoted survival, attenuated inﬂammation, and reduced the lung cytokine storm induced by the virus. In another study, the mean expression Biomolecules 2023, 13, 1681 12 of 15 12 of 15 level of miR-155 was signiﬁcantly higher in SARS-CoV-2-infected cell lines compared to control cell lines, suggesting that miR-155 plays a critical role in respiratory viral diseases by modulating antiviral responses, including inﬂammatory and immune responses [28]. y g p g y p The expression level of miR-146a exhibited a signiﬁcant increase in A549 cells that were infected with the inﬂuenza virus. This upregulation of miR-146a played a role in promoting viral replication by inhibiting the production of interferon type I [33]. Moreover, it has been observed that elevating the expression level of miR-146a can mitigate lung cell damage by suppressing inﬂammatory responses [34]. Regarding miR-21, a study by Nersisyan et al. 4. Discussion Further research is needed to elucidate the exact role of miR-146b in COVID-19 pathogenesis. In summary, the differential expression patterns of miRNAs in COVID-19 patients reﬂect their involvement in inﬂammatory and immune responses, as well as viral replication and survival. These miRNAs show potential as biomarkers for disease severity and could provide insights into the underlying mechanisms of COVID-19 pathogenesis. However, it is important to note that further validation studies are necessary to conﬁrm these ﬁndings and establish the clinical utility of miRNAs as diagnostic or prognostic markers for COVID-19. In the severe COVID-19 group, we observed signiﬁcant upregulation of miR-21, miR- 155, miR-let-7, miR-146a, and miR-223, as discussed earlier. These speciﬁc miRNAs exhib- ited signiﬁcant positive correlations with platelets, C-reactive protein (CRP), hemoglobin (HB %), urea, creatinine, and D-dimer levels. Additionally, they showed signiﬁcant nega- tive correlations with red blood cells (RBCs) and fasting blood sugar (FBS) levels. In the moderate COVID-19 group, we found signiﬁcant upregulation of miR-21, miR-155, miR-let- 7, and miR-146a. These miRNAs displayed signiﬁcant positive correlations with creatinine levels and signiﬁcant negative correlations with RBCs. Within the mild COVID-19 group, Biomolecules 2023, 13, 1681 13 of 15 13 of 15 we observed signiﬁcant upregulation of miR-21, miR-155, and miR-146a. These miRNAs demonstrated signiﬁcant positive correlations with creatinine and D-dimer levels, and signiﬁcant negative correlations with CRP, FBS, HB%, and RBCs. These ﬁndings partially align with a previous study on COVID-19 patients, which identiﬁed a signiﬁcant correlation between elevated expression levels of miR-155 and clinicopathological features such as increased white blood cell (WBC) and neutrophil counts, as well as a decreased lymphocyte count [40,41]. Another study also reported a signiﬁcant correlation between creatinine, urea levels, and differentially expressed miRNAs, suggesting the involvement of these miRNAs in the regulation of kidney function. It is well known that COVID-19 can lead to kidney injury [42]. y j y Furthermore, our ROC curve analysis demonstrated that miR-155, miR-let-7, and miR-223 exhibited high sensitivity and speciﬁcity, indicating their potential as biomarkers for distinguishing COVID-19 patients from healthy individuals. Adding miR-21 to these three miRNAs could be used as a biomarker for monitoring severe COVID-19 status, while adding miR-223 could be useful for monitoring moderate COVID-19, and adding miR-146 could be beneﬁcial for monitoring mild COVID-19. 4. Discussion In conclusion, this study was the ﬁrst to report differentially expressed miRNA proﬁles, including miR-21, miR-155, miR-let-7b, miR-146a, miR-146b, miR-223, and miR-342, in various COVID-19 severity groups and a healthy control group. These ﬁndings contribute to our understanding of the role of miRNAs in COVID-19 and suggest their potential as biomarkers for COVID-19 monitoring. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom13121681/s1, Figure S1: curve analysis using Serum, Urine and Nasopharyngeal miR-21, miR-146a, miR-146b, miR-155, let-7b, miR-223 and miR-342 for discrim- inating control, mild, moderate and severe COVID-19 patients. Author Contributions: Conceptualization, A.K.A. and R.S.; methodology, A.K.A., N.A., A.A. and N.F.E.; software, N.F.E. and A.T.Q.; validation, A.T.Q., F.M.A. and N.A.; formal analysis, N.A., R.S. and F.M.A.; investigation, A.K.A., R.S., D.E.-G.K. and T.A.; resources, R.S. and N.A.; data curation, S.B.A.-G.; writing—original draft preparation, S.B.A.-G., R.S., N.A., N.A. and A.T.Q.; writing—review and editing, A.K.A., N.F.E., F.M.A. and D.E.-G.K.; visualization, T.A., S.B.A.-G. and R.S.; supervision, R.S.; project administration, A.K.A.; funding acquisition, R.S. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, A.K.A. and R.S.; methodology, A.K.A., N.A., A.A. and N.F.E.; software, N.F.E. and A.T.Q.; validation, A.T.Q., F.M.A. and N.A.; formal analysis, N.A., R.S. and F.M.A.; investigation, A.K.A., R.S., D.E.-G.K. and T.A.; resources, R.S. and N.A.; data curation, S.B.A.-G.; writing—original draft preparation, S.B.A.-G., R.S., N.A., N.A. and A.T.Q.; writing—review and editing, A.K.A., N.F.E., F.M.A. and D.E.-G.K.; visualization, T.A., S.B.A.-G. and R.S.; supervision, R.S.; project administration, A.K.A.; funding acquisition, R.S. All authors have read and agreed to the published version of the manuscript. Funding: The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number: IFP22UQU4331312DSR229. Institutional Review Board Statement: The ethics committee of the Ahmed Maher Teaching Hospital has authorized this study (approved number HAM00125). Informed Consent Statement: Participants’ consent was not sought for polyomavirus testing; there- fore, samples were de-identiﬁed and analyzed anonymously, with just their age and gender indicated. Data Availability Statement: The data generated and/or examined during this study are not publicly available due to an ongoing research endeavor but are available upon reasonable request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3. Cron, R.Q. COVID-19 cytokine storm: Targeting the appropriate cytokine. Lancet Rheumatol. 2021, 3, e236–e237. [CrossRef] 4. Jin, Y.; Ji, W.; Yang, H.; Chen, S.; Zhang, W.; Duan, G. Endothelial activation and dysfunction in COVID-19: From basic mechanisms to potential therapeutic approaches. 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https://openalex.org/W4388104395	https://eurekaunima.com/index.php/jpfunima/article/download/277/181	Indonesian	null	PENGEMBANGAN PERANGKAT PEMBELAJARAN BERBASIS MODEL EXPERIENTIAL LEARNING DENGAN PENDEKATAN KONSTRUKTIVISME DI SMA NEGERI 2 TONDANO	Charm Sains	2,023	cc-by	6,467	CHARM SAINS Volume 4 No 3 Hal: 142-153 CHARM SAINS Volume 4 No 3 Hal: 142-153 CHARM SAINS Volume 4 No 3 Hal: 142-153 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 ABSTRACT This study aims to develop leatning tools based on experiential learning models with constructivist approach so as to produce valid, practical and effective learning tools that can improve learning processes and outcomes in the material of momentum and impulse in class X B students of SMA Negeri 2 Tondano. The learning tools produced are teaching modules, teaching materials and Student Worksheets. The type of research used in this research is development research with the 4D research model, namely define, design, development and disseminate. The results of this study are: (1) the resulting device is "Valid" and can be used without revision; (2) the resulting device is practically based on the percentage results for the implementation of learning which is 94.9% and the results of the student response questionnaire stating that the learning process with the experiential learning model with a constructivism approach is practically used in class; (3) the resulting device is effective as seen from the acquisition of an N-Gain score of 0.7 with the "Moderate" criterion, the results of learning attitudes obtain an average percentage of 80.33, the results of learning skills obtain an average percentage of 78.48 both have "Good" criteria, and student process assessment with 73.79% (P1) and 84.49% (P2) with an average process of 79.14% with "Good" criteria. From the acquisition of learning outcomes and comparison of process assessment values (P1) & (P2) it is concluded that there is an increase in learning outcomes and processes for students using the resulting learning tools. Keywords: Experiential Learning, Constructivism Approach, Learning Devices, Learning Outcomes and Processes. ABSTRAK Penelitian ini bertujuan untuk mengembangkan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme sehingga menghasilkan perangkat pembelajaran yang valid, praktis dan efektif yang dapat meningkatkan proses dan hasil belajar pada materi momentum dan impuls. Pada peserta didik kelas X B SMA Negeri 2 Tondano. Adapun perangkat pembelajaran yang dihasilkan adalah modul ajar, materi ajar dan LKPD. Jenis penelitian yang digunakan pada penelitian ini adalah penelitian pengembangan dengan model penelitian 4D yaitu define, design, development and desseminate. Hasil dari penelitian ini adalah: (1) perangkat yang dihasilkan adalah “Valid” dan dapat digunakan tanpa revisi; (2) perangkat yang dihasilkan praktis berdasarkan hasil persentase untuk keterlaksanaan pembelajaran yaitu 94,9% dan hasil angket respon peserta didik yang menyatakan bahwa proses pembelajaran dengan model experiential learning dengan pendekatan konstruktivisme praktis digunakan dalam kelas; (3) perangkat yang dihasilkan efektif dilihat dari perolehan skor N-Gain 0,7 dengan kriteria “Sedang”, hasil belajar sikap diperoleh rata-rata persentase yaitu 80,33, hasil belajar keterampilan diperoleh rata-rata presentase yaitu 78,48 keduanya memiliki kriteria “Baik”, dan penilaian proses peserta didik dengan 73,79% (P1) dan 84,49% (P2) dengan rata-rata proses 79,14% dengan kriteria “Baik”. Dari perolehan nilai hasil belajar dan perbandingan nilai penilaian proses (P1) & (P2) disimpulkan bahwa terdapat peningkatan hasil belajar dan proses pada peserta didik menggunakan perangkat pembelajaran yang dihasilkan. Kata kunci: Experiential Learning, Pendekatan Konstruktivisme, Perangkat Pembelajaran, Hasil dan Proses Belajar. PENGEMBANGAN PERANGKAT PEMBELAJARAN BERBASIS MODEL EXPERIENTIAL LEARNING DENGAN PENDEKATAN KONSTRUKTIVISME DI SMA NEGERI 2 TONDANO Heldi Ngkunda, Tineke Makahinda, Djeli Tulandi Fakultas Matematika, Ilmu Pengetahuan Alam dan Kebumian, Universitas Negeri Manado email: heldi16ag@gmail.com CHARM SAINS Volume 4 No 3 Hal: 142-153 CHARM SAINS Volume 4 No 3 Hal: 142-153 meningkatkan proses dan hasil belajar peserta didik sehingga tujuan pembelajaran dapat tercapai dengan maksimal Akan tetapi kualitas guru di Indonesia masih berada pada urutan ke 14 dari 14 negara berkembang dunia sesuai dengan data UNESCO dalam Global Education Monitoring (GEM) pada tahun 2016 (Eko Wahyudi dkk., 2022). Sementara itu, untuk meningkatkan kualitas pendidikan, tidak lepas dari peran penilaian akurat yang dapat mengukur hasil dari sebuah proses belajar (Shulhu Asysyifa dkk., 2019). Rendahnya kualitas guru ini tentu saja tidak dapat meningkatkan proses dan hasil belajar peserta didik di Indonesia. Hal ini terjadi karena guru melaksanakan pembelajaran dengan model konvensional dimana guru mengajar dengan berceramah, tanya jawab, metode diskusi dan penugasan (Devita & Budiyanto, 2022). Hasil penelitian sebelumnya menunjukkan bahwa pembelajaran dengan menggunakan model experiential learning dapat meningkatkan pemahaman belajar peserta didik materi Cahaya dan Sifat-Sifatnya dengan bukti peningkatan persentase ketuntasan belajar peserta didik berpredikat baik(Apriovilita Hariri & Yayuk, 2018) dan dalam penelitian lainnya ditemukan bahwa penerapan pendekatan konstruktivisme dalam pembelajaran menunjukkan peningkatan hasil belajar peserta didik dari yang terendah hingga mencapai kategori baik (Fitria dkk., 2021). p g Berdasarkan latar belakang tersebut dilakukan penelitian pengembangan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme untuk meningkatkan hasil belajar dan proses belajar peserta didik pada mata pelajaran IPA materi momentum dan impuls di SMA Negeri 2 Tondano. Penelitian ini berfokus untuk meningkatkan proses dan hasil belajar peserta didik karena kualitas pendidikan seharusnya mencakup pada dua aspek yakni aspek akademik dan keterampilan (Kholifah dkk., 2020) Berdasarkan observasi yang dilaksanakan di SMA Negeri 2 Tondano Kabupaten Minahasa yang dilakukan kepada peserta didik dan guru mata pelajaran IPA, diidentifikasi bahwa pembelajaran yang dilangsungkan menggunakan model konvensional sehingga peserta didik merasa bosan sehingga sulit memahami pelajaran IPA yang diberikan oleh guru. Hal ini sejalan dengan hasil penelitian bahwa peserta didik mengalami kesulitan pada saat menerima pembelajaran dengan model konvensional (Ginoga dkk., 2023). Guru diwajibkan memiliki keahlian dalam pengelolaan kelas. Berdasarkan latar belakang di atas, dirumuskan masalah secara umum “Bagaimana pengembangan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme dalam meningkatkan proses dan hasil belajar pada materi momentum dan impuls?”. Tujuan umum yang hendak dicapai dalam penelitian ini yaitu untuk mengembangkan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme sehingga menghasilkan perangkat pembelajaran yang valid, praktis dan efektif dan dapat meningkatkan proses dan hasil belajar pada materi momentum dan impuls. 1. PENDAHULUAN disiplin, berperan aktif dalam pelaksanaan kegiatan pembelajaran dan pendidikan yang efektif (Nurfatimah dkk., 2022). Guru yang berkualitas harus dapat melaksanakan proses pembelajaran dengan baik dan dapat Pendidikan dapat mengubah pola berpikir seseorang melakukan perbaikan dalam segala aspek kehidupan (Shulhu Asysyifa dkk., 2019). Guru sebagai salah satu aktor penting dalam 142 CHARM SAINS Volume 4 No 3 Hal: 142-153 Kolb dan Kolb pada tahun 2005 dalam (Mc Pherson-Geyser dkk., 2020), menyatakan bahwa model experiental learning adalah sebuah proses dimana sebuah data atau hal baru yang dipahami oleh peserta didik kemudian diubah melalui pengalaman. Pendekatan konstruktivisme adalah pendekatan dimana pengetahuan dan pemahaman peserta didik tidak diperoleh secara pasif melainkan secara aktif melaui pengalaman pribadi dan kegiatan percobaan (Muslikh dkk., 2022). Model Experiential Learning Model Experiential Learning Fisika adalah salah satu cabang ilmu pengetahuan yang mempelajari tentang sifat dan fenomena alam serta interaksi yang terjadi (Pakiding dkk., 2023). Dengan melibatkan peserta didik langsung dalam pembelajaran maka peserta didik dapat merasakan, memahami dan mengembangkan kemampuan yang dimilikinya sehingga dapat meningkatkan proses dan hasil belajar. Experiential learning terdiri dari dua suku kata yaitu “experiential” yang berarti pengalaman dan “learning” yang artinya pembelajaran. Model experiential learning adalah model yang menggunakan pengalaman sebagai media pembelajarannya. Experiential learning menjadikan pengalaman memiliki peran yang penting dalam sebuah proses belajar (Christiany Martono dkk., 2018). Afief Zuhryzal dalam (Sagitarini dkk., 2018) 143 CHARM SAINS Volume 4 No 3 Hal: 142-153 menggambarkan bahwa model experiential learning tidak hanya membagikan pengetahuan konsep saja melainkan mendorong pembangunan keterampilan melalui penugasan nyata sehingga terdapat umpan balik serta evaluasi antara hasil penerapan dengan hal apa yang seharusnya dilakukan. masalah dalam memecahkaan keterampilan untuk menerapkan ide-ide yang baru diperoleh dari pengalamannya (Gadola & Chindamo, 2019). Manfaat menggunakan model experiential learning adalah memberikan kesempatan kepada peserta didik untuk mengembangkan dan menerapkan konsep fisika yang sejatinya sangat erat dalam kehidupan sehari-hari. Experiential learning membantu peserta didik dalam mengaitkan materi pembelajaran dengan keadaan nyata, sehingga dengan pengalaman nyata tersebut peserta didik dapat mengingat dan memahami informasi yang ditemukan dalam pembelajaran. Abdul (2015:93) menegaskan bahwa model experiential learning adalah proses pembelajaran yang mengaktifkan peserta didik dalam membangun pengetahuan dan keterampilan melalui pengalamannya secara langsung sehingga pengalaman menjadi katalisator untuk menolong peserta didik dalam mengembangkan kemampuan proses belajarnya (Apriovilita Hariri & Yayuk, 2018). Experiential learning memiliki empat tahapan oleh Kolb (1984) yang digambarkan dalam siklus berikut: Hasil Belajar Hasil belajar adalah kemampuan yang didapatkan oleh peserta didik melalui kegiatan pembelajaran. Mulyono Abdurrahman (1993:31) dalam (Sulastri dkk., 2018)menjelaskan bahwa hasil belajar ialah kemampuan yang dimiliki seseorang setelah melaksanakan kegiatan belajar. Belajar adalah proses seseorang yang berupaya untuk memperoleh sebuah pengetahuan dan perubahan perilaku. Hasil belajar adalah perubahan tingkah laku dan keterampilan umum yang dimiliki seorang siswa setelah belajar, meliputi kognitif, emosional dan itu berupa keterampilan psikomotor. Model pengembangan 4D adalah model pengembangan berbagai macam media pembelajaran yang bersifat umum yang dikembangkan oleh Sivasailam Thiagarajan, Dorothy S. Semmel dan Melvyn I. Semmel (Arkadiantika dkk., 2020). Model pengembangan ini terdiri dari tahap define yaitu pendefinisian, design yaitu perancangan, development yaitu pengembangan dan disseminate yakni penyebaran. Model ini dipilih guna menghasilkan produk perangkat pembelajaran yang valid, praktis dan efektif berupa modul ajar, materi ajar dan LKPD berbasis experiential learning dengan pendekatan konstruktivisme. Manfaat hasil belajar adalah untuk mengetahui tingkat pencapaian kompetensi selama dan setelah proses pembelajaran berlangsung, sebagai umpan balik bagi peserta didik agar mengetahui kekuatan dan kelemahannya dalam proses pembelajaran, sebagai umpan balik bagi guru dalam memrefleksi diri. Pendekatan Konstruktivisme Pendekatan (approach) adalah seperangkat asumsi yang secara teoritis bisa dipertanggungjawabkan mengenai hakikat bahasa, hakikat pembelajaran dan hakikat pembelajaran yang digunakan untuk mencapai tujuan pedagogis, sementara konstruktivisme berarti bersifat membangun. Penelitian Piaget menyimpulkan bahwa pengetahuan dibangun dalam pikiran peserta didik. Piaget mengatakan bahwa pengetahuan dikonstruksi sambil mengendalikan pengalaman-pengalamannya yang terdiri atas struktur-struktur mental yang sudah ada padanya (Fitria dkk., 2021). Konstruktivisme merupakan teori belajar yang dijumpai dalam psikologi yang membalas mengenai bagaimana seseorang dapat menemukan pengetahuan belajar, karena hal itu memiliki relasi langsung dengan pendidikan. Sehingga dapat disimpulkan bahwa pendekatan konstruktivisme adalah pendekatan yang memberikan kebebasan kepada peserta didik untuk belajar menemukan sendiri kompetensi pengetahuannya untuk mengembangkan kemampuan pada dirinya dengan guru sebagai fasilitatornya. Pendekatan konstruktivisme dalam pembelajaran menekankan pada aktivitas peserta didik dalam mengkonstruksi pemahaman konseptualnya (Resbiantoro dkk., 2022). Prosedur dalam pendekatan konstruktivisme menurut Suprijono (2010:41) dalam (Maulana & Leonard, 2018) terdapat lima tahapan yaitu orientasi, elisitasi, rekonstruksi ide, aplikasi ide, dan review. Gambar 1. Tahapan Experiential Learning (Mc Pherson-Geyser dkk., 2020) Gambar 1. Tahapan Experiential Learning (Mc Pherson-Geyser dkk., 2020) Pengalaman konkrit (Concrete experience) adalah keadaan dimana peserta didik terlibat secara aktif dalam pengalaman. Pengamatan reflektif (Reflection Observation) dimana peserta didik haus mampu merefleksikan pengalamannya. Konseptualisasi abstrak (Abstract Conceptualization), peserta didik harus memiliki dan menggunakan keterampilan analisisnya untuk mengkonseptualisasikan pengalaman yang ditemukannya. Eksperimen aktif (Active Experimentation), peserta didik harus memiliki pengambilan keputusan dan Orientasi adalah tahap dimana guru memberikan kesempatan kepada peserta didik untuk dapat memperhatikan dan mengembangkan motivasi terhadap topik pembelajaran. Elisitasi merupakan kegiatan dimana guru memberikan bantuan kepada peserta didik untuk menggali ide-ide yang dimilikinya dengan memberi kesempatan kepada mereka mendiskusikan pengetahuan dasar atau ide mereka melalui tulisan yang dipresentasikan kepada seluruh peserta didik. 144 CHARM SAINS Volume 4 No 3 Hal: 142-153 CHARM SAINS Volume 4 No 3 Hal: 142-153 3. METODE PENELITIAN 3. METODE PENELITIAN Peserta didik selanjutnya akan melaksanakan klarifikasi ide dengan mengontraskan idenya dengan ide temannya yang lain melalui tahap rekonstruksi ide. Selanjutnya, ide dan pengetahuan yang telah terbentuk perlu diaplikasikan dalam berbagai situasi sehingga peserta didik dapat menemukan pengetahuan yang lengkap. Pengetahuan yang sudah lengkap lalu diaplikasikan dalam kehidupan sehari-hari, merevisi gagasannya dengan mengubah suatu keterangan menjadi lebih lengkap lalu hasil reviu akan dibandingkan dengan pengetahuan awal yang dimiliki oleh peserta didik. Metode penelitian yang digunakan dalam penelitian ini adalah penelitian dan pengembangan. Metode penelitian dan pengembangan memiliki beberapa jenis model salah satunya adalah model yang digunakan dalam penelitian dan pengembangan ini yaitu model 4D yang ditunjukkan dalam gambar berikut. Gambar 2. Model Pengembangan 4D (Maulida dkk., 2022) Gambar 2. Model Pengembangan 4D (Maulida dkk., 2022) 1) Tahap Pendefinisian (Define) 1) Tahap Pendefinisian (Define) Proses belajar adalah serangkaian aktivitas yang terjadi secara abstrak, karena terjadi secara mental dan dapat diamati hal jika terjadi perubahan perilaku dari seseorang yang berbeda dengan sebelumnya.Proses belajar merupakan aktivitas psikis dan mental melalui interaksi langsung dengan lingkungan sehingga menghasilkan perubahan (Salti dkk., 2023). Dalam psikologi belajar, proses diartikan sebagai cara atau langkah-langkah khusus yang dapat menimbulkan beberapa perubahan sehingga tercapainya hasil-hasil tertentu. Proses belajar terjadi antara guru dan peserta didik yang dipengaruhi oleh relasi yang ada dalam proses itu sendiri. Tahap ini merupakan tahap untuk mendefinisikan dan menetapkan syarat proses pembelajaran. Tahap define memiliki 5 langkah yaitu: Analisis Ujung Depan, Analisis Peserta Didik, Analisis Tugas, Analisis Materi, dan Perumusan Tujuan Pembelajaran. Teknik Pengumpulan Data g Teknik analisis data adalah proses penyederhanaan penyajian data dengan cara pengelompokkan menjadi satu sehingga mudah untuk dibaca (Pakiding dkk., 2023).Teknik pengumpulan data menggunakan teknik validasi perangkat, uji kepraktisan dan keefektifan perangkat dan dokumentasi. Adapun instrumen yang digunakan dalam penelitian ini adalah lembar validasi perangkat pembelajaran (modul ajar, materi ajar dan LKPD), lembar observasi keterlaksanaan pembelajaran, lembar angket peserta didik, 2) Tahap Perancangan (Design) Tahap ini bertujuan untuk merancang produk atau perangkat pembelajaran berupa modul ajar, materi ajar dan LKPD untuk menghasilkan contoh perangkat. Pada tahap ini dilakukan perancangan produk perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme dalam 4 langkah, yaitu: Penyusunan Tes, Pemilihan Media, Pemilihan Format, dan Rancangan Awal. 145 Teknik Analisis Data Teknik analisis data pada penelitian ini adalah yang pertama analisis validitas yaitu analisis data bersifat deskripsitif yang artinya mendeskripsikan saran dan masukan untuk ditindaklajuti dengan cara mengevaluasi dan memperbaiki kembali perangkat pembelajaran yang sudah dihasilkan (Monoarfa dkk., 2022). Perangkat pembelajaran akan divalidasi oleh validator melalui beberapa aspek diantaranya aspek kontruksi, isi, keterbacaan dan bahasa. Validator akan memberikan saran dan komentar melalui lembar yang telah disiapkan yang dinilai sesuai dengan tabel kriteria penilaian perangkat pembelajaran oleh Budiarso (2017:16). Tabel 1. Kriteria Penilaian Perangkat Tabel 1. Kriteria Penilaian Perangkat Pembelajaran (Monoarfa dkk., 2022) Pembelajaran (Monoarfa dkk., 2022) Pembelajaran (Monoarfa dkk., 2022) Interval Skor Kriteria Penilaian Keterangan 3,6 ≤ P < 4 Sangat Valid Dapat digunakan tanpa revisi 2,6 ≤ P < 3,5 Valid Dapat digunakan dengan sedikit revisi 1,6 ≤ P < 2,5 Kurang Valid Dapat digunakan dengan revisi banyak 1 ≤ P < 1,5 Tidak Valid Belum dapat digunakan dan masih memerlukan konsultasi Interval Skor Kriteria Penilaian Keterangan 4) Tahap Penyebaran (Desseminate) Setelah diujicobakan ke peserta didik, maka akan direvisi kembali sesuai dengan perbaikan dan kemudian perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme akan disebarluaskan. Akan tetapi penyebarluasan perangkat pembelajaran hanya dilakukan di sekolah yaitu kepada peserta didik dan kepada guru mata pelajaran IPA di SMA Negeri 2 Tondano untuk digunakan dalam pembelajaran. Penelitian ini dilakukan di SMA Negeri 2 Tondano. Waktu penelitian dilaksanakan pada semester ganjil 2023/2024. Subjek dalam penelitian ini adalah seluruh siswa kelas X B SMA Negeri 2 Tondano semester ganjil tahun ajaran 2023/2024 dengan jumlah siswa sebanyak 27 orang. Selanjutnya dilakukan uji n-gain, yaitu untuk mengukur peningkatan hasil belajar peserta didik dengan melihat perbedaan pada skor pretest dan skor posttest. Uji ini digunakan untuk mengetahui efektivitas pembelajaran menggunakan perangkat pembelajaran yang dikembangkan. 3) Tahap Pengembangan (Development) lembar penilaian proses, sikap, dan keterampilan peserta didik dan lembar penilaian hasil belajar. T k ik A li i D t ) p g g ( p ) Tahap ini bertujuan untuk menghasilkan produk atau perangkat pembelajaran. Tahap ini terdiri dari 2 langkah yaitu: Validasi Ahli, dan Uji Coba Produk. Pada tahap validasi, produk akan divalidasi oleh para ahli yaitu ahli materi dan ahli media sebelum diujicobakan ke peserta didik. Perangkat pembelajaran yang dibuat akan dinilai oleh dosen ahli untuk mengetahui kelayakan penggunaan. Setelah dilakukan validasi, perangkat pembelajaran akan direvisi sesuai dengan saran dan kritik dari para validator sehingga hasil revisi akan menghasilkan draft 2 yang akan diujicobakan ke peserta didik. Pada tahap uji coba produk, perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme akan diujicobakan ke peserta didik. Penelitian ini menggunakan uji coba terbatas dengan satu kelas saja tanpa pembanding. Uji coba tersebut dilakukan guna mengetahui kepraktisan, dan keefektifan perangkat pembelajaran yang dikembangkan. 4. HASIL DAN PEMBAHASAN Hasil Penelitian Pengembangan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme dilakukan untuk meningkatkan proses dan hasil belajar peserta didik pada materi momentum dan impuls di SMA Negeri 2 Tondano. Penelitian ini menghasilkan produk berupa modul ajar, 146 p c) Tahap Development (Pengembangan) Thiagarajan dalam (Witri dkk., 2018) menjelaskan bahwa tahap ini terdiri dari dua langkah yaitu: 5. Perumusan Tujuan Pembelajaran 5. Perumusan Tujuan Pembelajaran Tujuan yang lebih khusus didapatkan dari konversi analisis tugas dan analisis materi yaitu untuk meningkatkan proses dan hasil belajar peserta didik melalui pengembangan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme. 2. Pemilihan Media 2. Pemilihan Media Media yang digunakan adalah modul ajar yang di dalamnya telah tercantumkan materi ajar dan LKPD. Pada LKPD terdapat alat dan bahan berupa mistar, mobil mainan, timbangan dan stopwatch. 3. Analisis Tugas 3. Analisis Tugas Tahap ini dilakukan penyusunan desain pembelajaran mencakup tugas-tugas atau LKPD yang harus diselesaikan oleh peserta didik. JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 materi ajar dan LKPD yang valid, praktis dan efektif menurut model pengembangan 4D. materi ajar dan LKPD yang valid, praktis dan efektif menurut model pengembangan 4D. Tahap ini dilakukan untuk merancang perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme. Tahap ini dilakukan dengan langkah sebagai berikut: Tahap ini dilakukan untuk merancang perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme. 4. Rancangan Awal 4. Rancangan Awal 4. Analisis Materi Perangkat pembelajaran yang dirancang diberikan kepada dosen pembimbing guna diberikan masukan, saran dan kritik untuk direvisi sehingga menjadi draft 1 yang selanjutnya akan divalidasi dan diujicobakan pada tahap pengembangan. Perangkat pembelajaran materi momentum dan impuls yang meliputi: Modul, LKPD dan bahan Ajar berbasis model experiental learning dengan pendekatan konstruktivisme Analisis ini dilakukan dengan mengumpulkan dan memilah materi yang relevan lalu menyusunnya kembali menjadi sistematis. Aktivitas peserta didik dapat dilihat jika kegiatan belajar diikuti dengan aktivitas fisik (Widiyasari dkk., 2020). Oleh karena itu, materi yang digunakan guna meningkatkan proses dan hasil belajar peserta didik. Materi yang digunakan dalam pengembangan perangkat pembelajaran berbasis experiential learning adalah momentum dan impuls. 1. Penyusunan Tes 1. Penyusunan Tes Penyusunan tes berpatokkan dari analisis tugas analisis materi dan perumusan tujuan pembelajaran pada tahap pendefinisian sesuai dengan kisi-kisi yang telah disusun sehingga hasil dari posttest yang menunjukkan pencapaian tujuan pembelajaran dari peserta didik. 1. Analisis Ujung Depan Berdasarkan wawancara yang dilakukan, maka didapati bahwa guru mengalami kendala dalam proses belajar mengajar antara lain rendahnya hasil belajar peserta didik oleh karena penggunaan model dan pendekatan pembelajaran yang tidak sesuai. Wawancara dengan peserta didik juga ditemukan bahwa kebanyakan metode yang digunakan oleh guru adalah metode ceramah dan pemberian tugas (konvensional) sehingga peserta didik merasa jenuh saat pembelajaran berlangsung. Peserta didik juga mengharapkan interaksi belajar yang dinamis dimana terdapat komunikasi antara peserta didik dan guru. p g g a) Tahap Define (Pendefinisian) Tahap ini dilakukan dengan langkah sebagai berikut: Tahap ini dilakukan dengan langkah sebagai berikut: Tahap ini mendefinisikan mengenai informasi yang dibutuhkan dalam pelaksanaan penelitian. Tahap ini dilakukan dengan langkah berikut: 3. Pemilihan Format 3. Pemilihan Format Kegiatan pembelajaran berlangsung dengan menggunakan pembelajaran mandiri dan berkelompok dalam 2 kali pertemuan dengan menggunakan format perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme dilandaskan pada sintaks experiential learning dan pendekatan konstruktivisme dan mengacu pada kurikulum merdeka belajar sedangkan untuk isi pembelajaran berpedoman terhadap hasil analisis tugas, analisis materi dan perumusan tujuan pembelajaran yang telah ditetapkan pada tahap pendefinisian. 2. Analisis Peserta Didik Peserta didik di kelas X B SMA Negeri 2 Tondano memiliki beragam sifat kemampuan yang bersifat heterogen yaitu tinggi, sedang dan rendah. Tahap ini menjadi dasar penyusunan perangkat pembelajaran yang sesuai dengan akademik peserta didik (Witri dkk., 2018). g p b) Tahap Design (Perancangan) JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 CHARM SAINS Volume 4 No 3 Hal: 142-153 keterbacaan dan bahasa. Hasil penilaian dan masukkan akan direvisi kemudian akan menghasilkan draft 2 yang akan digunakan dalam uji coba. Kepraktisan pembelajaran juga dapat dilihat dari angket respon peserta didik. Pemberian skor untuk tiap pernyataan menggunakan skala Likert, yang terdiri dari 7 indikator mengenai pendapat peserta didik tentang model experiential learning dengan pendekatan konstruktivisme, kesan peserta didik terhadap model experiential learning dengan pendekatan konstruktivisme, perasaan peserta didik selama mengikuti pembelajaran dengan menggunakan perangkat pembelajaran dengan model experiential learning dengan pendekatan konstruktivisme,pendapat peserta didik menggunakan LKPD dan materi yang telah dikembangkan dalam pembelajaran, hasil belajar peserta didik setelah mengikuti pembelajaran dengan model experiential learning dengan pendekatan konstruktivisme, dan efektivitas penggunaan LKPD yang telah dikembangkan. Ketujuh indikator dituangkan kedalam 35 pernyataan dengan indikator positif dan negatif. Hasil analisis angket respon peserta didik secara keseluruhan disimpulkan bahwa mayoritas peserta didik menyatakan bahwa proses pembelajaran dengan menggunakan model experiential learning dengan pendekatan konstruktivisme praktis untuk digunakan di dalam kelas. Tabel 2. Skor Rerata Validasi Perangkat Tabel 2. Skor Rerata Validasi Perangkat Tabel 2. Skor Rerata Validasi Perangkat Pembelajaran Instrumen Nilai Rata-Rata Materi 3,85 Modul Ajar 3,82 LKPD 3,81 Tabel 2 menunjukkan perangkat pembelajaran dinyatakan valid oleh dua validator. Tabel 2. Skor Rerata Validasi Perangkat Pembelajaran 2. Uji Coba Produk 2. Uji Coba Produk Uji coba dilakukan untuk melihat kepraktisan dan keefektifan perangkat pembelajaran yang dikembangkan berbasis experiential learning dengan pendekatan konstruktivisme adalah kegiatan uji coba perangkat pembelajaran pada sasaran subjek yang sesungguhnya (Witri dkk., 2018). Uji kepraktisan ditentukan dari dua faktor yaitu keterlaksanaan pembelajaran yang menggunakan lembar observasi keterlaksanaan pembelajaran yang dibuat sesuai dengan sintaks pembelajaran model experiential learning dengan pendekatan konstruktivisme dan angket respon peserta didik. Uji keefektifan dapat dilihat melalui penilaian proses belajar peserta didik berdasarkan 9 indikator penilaian masing- masing memiliki 3 kategori yang dinilai. Berdasarkan penelitian dan pengamatan didapatkan persentase penilaian proses belajar peserta didik. Keefektifan pembelajaran juga dapat dilihat dari hasil belajar peserta didik. Blomm mengatakan bahwa hasil belajar merupakan perubahan perilaku yang mencakup 3 aspek yaitu kognitif, afektif dan psikomotorik sehingga analisis data hasil belajar peserta didik dalam penelitian ini dikategorikan ke dalam 3 kategori, yaitu analisis hasil belajar penilaian sikap, analisis hasil belajar penilaian keterampilan dan analisis hasil belajar penilaian pengetahuan peserta didik (Maulana & Leonard, 2018). Penilaian dilakukan dua kali pertemuan. titunjukkan pada tabel 4. Tabel 3. Persentase Keterlaksanaan Tabel 3. 1. Validasi Ahli Validasi dilakukan oleh dua orang dosen ahli fisika yaitu ahli media dan ahli materi. Validator menguji kelayakan perangkat pembelajaran baik modul ajar, LKPD dan materi ajar dari 4 syarat yaitu konstruksi, isi, b) Tahap Design (Perancangan) 147 CHARM SAINS Volume 4 No 3 Hal: 142-153 Sikap 1 76,71% Baik 2 83,95% Baik Rata-Rata 80,33% Baik Ketrampilan 1 72,48% Baik 2 84,47% Baik Rata-Rata 78,48% Baik sehingga perangkat pembelajaran dikatakan efektif. d) Tahap Desseminate (Penyebaran) Penyebaran produk berupa perangkat pembelajaran yang meliputi modul ajar, materi ajar dan LKPD dilakukan di SMA Negeri 2 Tondano dengan cara diberikan kepada guru mata pelajaran IPA dan kepada peserta didik untuk dapat digunakan dalam pembelajaran IPA, khususnya materi momentum dan impuls. Pembahasan Penggunaan perangkat pembelajaran model experiential learning dengan pendekatan konstruktivisme membuat peserta didik aktif dalam mengikuti pembelajaran. Penggunaan perangkat juga membuat hasil belajar sikap dan keterampilan peserta didik meningkat sehingga dapat disimpulkan bahwa perangkat pembelajaran yang dikembangkan dapat digunakan. Tujuan dari penelitian ini adalah untuk mengembangkan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme sehingga menghasilkan perangkat pembelajaran yang valid, praktis dan efektif dalam meningkatkan proses dan hasil belajar pada materi momentum dan impuls. Perangkat pembelajaran yang dihasilkan terdiri dari materi ajar, modul ajar dan LKPD. g 1) Hasil Belajar Pengetahuan 1) Hasil Belajar Pengetahuan Hasil belajar pengetahuan dilakukan dengan menggunakan lembar penilaian tes hasil belajar pretest dan posttest. Tes disusun berdasarkan indikator soal sebanyak 5 butir soal. Tingkatan ranah kognitif tes disusun sesuai 3 tingkatan yaitu C2 (pemamahaman), C3 (penerapan) dan C4 (analisis). Terdapat peningkatan hasil belajar ranah kognitif dengan nilai 0,7 pada C2 dengan kriteria “Sedang”, 0,8 pada C3 dengan kriteria “Tinggi” dan 0,6 pada C4 dengan kriteria “Sedang”. Tahap awal dari penelitian ini yaitu define studi pendahuluan yang dilakukan guna mempersiapan tahap design dan development. Tahap awal terdiri dari 4 langkah, yaitu analisis awal akhir, analisis peserta didik , analisis tugas,analisis materi dan perumusan tujuan pembelajaran. Tahap pertama, analisis awal akhir dilakukan observasi guna mengumpulkan permasalahan yang berhubungan dengan penelitian. Tahap ini dilaksanakan dengan wawancara kepada guru dan peserta didik mengenai pembelajaran fisika di dalam kelas. Umumnya perserta didik menjawab bahwa pelajaran fisika di kelas membosankan karena sistem penugasan yang dilakukan oleh guru dan pembelajaran konvensional yang sering dilakukan di dalam kelas. Guru juga melaksanakan pembelajaran menggunakan model dan pendekatan pembelajaran yang belum sesuai. Tahap kedua yaitu analisis peserta didik dimana peserta didik dibagi berdasarkan karakteristik dan tingkatan kemampuan peserta didik di kelas XB SMA Negeri 2 Tondano dengan kemampuan peserta didik yang memiliki sifat heterogen yaitu tinggi, sedang, dan rendah yang menjadi panduan dalam menyusun perangkat pembelajaran yang sesuai dengan tingkatan kemampuan akademik peserta didik. JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 CHARM SAINS Volume 4 No 3 Hal: 142-153 Persentase Keterlaksanaan Pembelajaran Pertemuan Persentase (%) Kriteria 1 93,1% Sangat Baik 2 96,5% Sangat Baik Rata-Rata 94,9% Sangat Baik Tabel 3. Persentase Keterlaksanaan Pembelajaran Kesimpulan yang ditemukan dari tabel 3 adalah pembelajaran yang dilaksanakan telah sesuai dengan modul ajar yang dikembangkan dan direncanakan sehingga efektif untuk digunakan di dalam kelas. Gambar 2. Pembelajaran Menggunakan Experiential Learning dan Pendekatan Konstruktivisme Gambar 2. Pembelajaran Menggunakan Experiential Learning dan Pendekatan Konstruktivisme Tabel 4. Hasil Penilaian Proses, Sikap dan Tabel 4. Hasil Penilaian Proses, Sikap dan Keterampilan Indikator Pert. Persentase (%) Kriteria Proses 1 73,79% Baik 2 84,49% Baik Rata-Rata 79,14% Baik Gambar 2. Pembelajaran Menggunakan Experiential Learning dan Pendekatan Konstruktivisme 148 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 pembelajaran yang didalamnya mencakup tugas-tugas atau LKPD yang harus diselesaikan oleh peserta didik. Tahap keempat yaitu analisis materi, tahap mengidentifikasi materi yang akan digunakan dan dikembangkan dalam penelitian. Analisis ini dilakukan dengan mengumpulkan dan memilah materi yang relevan kemudian menyusunnya kembali dengan sistematis yitu momentum dan impuls. Tahap kelima adalah perumusan tujuan pembelajaran yang dikonversikan dari analisis tugas dan analisis materi sehingga menjadi tujuan yang lebih khusus yaitu meningkatkan proses dan hasil belajar peserta didik dengan mengembangkan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme sebagai fasilitas belajar bagi peserta didik kelas XB di SMA Negeri 2 Tondano. learning dengan pendekatan konstruktivisme dalam meningkatkan proses dan hasil belajar peserta didik. 1) Validitas Perangkat Pembelajaran Valid dan tidaknya perangkat pembelajaran yang dikembangkan telah melalui tanggapan para ahli. Terdapat lima aspek penilaian yang dinilai yaitu konstruksi, isi, keterbacaan, bahasa dan penampilan untuk setiap instrumen perangkat pembelajaran yang dikembangkan mencakup materi ajar, modul ajar dan LKPD. Hasil validasi ahli materi pada validasi pertama adalah materi ajar valid digunakan dengan revisi kecil. Komentar dan saran dari validator ahli materi menjadi bahan perbaikan dan revsi oleh peneliti sehingga pada validasi akhir materi valid digunakan tanpa revisi. Modul ajar yang dikembangkan menggunakan model experiential learning dengan menggunakan pendekatan konstruktivisme divalidasi mencakup 2 kali pertemuan oleh validator ahli media dengan hasil validasi pertama valid dengan revisi kecil. Komentar dan saran dari validator kemudian dijadikan acuan sebagai bahan revisi sehingga pada validasi akhir modul ajar valid digunakan tanpa revisi. Tahap design, dilakukan dalam merancang perangkat pembelajaran materi momentum dan impuls berbasis experiential learning dengan pendekatan konstruktivisme. Tahap ini terdiri dari 3 langkah yaitu penyusunan tes agar mengetahui pemahaman peserta didik dalam pembelajaran. Langkah kedua adalah pemilihan media yaitu modul ajar yang didalamnya terdapat materi yang dan LKPD yang telah dikembangkan berbasis model experiential learning dengan pendekatan konstruktivisme. Selanjutnya bahan yang digunakan untuk LKPD adalah bahan praktikum:1) Mistar 2) Stopwatch 3) Timbangan 4) Mobil mainan. Langkah ketiga adalah pemilihan format dimana kegiatan pembelajaran berlangsung dengan menggunakan pembelajaran mandiri dan berkelompok dalam 2 kali pertemuan dengan menggunakan sintaks model experiential learning dengan pendekatan konstruktivisme mengacu pada kurikulum merdeka belajar. Hasil validasi ahli media pada LKPD adalah valid dengan revisi besar. Komentar dan saran dari validator kemudian dijadikan acuan perbaikan sehingga pada validasi akhir LKPD dinyatakan valid dan dapat digunakan tanpa revisi. CHARM SAINS Volume 4 No 3 Hal: 142-153 Tahap ketiga yaitu analisis tugas yaitu melakukan pembuatan desain pembelajaran yang dapat mengembangkan keterampilan afektif, psikomotor dan kognitif peserta didik dalam Tabel 5. Skor N-Gain Hasil Belajar Peserta j Didik Pertemuan 1 (Pretest) Pertemuan 2 (Posttest) N- Gain Kriteria 3 71,11 0,7 Sedang Nilai rata-rata pada pertemuan 1 adalah 3, karena peserta didik belum mempelajari materi momentum dan impuls. Pada pertemuan 2 diperoleh nilai rata-rata 71,11 karena telah dilakukan pembelajaran menggunakan perangkat pembelajaran berbasis model experiential learning dengan pendekatan konstruktivisme. Dari nilai tersebut diperoleh skor Gain sebesar 0,7 dengan kriteria “Sedang”. Hasil penelitian serupa yang menjelaskan mengenai model experiential learning juga memperoleh peningkatan kemampuan peserta didik dalam berpikir dengan nilai Gain sebesar 0,67 dengan kriteria “Sedang” (Hajjah dkk., 2022). Dari nilai pretest dan posttest juga didapatkan kesimpulan bahwa dengan menggunakan perangkat pembelajaran berbasis model model experiential learning dengan pendekatan konstruktivisme dapat meningkatkan hasil belajar peserta didik 149 CHARM SAINS Volume 4 No 3 Hal: 142-153 CHARM SAINS Volume 4 No 3 Hal: 142-153 experiential learning dengan pendekatan konstruktivisme pada materi momentum dan impuls di kelas X SMA Negeri 2 Tondano adalah praktis. Dengan demikian kriteria kepraktisan telah terpenuhi. pertemuan pertama dan kedua. Pada pertemuan pertama dilaksanakan tes 1 sebelum peneliti mengimplementasikan model dan tes 2 dilaksanakan sesudah implementasi model. Berdasarkan hasil analisis yang dilakukan, diperoleh rata-rata tes hasil belajar pada pertemuan pertama adalah 3% dan untuk pertemuan kedua setelah implementasi model adalah 71,11%. Dari data tersebut dapat ditentukan skor N Gain sebesar 0,7 dengan kriteria “Sedang”. Pada uji N Gain untuk tiap indikator didapati skor sebesar 0,7 (C2) dengan kriteria “Sedang”, 0,8 (C3) dengan kriteria “Tinggi” dan 0,6 (C4) dengan kriteria “Sedang”. Dari hasil tersebut ditemukan bahwa terdapat peningkatan hasil belajar pada peserta didik sehingga perangkat pembelajaran yang dikembangkan dengan menggunakan model experiential learning dengan pendekatan konstruktivisme dapat dikatakan efektif. 3) Keefektifan Perangkat Pembelajaran ) g j Perangkat pembelajaran yang dihasilkan dari pengembangan model experiential learning dengan pendekatan konstruktivisme dapat dikatakan efektif didasari oleh penilaian proses belajar peserta didik dan hasil belajar peserta didik. Berdasarkan hasil analisis penilaian proses peserta didik, terdapat nilai persentase pertemuan pertama 73,79% dan pertemuan kedua 84,49% dan diperoleh rata- rata 79,14 dengan kriteria “Baik”, sehingga perangkat pembelajaran menggunakan model experiential learning dengan pendekatan konstruktivisme yang dikembangkan adalah efektif ditinjau dari penilaian proses peserta didik. Dari perbandingan hasil penilaian proses juga dapat disimpulkan bahwa terdapat peningkatan proses belajar peserta didik pada pertemuan pertama dan kedua, sehingga dapat disimpulkan bahwa dengan menggunakan perangkat pembelajaran model experiential learning dengan pendekatan konstruktivisme terdapat perolehan proses belajar peserta didik “Meningkat”. Hal ini sejalan dengan hasil penelitian yang menjelaskan bahwa pendekatan konstruktivisme dapat meningkatkan proses dan hasil belajar peserta didik (Ilham Dhani dkk., 2022). Hasil penelitian ini diperkuat dengan hasil penelitian sebelumnya yang menemukan bahwa penggunaan perangkat model experiential learning dapat membuat peserta didik aktif di dalam kelas dan pembelajaran menjadi efektif (Sagitarini dkk., 2018). Dari penilaian hasil belajar kognitif juga didapatkan bahwa adanya peningkatan hasil belajar peserta didik setelah menggunakan perangkat pembelajaran berbasis experiential learning dengan pendekatan konstruktivisme. Tahap desseminate, adalah tahapan paling akhir dari penelitian pengembangan ini yang berguna untuk mempromosikan hasil pengembangan (Pakiding dkk., 2023). Hasil dari penelitian pengembangan ini adalah perangakat pembelajaran bebasis model experiential learning dengan pendekatan konstruktivisme yang valid, praktis, dan efektif digunakan dalam pembelajaran. JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 JURNAL PENDIDIKAN FISIKA E-ISSN 2722-5860 25 Oktober 2023 2) Kepraktisan Perangkat Pembelajaran Kepraktisan dari perangkat pembelajaran yang dikembangkan dapat dilihat dari observasi keterlaksanaan pembelajaran, serta angket respon peserta didik. Berdasarkan pengamatan yang dilakukan oleh pengamat terhadap pembelajaran, ditemukan bahwa keterlaksanaan pembelajaran sesuai dengan langkah-langkah pada modul pada pertemuan pertama adalah 93,1% dan pertemuan kedua adalah 96,5%, sehingga mendapatkan rata-rata persentase keterlaksanaan pembelajaran 94,9% dengan kriteria “Sangat Baik” berdasarkan kriteria menurut (Sukardi 2013:201) (Monoarfa dkk., 2022). Tahap development adalah pengembangan. Penyusunan produk awal didapatkan berdasarkan hasil studi literatur yang merupakan kesimpulan yang bersifat teoritis dan hasil penelitian terdahulu dan juga data studi lapangan sehingga menghasilkan draf 1. Draf 1 lalu divalidasi oleh 2 validator yaitu ahli media dan ahli materi. Hasil validasi kemudian direvisi oleh peneliti dan menghasilkan draf 2 yang digunakan pada uji coba terbatas dan tahap uji coba. Tahap uji coba dilaksanakan untuk menguji kelayakan dari perangkat pembelajaran berbasis model experiential Kepraktisan juga ditentukan dari angket respon peserta didik. Pada lembar angket terdiri dari 35 pernyataan dari 7 indikator berbeda. Hasil perolehan respon peserta didik didapatkan bahwa pengembangan perangkat pembelajaran dengan menggunakan model 150 CHARM SAINS Volume 4 No 3 Hal: 142-153 Perangkat pembelajaran berupa modul ajar, materi ajar, dan LKPD kemudian disebarluaskan kepada para peserta didik di SMA Negeri 2 Tondano dan kepada guru mata pelajaran fisika untuk digunakan dalam pembelajaran di sekolah. Keefektifan perangkat dapat pula ditinjau dari hasil belajar peserta didik dari segi afektif, psikomotor dan kognitif. Dari analisis data yang diperoleh persentase hasil belajar penilaian sikap (afektif) peserta didik pada pertemuan pertama adalah 76,71% dan pertemuan kedua 83,95% dengan rata-rata 80,33% berkriteria “Baik”. Hasil belajar keterampilan peserta didik pada pertemuan pertama diperoleh nilai persentase sebesar 72,48% dan pertemuan kedua sebesar 84,47% dengan nilai rata-rata 78,48% dengan kriteria “Baik” berdasarkan (Sukardi 2013:201) (Monoarfa dkk., 2022). Nilai tersebut menunjukkan bahwa perangkat pembelajaran model experiential learning dengan pendekatan konstruktivisme sangat efektif untuk digunakan ditinjau dari sikap dan keterampilan peserta didik. 6. REFERENSI Apriovilita Hariri, C., & Yayuk, E. (2018). Penerapan Model Experiential Learning untuk Meningkatkan Pemahaman Materi Cahaya dan Sifat-Sifatnya Siswa Kelas 5 SD. Jurnal Pendidikan dan Kebudayaan , 8(1), 1–15. www.diknas.net Ginoga, S., Silangen, P. M., & Polii, J. (2023). Penerapan Model Problem Based Learning Secara Daring dalam Meningkatkan Hasil Belajar Fisika Materi Elastisitas dan Hukum Hooke pada Siswa Kelas XI SMA Negeri 1 Tondano. CHARM SAINS: Jurnal Pendidikan Fisika, 4(1), 105–111. Arkadiantika, I., Ramansyah, W., Effindi, M. A., & Dellia, P. (2020). Pengembangan Media Pembelajaran Virtual Reality pada Materi Pengenalan Termination dan Splicing Fiber Optic. 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https://openalex.org/W4243886542	https://www.qeios.com/read/X0P9GF/pdf	English	null	Electrolytes	Definitions	2,020	cc-by	75	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Electrolytes National Cancer Institute National Cancer Institute Qeios ID: X0P9GF · https://doi.org/10.32388/X0P9GF Source National Cancer Institute. Electrolytes. NCI Thesaurus. Code C464. Substances that ionize into anions and cations in body fluids. Electrolytes regulate nerve and muscle function and water distribution between cells, plasma, and interstitial fluid. Imbalances can cause serious physiologic effects, including seizures, cardiac arrhythmias and convulsions. Qeios ID: X0P9GF · https://doi.org/10.32388/X0P9GF 1/1
https://openalex.org/W2101871313	https://zenodo.org/records/1234679/files/article.pdf	English	null	Pacific Interdecadal Climate Variability: Linkages between the Tropics and the North Pacific during Boreal Winter since 1900	Journal of climate	2,004	public-domain	12,647	Corresponding author address: Dr. Clara Deser, Climate and Glob- al Dynamics Division, NCAR, P.O. Box 3000, Boulder, CO 80307. E-mail: cdeser@ucar.edu ABSTRACT This study examines the tropical linkages to interdecadal climate ﬂuctuations over the North Paciﬁc during boreal winter through a comprehensive and physically based analysis of a wide variety of observational datasets spanning the twentieth century. Simple difference maps between epochs of high sea level pressure over the North Paciﬁc (1900–24 and 1947–76) and epochs of low pressure (1925–46 and 1977–97) are presented for numerous climate variables throughout the tropical Indo-Paciﬁc region, including rainfall, cloudiness, sea surface temperature (SST), and sea level pressure. The results support the notion that the Tropics play a key role in North Paciﬁc interdecadal climate variability. In particular, SST anomalies in the tropical Indian Ocean and southeast Paciﬁc Ocean, rainfall and cloudiness anomalies in the vicinity of the South Paciﬁc convergence zone, stratus clouds in the eastern tropical Paciﬁc, and sea level pressure differences between the tropical southeast Paciﬁc and Indian Oceans all exhibit prominent interdecadal ﬂuctuations that are coherent with those in sea level pressure over the North Paciﬁc. The spatial patterns of the interdecadal tropical climate anomalies are compared with those associated with ENSO, a predominantly interannual phenomenon; in general, the two are similar with some differences in relative spatial emphasis. Finally, a published 194-yr coral record in the western tropical Indian Ocean is shown to compare favorably with the twentieth-century instrumental records, indicating the potential for extending knowledge of tropical interdecadal climate variability to earlier time periods. 3109 3109 D E S E R E T A L . 15 AUGUST 2004 1. Introduction Paciﬁc; opposite conditions obtain for positive values. The NPI also provides a proxy record of the Paciﬁc– North American (PNA) teleconnection pattern evident in the middle troposphere (Trenberth and Hurrell 1994). A striking feature of the NPI is the occurrence of ex- tended periods, two to three decades in duration, with predominantly positive (1900–24 and 1947–76) or neg- ative values (1925–46 and 1977–2003). In addition to the interdecadal ﬂuctuations associated with the phase transitions in 1925, 1947, and 1977, the NPI also ex- hibits considerable high-frequency variability: approx- imately 75% of the variance of the NPI occurs at periods shorter than 20 yr (not shown). Paciﬁc; opposite conditions obtain for positive values. The NPI also provides a proxy record of the Paciﬁc– North American (PNA) teleconnection pattern evident in the middle troposphere (Trenberth and Hurrell 1994). A striking feature of the NPI is the occurrence of ex- tended periods, two to three decades in duration, with predominantly positive (1900–24 and 1947–76) or neg- ative values (1925–46 and 1977–2003). In addition to the interdecadal ﬂuctuations associated with the phase transitions in 1925, 1947, and 1977, the NPI also ex- hibits considerable high-frequency variability: approx- imately 75% of the variance of the NPI occurs at periods shorter than 20 yr (not shown). Numerous studies have documented interdecadal var- iations in climate over the North Paciﬁc, including Tren- berth and Hurrell (1994), Mantua et al. (1997, hereafter M97), Minobe (1997), Zhang et al. (1997), Dettinger et al. (2000), Chao et al. (2000), and Mantua and Hare (2002). These interdecadal climate ﬂuctuations are char- acterized by changes in the intensity of the large-scale atmospheric circulation over the North Paciﬁc during winter, with impacts upon physical and biological con- ditions in the North Paciﬁc Ocean and consequences for climate downstream over North America, including air temperature, precipitation, streamﬂow, and vegetation (Cayan et al. 2001). An index of the strength of the wintertime atmospheric circulation over the North Pa- ciﬁc is shown in Fig. 1 for the period 1899–2003. This ‘‘North Paciﬁc Index’’ (NPI; see Trenberth and Hurrell 1994) is the area-averaged sea level pressure (SLP) for the region 308–658N, 1608–1408W. 1. Introduction The shaded curve is the raw time series and the thick black curve is a low-pass-ﬁltered version using a weighted seven-point running mean. FIG. 2. The (top) inverted NPI, (middle) inverted SOI, and (bottom) eastern equatorial Paciﬁc SST (the CTI) during 1900–96, normalized by their respective std devs. The dashed vertical lines denote regime transitions identiﬁed on the basis of the NPI (1925, 1947, and 1977). This ﬁgure is reproduced from M97. climate noise due to the integrating effect of stochastic atmospheric variability by the North Paciﬁc Ocean mixed layer (Pierce et al. 2001; Newman et al. 2003) and the thermocline (Frankignoul et al. 2000). Note that the Tropics play an active role in some theories but not in others. was presented by Minobe (1997), who showed that an- nually averaged SST anomalies in the tropical Indian Ocean vary coherently with the NPI on interdecadal time scales. A more systematic investigation of the re- lationship between the North Paciﬁc and the Tropics on time scales longer than ;6 yr was presented by Zhang et al. (1997). They used linear regression analysis to document the annually averaged SST and SLP anomaly patterns associated with the dominant mode of global SST variability (a structure that resembles the 1977 cli- mate transition) after the effects of global warming and interannual ENSO ﬂuctuations are removed. They char- acterized the patterns as ‘‘ENSO-like’’ in view of their general similarity to those associated with the ENSO cycle. Due to the statistical nature of their approach, Zhang et al.’s analysis provides a general description of Paciﬁc low-frequency variability weighted toward the data-rich post–World War II period; it does not provide a speciﬁc description of each of the climate transitions individually. What do observational studies indicate about the role of the Tropics in North Paciﬁc interdecadal climate var- iability? Many have shown that the 1977 climate tran- sition in the North Paciﬁc is evident in the tropical Pa- ciﬁc where it resembles the pattern associated with the warm phase of the El Nin˜o–Southern Oscillation (ENSO) phenomenon (e.g., Nitta and Yamada 1989; Trenberth and Hurrell 1994; Graham et al. 1994; Hough- ton et al. 1996; M97; Zhang et al. 1997; Garreaud and Battisti 1999). Less is known about the tropical sig- nature of the earlier climate transitions in 1925 and 1947. 1. Introduction Negative values cor- respond to a deeper-than-normal Aleutian low pressure system, accompanied by enhanced westerly winds across the central North Paciﬁc and strengthened south- erly (northerly) ﬂow over the eastern (western) North A striking feature of the NPI is the occurrence of ex- tended periods, two to three decades in duration, with predominantly positive (1900–24 and 1947–76) or neg- ative values (1925–46 and 1977–2003). In addition to the interdecadal ﬂuctuations associated with the phase transitions in 1925, 1947, and 1977, the NPI also ex- hibits considerable high-frequency variability: approx- imately 75% of the variance of the NPI occurs at periods shorter than 20 yr (not shown). y What are the origins of interdecadal ﬂuctuations in the NPI? Several hypotheses have been proposed, in- cluding coupled ocean–atmosphere interactions within the North Paciﬁc (Latif and Barnett 1996; Robertson 1996) and with extensions to the tropical Paciﬁc via an atmospheric bridge (Barnett et al. 1999); air–sea inter- action within the subtropical Paciﬁc, with atmospheric teleconnections to the North Paciﬁc as a by-product (Knutson and Manabe 1998; Kleeman et al. 1999; McPhaden and Zhang 2002; Nonaka et al. 2002; Vimont et al. 2001; Liu et al. 2002); extratropical–tropical in- teractions via an equatorward subsurface oceanic path- way and a return atmospheric bridge (Gu and Philander 1997; Deser et al. 1996; Schneider et al. 1999); and q 2004 American Meteorological Society J O U R N A L O F C L I M A T E 3110 VOLUME 17 FIG. 1. The NPI from 1900 to 2003, normalized by its std dev (2.3 hPa). The NPI is deﬁned as the winter mean (Dec–Mar) SLP anomaly averaged over the region 308–658N, 1608–1408W. The shaded curve is the raw time series and the thick black curve is a low-pass-ﬁltered version using a weighted seven-point running mean. FIG. 2. The (top) inverted NPI, (middle) inverted SOI, and (bottom) eastern equatorial Paciﬁc SST (the CTI) during 1900–96, normalized by their respective std devs. The dashed vertical lines denote regime transitions identiﬁed on the basis of the NPI (1925, 1947, and 1977). This ﬁgure is reproduced from M97. FIG. 1. The NPI from 1900 to 2003, normalized by its std dev (2.3 hPa). The NPI is deﬁned as the winter mean (Dec–Mar) SLP anomaly averaged over the region 308–658N, 1608–1408W. 1. Introduction While this dataset has the advantage of increased spatial coverage and smoother ﬁelds than in the COADS, it may also introduce climate signals into areas devoid of observations or with limited data coverage. We have veriﬁed that our ﬁndings based upon the Kaplan et al. dataset are conﬁrmed by the COADS. y For air temperature, we use the University of East Anglia’s Climate Research Unit (CRU) dataset (Jones 1994), archived on a 58 3 58 latitude–longitude grid based upon land stations and ships of opportunity. For SLP, we make use of the COADS and an updated version of Trenberth and Paolino (1980). The latter is based upon daily historical analyses for the Northern Hemi- sphere poleward of 208N on a 58 3 58 latitude–longitude grid. For precipitation, we use the gridded dataset (2.58 latitude 3 3.758 longitude) from the University of East Anglia’s CRU (Hulme et al. 1998) based upon land station records. We also make use of total cloud amount from the COADS as a proxy for precipitation over the oceans in regions where rainfall-producing cloud sys- tems occur. (All datasets are available online at http:// www.cgd.ucar.edu/cas/guide/.) The paper is organized as follows: The datasets and analysis procedures are described in section 2. Epoch difference maps associated with the climate transitions in 1925, 1947, and 1977 are presented in section 3 along with selected regional time series. The results are sum- marized and discussed in section 4, including a com- parison to ENSO and a 194-yr paleoclimate reconstruc- tion based upon a tropical Indian Ocean coral record (Cole et al. 2000). g g We formed monthly anomalies by subtracting the long-term monthly means from each calendar month’s mean. Winter (December–March) means were computed from the monthly anomalies, requiring a minimum of 1 month to deﬁne a winter average. To improve the readability of the maps shown for COADS variables (SLP and cloudiness) and precipitation, linear interpo- lation (across gaps not exceeding three points in latitude and ﬁve points in longitude) and weighted binomial smoothing (three points in latitude and nine points in longitude) were applied. All regional time series are based on the raw (uninterpolated and unsmoothed) data. We formed epoch difference maps requiring a minimum of 20% of the years within each period to have obser- vations. All data were detrended prior to computation of the epoch difference maps. 1. Introduction This choice is dictated by the season of stron- gest atmospheric circulation variability over the North Paciﬁc (cf. Trenberth and Hurrell 1994) and established physical mechanisms for atmospheric teleconnections forced by deep convection in the Tropics (cf. Trenberth et al. 1998; Alexander et al. 2002). A suite of selected time series for key regions and climate parameters is also developed to further characterize and synthesize the tropical interdecadal variability that is coherent with the NPI. Finally, we compare the patterns of tropical climate anomalies on interannual (ENSO) and interde- cadal time scales, as in Garreaud and Battisti (1999; note that their analysis is limited to the post-1958 period and considers only annual averages). As far as we are aware, our study is the ﬁrst to examine, in a compre- hensive and systematic manner, the tropical Indo-Paciﬁc climate anomalies associated with interdecadal varia- tions in the NPI during boreal winter. features that are common to all three and those that are speciﬁc to a single case. All of the analyses are for the boreal winter season, December–March, unless noted otherwise. This choice is dictated by the season of stron- gest atmospheric circulation variability over the North Paciﬁc (cf. Trenberth and Hurrell 1994) and established physical mechanisms for atmospheric teleconnections forced by deep convection in the Tropics (cf. Trenberth et al. 1998; Alexander et al. 2002). A suite of selected time series for key regions and climate parameters is also developed to further characterize and synthesize the tropical interdecadal variability that is coherent with the NPI. Finally, we compare the patterns of tropical climate anomalies on interannual (ENSO) and interde- cadal time scales, as in Garreaud and Battisti (1999; note that their analysis is limited to the post-1958 period and considers only annual averages). As far as we are aware, our study is the ﬁrst to examine, in a compre- hensive and systematic manner, the tropical Indo-Paciﬁc climate anomalies associated with interdecadal varia- tions in the NPI during boreal winter. ﬁcial rise in SST around 1940 associated with the change in measurement technique from bucket to en- gine-intake temperatures. In the Kaplan et al. analyses, missing grid squares have been ﬁlled in using an (em- pirical orthogonal function) EOF-based technique. 1. Introduction The statistical signiﬁcance of the epoch difference values is assessed by means of a Student’s t test taking into account serial correlation (Zwiers and von Storch 1995; maps of the Student’s t values may be viewed online at http://www.cgd.ucar. edu/;cdeser/PDVsupﬁg.html for each epoch difference ﬁeld). When discussing the epoch difference maps, we emphasize mainly those features that are signiﬁcant at the 95% conﬁdence level. 1. Introduction Figure 2, reproduced from M97, compares the NPI with two canonical indices of ENSO: an equatorial Paciﬁc sea surface temperature (SST) index (the average of SST anomalies in the region 68N–68S, 1808–908W) from which the global mean SST anomalies have been subtracted and a Southern Oscillation index (SOI) [the SLP difference between the tropical Indian/western Pa- ciﬁc and the remaining oceanic areas (see M97 for de- tails)]. While the 1977 transition is evident in both ENSO records, the 1925 and 1947 shifts are not as clear. For example, the 1925 transition is absent from the SOI (which remains predominantly positive throughout the ﬁrst half of the twentieth century), and the 1947 shift is absent from the SST index. Furthermore, the trend of the SST index during the ﬁrst half of the twentieth cen- tury is in the opposite sense to that of the NPI. These discrepancies raise the question, Was the 1977 transition unique in terms of its connection to the tropical Paciﬁc as measured by the canonical ENSO indices, or are there other regions of the Tropics that exhibit a more consis- tent linkage to the NPI during the twentieth century? P li i id f b t t i l ti In this study, we revisit the issue of tropical linkages to North Paciﬁc interdecadal climate variability through a comprehensive and physically based analysis of a wide variety of observational datasets spanning the twentieth century. As mentioned earlier, elucidating the role of the Tropics is important from the standpoint of provid- ing guidance for mechanistic studies of Paciﬁc inter- decadal climate variability. We present simple differ- ence maps between epochs of high (1900–24 and 1947– 76) and low (1925–46 and 1977–97) NPI for numerous climate variables in the tropical Indo-Paciﬁc region, in- cluding SST, SLP, air temperature, precipitation, and cloudiness, emphasizing physical consistency among the various climatic parameters to build conﬁdence in the results. Comparing the three epoch differences (1900–24 minus 1925–46; 1947–76 minus 1925–46; and 1947–76 minus 1977–97) allows us to assess those Preliminary evidence for a robust tropical connection 3111 D E S E R E T A L . 15 AUGUST 2004 features that are common to all three and those that are speciﬁc to a single case. All of the analyses are for the boreal winter season, December–March, unless noted otherwise. 2. Data and methods Our strategy is to examine a suite of independent climate variables for collective evidence of coherent low-frequency variability over the Indo-Paciﬁc sector. We examine the period 1900–97 for all variables, al- though some datasets may nominally extend further back in time and some may be updated beyond 1997. Our choice of 1900 as the starting year for the analysis is based upon data coverage considerations (cf. Wood- ruff et al. 1987). For SST, we make use of two archives: the Compre- hensive Ocean–Atmosphere Data Set (COADS; Wood- ruff et al. 1987) and the Lamont-Doherty Earth Obser- vatory analyses (Kaplan et al. 1998). The COADS ar- chive contains monthly data assembled into 28 latitude 3 28 longitude grid squares over the global oceans dat- ing back to 1854 with a minimum of processing (e.g., standard quality control procedures but no ﬁlling in of missing data). The Kaplan et al. dataset contains month- ly data on a 58 latitude 3 58 longitude grid over the global oceans dating back to 1871 with substantially more processing than in COADS. The input data to the Kaplan et al. analyses are from the Met Ofﬁce historical SST dataset (Folland and Parker 1995), which are sim- ilar to the COADS except that additional corrections have been applied to empirically account for the arti- Simple linear correlation analysis is used to quantify the relationship between two time series. The statistical signiﬁcance of the resulting correlation coefﬁcient is assessed according to a one-tailed Student’s t test taking into account temporal autocorrelation following Tren- berth (1984). A correlation coefﬁcient that exceeds 0.36 in absolute value is signiﬁcantly different from 0 at the 3112 J O U R N A L O F C L I M A T E VOLUME 17 FIG. 3. Epoch difference maps of winter SLP (contours) and land precipitation (color shading) for high-minus-low NPI regimes (years as indicated above each panel). The contour interval for SLP is 1 hPa and negative contours are dashed. The precipitation scale (color bar at bottom of ﬁgure) is in units of mm day21. FIG. 3. Epoch difference maps of winter SLP (contours) and land precipitation (color shading) for high-minus-low NPI regimes (years as indicated above each panel). The contour interval for SLP is 1 hPa and negative contours are dashed. The precipitation scale (color bar at bottom of ﬁgure) is in units of mm day21. 2. Data and methods 95% conﬁdence level for data that have been temporally smoothed with a three-point binomial ﬁlter. All of the correlation coefﬁcients cited in this study exceed this value unless otherwise noted and are maximum at 0 lag. with maximum values ;4 hPa in the vicinity of the Aleutian Islands. Some differences are also apparent, particularly in the orientation of the anomalous ridge over the eastern North Paciﬁc, which shows a pro- nounced southeastward extension in the most recent ep- och difference map compared to the early one. The pre- cipitation ﬁelds are similar for the three epoch differ- ences, with positive values along the Asian coast (Japan and the Kamchatka peninsula), negative values in south- ern Alaska and the southwestern United States, and pos- itive values over the Paciﬁc Northwest. The similarity among the maps, particularly the ﬁrst and last, which contain no data in common, provides support for the robustness of the precipitation patterns. The banded structure over North America is similar to that docu- mented in Dettinger et al. (2000) based upon station records of precipitation and streamﬂow. The precipita- a. Extratropics This result is consistent with the notion that the response time for extratropical SST anomalies to atmospheric forcing is on the order of 1 month (Frankignoul 1985; Deser and Timlin 1997) and that once created, SST anomalies per- sist for many months due to the thermal inertia asso- ciated with a deep winter mixed layer (cf. Deser et al. 2003). However, epoch difference maps based upon De- cember–March look very similar to those based upon the extended winter season (not shown). The SST epoch differences (Fig. 6) exhibit positive values over the cen- tral and western Paciﬁc centered along ;408N and neg- ative values in the eastern and far northern Paciﬁc, sim- ilar to the leading EOF of monthly SST anomalies shown in M97. Some differences are apparent between the ﬁrst and last epoch difference maps, particularly in the Gulf of Alaska, where negative values are found in the early one and positive values in the later one. Note the overall similarity between the SST and marine air temperature patterns shown in Fig. 5. FIG. 4. Selected regional climate records over the North Paciﬁc and adjacent continents during 1899–2002 (1899–1998 for precipi- tation): the inverted NPI, the winter precipitation difference between coastal Alaska and Japan, winter surface air temperature anomalies over northwestern Canada and Alaska, and the leading PC time series of monthly SST anomalies over the North Paciﬁc from M97 averaged for the winter–spring season (see text for precise deﬁnitions). All records are normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents 1 std dev. tion patterns are understandable in the context of the large-scale atmospheric circulation changes. For ex- ample, a weak Aleutian low (anomalously high SLP) is accompanied by reduced low-level southerly ﬂow over the eastern North Paciﬁc that decreases the moisture ﬂux into coastal Alaska, consistent with the precipitation re- sponse. Similarly, anomalous southerly ﬂow over the western North Paciﬁc associated with a weak Aleutian low enhances the moisture ﬂux into coastal Asia, in agreement with the precipitation signal there. The weak amplitude of the precipitation anomalies over the west- ern United States in the earliest epoch difference map compared to the later ones may be related to the lack of an appreciable SLP signal over the eastern North Paciﬁc in the former. The January–May PDO record (obtained online at http://tao.atmos.washington.edu/pdo) is shown in Fig. 4. a. Extratropics Atmospheric circulation variations associated with the NPI have important consequences for climate over the North Paciﬁc and adjacent continents, as discussed in the introduction. Figure 3 shows epoch difference maps of winter SLP and precipitation for high-minus- low NPI regimes: 1900–24 minus 1925–46; 1947–76 minus 1925–46; and 1947–76 minus 1977–97. As ex- pected, positive SLP values extend over a broad region of the North Paciﬁc in all three epoch difference maps, 3113 D E S E R E T A L . 15 AUGUST 2004 FIG. 4. Selected regional climate records over the North Paciﬁc and adjacent continents during 1899–2002 (1899–1998 for precipi- tation): the inverted NPI, the winter precipitation difference between coastal Alaska and Japan, winter surface air temperature anomalies over northwestern Canada and Alaska, and the leading PC time series of monthly SST anomalies over the North Paciﬁc from M97 averaged for the winter–spring season (see text for precise deﬁnitions). All records are normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents 1 std dev. individual station records. The western North Paciﬁc Ocean, on the other hand, is relatively warm, mainly in the region of reduced westerly wind strength, consistent with an equilibrated response to the underlying SST change (Fig. 6) due to diminished upward turbulent en- ergy ﬂuxes at the sea surface and cold advection by anomalous Ekman currents (cf. Miller et al. 1994). The time series of air temperature over Alaska and north- western Canada (518–708N, 1758–1008W) is shown in Fig. 4. Like the precipitation difference record, it ex- hibits strong support for the interdecadal regimes evi- dent in the NPI; the correlation coefﬁcient between the air temperature record and the NPI is 0.74 based upon data smoothed with a three-point binomial ﬁlter and is maximum at 0 lag. Epoch difference maps for SST based upon the ex- tended winter season, January–May, are shown in Fig. 6. We use the extended winter season for SST in view of the fact that the correlation between the leading prin- cipal component time series of monthly SST anomalies over the North Paciﬁc [termed the Paciﬁc (inter) decadal oscillation (PDO) by M97], and the winter NPI is stron- gest during these months (not shown). a. Extratropics This time series exhibits a greater proportion of in- terdecadal compared to interannual variability than do the atmospheric indices shown in the ﬁgure, consistent with the greater thermal inertia of the ocean mixed layer compared to the atmosphere (cf. Frankignoul and Has- selmann 1977; Deser et al. 2003). With the exception of the ﬁrst decade of the twentieth century, the SST record is highly coherent with each of the atmospheric indices; the correlation coefﬁcient between the PDO re- cord and the NPI is 0.64 (maximum at zero lag; 0.78 for the period 1911–97). The origin of the discrepancy between the SST and atmospheric records during the ﬁrst decade of the twentieth century requires further study; however, it is unlikely that the atmospheric re- The time series of the precipitation difference be- tween Alaska (558–608N, 1608–1358W) and Japan (32.58–47.58N, 1308–1468E) is shown in Fig. 4 together with the NPI. The precipitation record exhibits a high degree of similarity to the NPI; their correlation coef- ﬁcient is 0.66 based upon data smoothed with a three- point binomial ﬁlter and is a maximum at 0 lag. The interdecadal regimes evident in the NPI are also ap- parent in the precipitation difference record, demon- strating the impact of large-scale atmospheric circula- tion variations upon climate on both sides of the Paciﬁc. Figure 5 shows epoch difference maps for winter air temperature and SLP. Interior Alaska and northwestern Canada are cold when the Aleutian low is weak, con- sistent with the results of Minobe (1997) based upon Figure 5 shows epoch difference maps for winter air temperature and SLP. Interior Alaska and northwestern Canada are cold when the Aleutian low is weak, con- sistent with the results of Minobe (1997) based upon 3114 J O U R N A L O F C L I M A T E VOLUME 17 FIG. 5. As in Fig. 3 but for winter SLP (contours) and surface air temperature (color shading). The air temperature scale (color bar at bottom of ﬁgure) is in units of 8C. FIG. 5. As in Fig. 3 but for winter SLP (contours) and surface air temperature (color shading). The air temperature scale (color bar at bottom of ﬁgure) is in units of 8C. a. Extratropics the NPI and boreal winter SST anomalies based upon the period 1900–97 using low-pass-ﬁltered data to em- phasize ﬂuctuations with periods greater than ;9 yr (the results are not unduly sensitive to the low-pass ﬁlter used, provided that the cutoff is at periods longer than a few years). The strongest positive correlations (r . 0.6) are found in the tropical Indian Ocean extending into the far western Paciﬁc and outside of the equatorial zone in the eastern tropical Paciﬁc. The correlations within the Indian Ocean reach peak values of 0.8 along ;108N. Weak and insigniﬁcant correlations are found along the equator east of 1808. cords are erroneous in light of their mutual consistency. More likely, in our view, is that the SST index contains spurious signals due to the paucity of data during this period, a speculation that deserves further investigation. The 1925 and 1947 climate transitions in the NPI appear to be preceded by shifts in the PDO index by a couple of years. We caution against attaching too much sig- niﬁcance to this apparent lead of the ocean relative to the atmosphere since 1) the 1977 transition is simul- taneous and 2) the NPI and PDO correlate most strongly at 0 lag (not shown). Another view of the SST anomaly patterns associated with interdecadal variations in the NPI is obtained from epoch difference maps based on Kaplan et al. (1998) data (Fig. 8, left). Negative values are found over most of the tropical Indo-Paciﬁc in all three epoch differences except for weak positive values along the equator be- b. Tropical linkages For the same reason, the earliest epoch difference map based upon (top right) COADS excludes data from the period 1940–46. FIG. 8. Epoch difference maps of boreal winter SST from (left) the Kaplan et al. (1998) dataset and (right) COADS. The scale (color bar at bottom of ﬁgure) is in units of 8C. Note that the COADS data are not interpolated (white areas denote missing data). The middle panel has been omitted for COADS due to the artiﬁcial increase in SSTs between 1940 and 1941 resulting from a change in observing practice. For the same reason, the earliest epoch difference map based upon (top right) COADS excludes data from the period 1940–46. tween the international date line and the coast of South America in the earliest one. Note that although the mag- nitudes of the SST differences in the Indian Ocean are relatively small (;0.2 K), they are highly signiﬁcant statistically due to the relatively weak background in- terannual variability. The opposing sign of the equa- torial east Paciﬁc ‘‘cold tongue’’ SST anomalies be- tween the ﬁrst and last epoch differences results in weak correlations with the NPI for the twentieth century as a whole (recall Fig. 7). Other seasons also exhibit weak positive values in the eastern equatorial Paciﬁc for the earliest epoch difference (not shown). The COADS (Fig. 8, right) conﬁrms the results based upon Kaplan et al. (1998), indicating that this feature is not due to the way in which the Kaplan et al. SST data were processed (recall section 2). Further support for the credibility of the change in sign of the SST anomalies in the equatorial cold tongue between the ﬁrst and last epoch differences is the similar behavior of SLP anomalies at Darwin, Australia (not shown), a reliable index of the Southern Oscillation (cf. Wright et al. 1988; Deser and Wallace 1990). Indeed, the Darwin SLP record correlates with the cold tongue SST index (CTI; SST anomalies av- eraged over the region 58N–58S, 1808–1108W; see Deser and Wallace 1990) at a level of 0.87 based upon averages for boreal winter during 1900–46 (0.85 during 1947– 97), attesting to the ﬁdelity of both records. tween the international date line and the coast of South America in the earliest one. b. Tropical linkages 1) SST To identify areas in the Tropics where SSTs vary co- herently with the NPI on time scales longer than inter- annual, we show in Fig. 7 the correlation map between 3115 D E S E R E T A L . 15 AUGUST 2004 FIG. 6. As in Fig. 3 but for winter SLP (contours) and winter–spring (Dec–Mar) SST (color shading). The SST scale (color bar at bottom of ﬁgure) is in units of 8C. FIG. 6. As in Fig. 3 but for winter SLP (contours) and winter–spring (Dec–Mar) SST (color shading). The SST scale (color bar at bottom of ﬁgure) is in units of 8C. FIG. 7. Correlation map between the inverted NPI and boreal winter SST anomalies in the tropical Indo-Paciﬁc, based on 9-yr low-pass-ﬁltered data during the period 1900–97. The color bar shows the scale for the correlation coefﬁcients. FIG. 7. Correlation map between the inverted NPI and boreal winter SST anomalies in the tropical Indo-Paciﬁc, based on 9-yr low-pass-ﬁltered data during the period 1900–97. The color bar shows the scale for the correlation coefﬁcients. FIG. 7. Correlation map between the inverted NPI and boreal winter SST anomalies in the tropical Indo-Paciﬁc, based on 9-yr low-pass-ﬁltered data during the period 1900–97. The color bar shows the scale for the correlation coefﬁcients. 3116 J O U R N A L O F C L I M A T E VOLUME 17 FIG. 8. Epoch difference maps of boreal winter SST from (left) the Kaplan et al. (1998) dataset and (right) COADS. The scale (color bar at bottom of ﬁgure) is in units of 8C. Note that the COADS data are not interpolated (white areas denote missing data). The middle panel has been omitted for COADS due to the artiﬁcial increase in SSTs between 1940 and 1941 resulting from a change in observing practice. For the same reason, the earliest epoch difference map based upon (top right) COADS excludes data from the period 1940–46. FIG. 8. Epoch difference maps of boreal winter SST from (left) the Kaplan et al. (1998) dataset and (right) COADS. The scale (color bar at bottom of ﬁgure) is in units of 8C. Note that the COADS data are not interpolated (white areas denote missing data). The middle panel has been omitted for COADS due to the artiﬁcial increase in SSTs between 1940 and 1941 resulting from a change in observing practice. 2) PRECIPITATION Although SST changes over the tropical Indian and southeastern tropical Paciﬁc Oceans are coherent with interdecadal variations in the NPI, SST anomalies per se do not force atmospheric teleconnections to higher latitudes; the release of latent heat in precipitating deep convection is the direct forcing mechanism. Therefore, we examine the tropical rainfall changes associated with interdecadal variations in the NPI using station data over land and cloudiness as a proxy over the oceans. As background to the epoch difference maps, the long-term mean precipitation distribution during boreal winter based upon satellite measurements (Xie and Arkin 1997) for the period 1979–97 is shown in Fig. 10 (top). The highest rainfall amounts occur over the southwestern tropical Paciﬁc within the South Paciﬁc convergence zone (SPCZ), along ;78N within the Paciﬁc Intertrop- b. Tropical linkages Each tick mark on the ordinate represents one std dev. The numeral to the right of each tropical index represents its correlation coefﬁcient with the inverted NPI. FIG. 9. Selected regional climate anomaly records over the tropical Indo-Paciﬁc for boreal winter during 1900–97: the inverted NPI (through 2000), tropical Indian Ocean SST, southeast tropical Paciﬁc SST, rainfall south of the SPCZ, rainfall within the SPCZ, central Paciﬁc cloudiness, and tropical Indian Ocean minus southeast Paciﬁc SLP. Regions are deﬁned in the text and shown in Figs. 11–13. All records are normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents one std dev. The numeral to the right of each tropical index represents its correlation coefﬁcient with the inverted NPI. p p We formed two regional precipitation indices for the tropical western Paciﬁc, choosing areas with consistent signals in all three epoch difference maps (see Fig. 11 for regional outlines); these indices are shown in Fig. 9. The precipitation index for the poleward ﬂank of the SPCZ (‘‘SPCZ Rain S’’) is highly correlated with the NPI (r 5 0.69) and exhibits clear evidence of regime transitions in 1947 and 1977, while the 1925 transition occurred a couple of years in advance (note that this index extends back to 1908 only). The precipitation in- dex for the main branch of the SPCZ (‘‘SPCZ Rain’’), which extends back to 1929 only, also shows coherent behavior with the NPI (r 5 0.43), although the 1947 transition occurred several years too early. b. Tropical linkages Note that although the mag- nitudes of the SST differences in the Indian Ocean are relatively small (;0.2 K), they are highly signiﬁcant statistically due to the relatively weak background in- terannual variability. The opposing sign of the equa- torial east Paciﬁc ‘‘cold tongue’’ SST anomalies be- tween the ﬁrst and last epoch differences results in weak correlations with the NPI for the twentieth century as a whole (recall Fig. 7). Other seasons also exhibit weak positive values in the eastern equatorial Paciﬁc for the earliest epoch difference (not shown). The COADS (Fig. 8, right) conﬁrms the results based upon Kaplan et al. (1998), indicating that this feature is not due to the way in which the Kaplan et al. SST data were processed (recall section 2). Further support for the credibility of the change in sign of the SST anomalies in the equatorial cold tongue between the ﬁrst and last epoch differences is the similar behavior of SLP anomalies at Darwin, Australia (not shown), a reliable index of the Southern Oscillation (cf. Wright et al. 1988; Deser and Wallace 1990). Indeed, the Darwin SLP record correlates with ), g y Figures 7 and 8 indicate that SST ﬂuctuations in the tropical Indian Ocean and the eastern Paciﬁc north and south of the equator are associated with low-frequency variations in the NPI. The SST anomaly time series for the tropical Indian (108S–208N, 508–1258E) and south- east tropical Paciﬁc (58–208S, 1408–908W) are shown in Fig. 9, together with the NPI (the northeast tropical Paciﬁc is similar to the southeast record, but is less highly correlated with the NPI, and the interdecadal regimes are less pronounced; not shown). Both records correspond well with the NPI, with correlations of 0.67 and 0.65, respectively, based upon data smoothed with a three-point binomial ﬁlter (the correlations are a max- imum at 0 lag). The interdecadal regimes evident in the NPI are apparent in the tropical SST time series except for the early portion of the ﬁrst epoch in the southeast Paciﬁc record. 3117 D E S E R E T A L . 15 AUGUST 2004 FIG. 9. Selected regional climate anomaly records over the tropical Indo-Paciﬁc for boreal winter during 1900–97: the inverted NPI (through 2000), tropical Indian Ocean SST, southeast tropical Paciﬁc SST, rainfall south of the SPCZ, rainfall within the SPCZ, central Paciﬁc cloudiness, and tropical Indian Ocean minus southeast Paciﬁc SLP. b. Tropical linkages Regions are deﬁned in the text and shown in Figs. 11–13. All records are normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents one std dev. The numeral to the right of each tropical index represents its correlation coefﬁcient with the inverted NPI. ical convergence zone (ITCZ), and over the equatorial Indian Ocean, with maximum values (.12 mm day21) near 108S, 1708E. The long-term mean cloud amount distribution from COADS for the same period is shown in Fig. 10 (bottom). In general, the regions of high rain- fall correspond to areas of high total cloud cover (values .5 octas). In addition, large cloud amounts occur west of North and South America and west of Australia, areas dominated by shallow nonprecipitating clouds (Klein and Hartmann 1993), and poleward of 308N. Epoch difference maps for land station precipitation are shown in Fig. 11. Coherent rainfall changes are ev- ident in the Tropics where data exist. In the most recent epoch difference, negative values are found within and equatorward of the mean axis of the SPCZ and within the ITCZ over the central Paciﬁc, and positive values obtain on the poleward ﬂanks of the SPCZ and ITCZ (the latter extends westward to the southern tip of India). Over South and Central America, negative values are found between 08 and 208S, and positive values are found between 08 and 158N. Positive values are evident in the Hawaiian Islands. The middle epoch difference is similar to the latest one except for the region between southern India and the Philippines where values are near zero. Data coverage in the earliest epoch difference is severely limited, but positive values are still evident over the western Paciﬁc, in keeping with the two later maps. However, stations from the southern tip of India eastward to the Philippines exhibit strongly negative values in the earliest epoch difference, in contrast to the positive values found in the recent epoch difference. FIG. 9. Selected regional climate anomaly records over the tropical Indo-Paciﬁc for boreal winter during 1900–97: the inverted NPI (through 2000), tropical Indian Ocean SST, southeast tropical Paciﬁc SST, rainfall south of the SPCZ, rainfall within the SPCZ, central Paciﬁc cloudiness, and tropical Indian Ocean minus southeast Paciﬁc SLP. Regions are deﬁned in the text and shown in Figs. 11–13. All records are normalized and smoothed with a three-point binomial ﬁlter. 3) MARINE CLOUDINESS To extend the tropical rainfall analysis to oceanic ar- eas where direct measurements are lacking, we employ reports of total cloudiness from COADS as qualitative proxy records of precipitation. Figure 12 shows a side- by-side comparison of the epoch difference maps for land-based precipitation and ship-based cloudiness in the Tropics during December–March. In regions dom- inated by deep convective clouds such as the conver- gence zones, we expect a correspondence between pre- cipitation and cloud cover; in areas where nonprecipi- VOLUME 17 J O U R N A L O F C L I M A T E FIG. 10. (top) Climatological distribution of boreal winter precipitation (mm day 21) based upon satellite measurements during the period 1979–97. The contour interval is 2 mm day 21, with values exceeding 4 mm day21 shaded (see color bar at right). (bottom) Climatological distribution of total cloud amount (octas) over the oceans during boreal winter based upon COADS during 1979–97. The contour interval is 0.5 (0.25) octas for values less than (greater than) 5 octas, with values exceeding 5 octas shaded (see color bar at right). 3118 J O U R N A L O F C L I M A T E VOLUME 17 FIG. 10. (top) Climatological distribution of boreal winter precipitation (mm day 21) based upon satellite measurements during the period 1979–97. The contour interval is 2 mm day 21, with values exceeding 4 mm day21 shaded (see color bar at right). (bottom) Climatological distribution of total cloud amount (octas) over the oceans during boreal winter based upon COADS during 1979–97. The contour interval is 0.5 (0.25) octas for values less than (greater than) 5 octas, with values exceeding 5 octas shaded (see color bar at right). (r 5 0.57) and exhibits the regime transitions of 1925, 1947, and 1977. Only the ﬁrst few years of the record (1903–06; data are missing before 1903) are inconsistent with the NPI (but consistent with the southeastern trop- ical Paciﬁc SST index). tating (e.g., low stratiform) clouds prevail, such as off the west coasts of South and North America, no such correspondence is expected. The epoch difference maps of marine cloudiness generally support and extend the land-based rainfall results. 3) MARINE CLOUDINESS In particular, reduced cloud- iness over the central equatorial Paciﬁc and enhanced cloudiness over the western equatorial Paciﬁc and along the poleward ﬂank of the SPCZ in the two recent maps corroborate the station rainfall changes. The enhanced cloud cover over the western Paciﬁc extends into the eastern Indian Ocean. Increased cloudiness is also found over the eastern tropical Paciﬁc and west of Australia, regions dominated by low-stratiform cloudiness. Posi- tive cloudiness values in the vicinity of the Hawaiian Islands corroborate the land-based rainfall measure- ments. tating (e.g., low stratiform) clouds prevail, such as off the west coasts of South and North America, no such correspondence is expected. The epoch difference maps of marine cloudiness generally support and extend the land-based rainfall results. In particular, reduced cloud- iness over the central equatorial Paciﬁc and enhanced cloudiness over the western equatorial Paciﬁc and along the poleward ﬂank of the SPCZ in the two recent maps corroborate the station rainfall changes. The enhanced cloud cover over the western Paciﬁc extends into the eastern Indian Ocean. Increased cloudiness is also found over the eastern tropical Paciﬁc and west of Australia, regions dominated by low-stratiform cloudiness. Posi- tive cloudiness values in the vicinity of the Hawaiian Islands corroborate the land-based rainfall measure- ments. 5) AN OPTIMAL TROPICAL INDEX 97), including a change in the sign of the SST anomalies in the equatorial Paciﬁc cold tongue, a westward shift (;508 of longitude) of the nodal line between positive and negative SLP anomalies such that Darwin, Australia is no longer located within the western pole of the Southern Oscillation, and a change in the sign of the rainfall/cloudiness anomalies over Borneo, the Philip- pines, and the South China Sea. These differences high- light that with only three realizations of interdecadal variability to study, it is problematic to characterize any single case as ‘‘canonical.’’ These differences also un- derscore that conventional ENSO indices such as the Darwin SOI and CTI fail as robust metrics of the in- terdecadal variability. Speciﬁcally, neither Darwin SLP nor the CTI exhibit regime transitions in 1925 and 1947 that characterize the NPI, although they do portray the 1977 shift (Fig. 16; note that the Darwin SOI and CTI records in this ﬁgure differ from the versions of the SOI 97), including a change in the sign of the SST anomalies in the equatorial Paciﬁc cold tongue, a westward shift (;508 of longitude) of the nodal line between positive and negative SLP anomalies such that Darwin, Australia is no longer located within the western pole of the Southern Oscillation, and a change in the sign of the rainfall/cloudiness anomalies over Borneo, the Philip- pines, and the South China Sea. These differences high- light that with only three realizations of interdecadal variability to study, it is problematic to characterize any single case as ‘‘canonical.’’ These differences also un- derscore that conventional ENSO indices such as the Darwin SOI and CTI fail as robust metrics of the in- terdecadal variability. Speciﬁcally, neither Darwin SLP nor the CTI exhibit regime transitions in 1925 and 1947 that characterize the NPI, although they do portray the 1977 shift (Fig. 16; note that the Darwin SOI and CTI records in this ﬁgure differ from the versions of the SOI 4) SLP Note that this index differs from the canonical SOI, conventionally deﬁned as the SLP difference be- tween Darwin (128S, 1318E) and Tahiti (178S, 1498W) or regional averages centered around these stations (cf. Deser and Wallace 1990). The new SLP index, shown as the last record in Fig. 9, correlates well with the NPI (r 5 0.52) and exhibits ‘‘regime transitions’’ in 1925 and 1977 (another shift occurs in 1945, two years before that in the NPI). However, the SLP record is positive during 1900–06, in disagreement with the NPI and the Indian Ocean SST index but consistent with the central Paciﬁc cloud and southeastern tropical Paciﬁc SST time series. patterns, we formed an index of the difference in SLP between the southeastern tropical Paciﬁc (78–298S, 1678–898W) and south Indian (78–298S, 578–1298E) Oceans. Note that this index differs from the canonical SOI, conventionally deﬁned as the SLP difference be- tween Darwin (128S, 1318E) and Tahiti (178S, 1498W) or regional averages centered around these stations (cf. Deser and Wallace 1990). The new SLP index, shown as the last record in Fig. 9, correlates well with the NPI (r 5 0.52) and exhibits ‘‘regime transitions’’ in 1925 and 1977 (another shift occurs in 1945, two years before that in the NPI). However, the SLP record is positive during 1900–06, in disagreement with the NPI and the Indian Ocean SST index but consistent with the central Paciﬁc cloud and southeastern tropical Paciﬁc SST time series. 4) SLP Epoch difference maps for SLP are shown in Fig. 13. The tropical mean anomaly (258N–258S) has been re- moved from each grid box to enhance the zonal asym- metries. The most recent epoch difference exhibits be- low-normal values over the Indian and western Paciﬁc Oceans, and above-normal values over the eastern two- thirds of the Paciﬁc, reminiscent of the spatial pattern associated with the Southern Oscillation (cf. Trenberth and Caron 2000). The middle epoch difference is gen- erally similar to the later one, except over the northern Indian Ocean and South China Sea, where positive val- ues are found in place of negative ones. Although noisier due to reduced data coverage, the early epoch difference resembles the middle one with the notable exception of the area around Australia (;108–208S, 1158–1708E), where positive values are found in place of negative ones. Thus, the eastern pole of the Southern Oscillation appears to extend considerably farther west in the early epoch difference compared to the later ones, consistent with the Darwin (128S, 1318E) SLP record discussed above. The earliest epoch difference is noisier than the other two due to reduced data coverage. Nonetheless, many of the same patterns are discernible, including the op- positely signed anomalies on either side of the equator in the central Paciﬁc, some evidence of enhanced cloud cover over the eastern tropical Paciﬁc, and increased cloudiness over the equatorial Indian Ocean. Dimin- ished cloudiness is found over Indonesia and the South China Sea, roughly consistent with the land-based rain- fall results. The time series of the difference between cloudiness anomalies north and south of the equator in the central Paciﬁc (regions are outlined in Fig. 12) is shown in Fig. 9. This cloudiness index is well correlated with the NPI Taking into account the nonstationarity of the SLP 3119 D E S E R E T A L . 15 AUGUST 2004 FIG. 11. Epoch difference maps of boreal winter precipitation over land. The precipitation scale (color bar at bottom of ﬁgure) is in units of mm day21. The black contours denote the 7.5 and 12 mm day21 contours from the climatology. Rectangles outline regions used for the time series shown in Fig. 9. patterns, we formed an index of the difference in SLP between the southeastern tropical Paciﬁc (78–298S, 1678–898W) and south Indian (78–298S, 578–1298E) Oceans. 5) AN OPTIMAL TROPICAL INDEX Figure 9 provides collective evidence for a robust tropical linkage to North Paciﬁc interdecadal climate variations during boreal winter based upon a diverse set of independent parameters spanning a broad region of the Indo-Paciﬁc. To summarize the suite of tropical cli- mate records shown in Fig. 9, we computed the leading EOF of the six normalized time series (note that the NPI is not included in the EOF analysis). The leading principal component (PC) time series, which accounts for 64% of the variance compared to 16% for the second PC, is shown in Fig. 14 together with the NPI (the six input time series contribute nearly equally to the leading EOF). All three regime transitions evident in the NPI (1925, 1947, and 1977) are present in the leading PC of the tropical records. The correlation coefﬁcient be- tween the two time series is 0.72, based upon data smoothed with a three-point binomial ﬁlter; that is, half of the variance of the NPI at periods longer than ;3 yr is coherent with the leading tropical PC time series. FIG. 11. Epoch difference maps of boreal winter precipitation over land. The precipitation scale (color bar at bottom of ﬁgure) is in units of mm day21. The black contours denote the 7.5 and 12 mm day21 contours from the climatology. Rectangles outline regions used for the time series shown in Fig. 9. Power spectra of the detrended NPI and the leading tropical PC time series are shown in Fig. 15. Both spec- tra exhibit enhanced variance at the lowest frequencies (periods . ;20 yr) and a deﬁcit of power around 8– 14 yr. The tropical PC power spectrum also exhibits an enhancement of power at interannual frequencies (;4– 7 yr). The increased power at interdecadal periods in both records is signiﬁcant at the 95% conﬁdence level based upon a ﬁrst-order autoregressive model null hy- pothesis. The cross-spectrum between the two records (Fig. 15, bottom) exhibits high squared coherence (;0.8) at periods .20 yr and between 4 and 7 yr [values exceeding 0.6 (0.75) are statistically signiﬁcant at the 95% (99%) conﬁdence level]. 4. Discussion The earliest epoch difference (1900–24 minus 1925– 46) exhibits some notable distinctions from the later two (1947–76 minus 1925–46 and 1947–76 minus 1977– 3120 J O U R N A L O F C L I M A T E VOLUME 17 FIG. 12. As in Fig. 11 but for (left) land station precipitation and (right) marine cloudiness. The cloudiness (precipitation) color bar is in units of octas (mm day 21). Rectangles outline regions used for the time series shown in Fig. 9. FIG. 12. As in Fig. 11 but for (left) land station precipitation and (right) marine cloudiness. The cloudiness (precipitation) color bar is in units of octas (mm day 21). Rectangles outline regions used for the time series shown in Fig. 9. and CTI shown in Fig. 2 from M97). With regard to the change in sign of the rainfall anomalies over the region encompassing Borneo, the Philippines, and the South China Sea between the early and recent epoch differences, it is interesting to note that this change co- incides with an alteration of the SLP anomaly signal over the eastern North Paciﬁc (recall Fig. 3); whether the two are causally related remains to be elucidated. FIG. 13. As in Fig. 11 but for boreal winter SLP. The SLP color bar is in units of hPa. Rectangles outline regions used for the time series shown in Fig. 9. FIG. 14. (top) The inverted NPI and (bottom) the optimal tropical index (see text for deﬁnition) during 1900–97. Both records have been normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents 1 std dev. The numeral to the right of the tropical index represents its correlation coefﬁcient with the inverted NPI. and CTI shown in Fig. 2 from M97). With regard to the change in sign of the rainfall anomalies over the region encompassing Borneo, the Philippines, and the South China Sea between the early and recent epoch differences, it is interesting to note that this change co- incides with an alteration of the SLP anomaly signal over the eastern North Paciﬁc (recall Fig. 3); whether the two are causally related remains to be elucidated. Given the evidence for a tropical Indo-Paciﬁc linkage to interdecadal climate variations over the North Paciﬁc, it is natural to inquire whether there are any distinctions between these connections and those that occur in as- FIG. 13. 4. Discussion the anomaly ﬁelds upon the CTI during 1947–97, and the interdecadal patterns are those associated with the 1977 transition. The two SST anomaly patterns (Fig. 17, top) are broadly similar, with positive values throughout much of the tropical Indian and Paciﬁc Oceans and a dipole pattern in the North Paciﬁc with positive values in the east and negative values in the center. Some differences are also apparent, however; compared to ENSO, the epoch difference is character- ized by a more equal weighting of the tropical Indian and Paciﬁc anomalies, a broader meridional extent of the eastern equatorial Paciﬁc anomalies, and a more equal weighting between the North Paciﬁc and the Trop- ics. These results are in agreement with the ﬁndings of Garreaud and Battisti (1999) based upon annual aver- ages. However, note that in the earliest epoch difference, the SST anomalies along the equatorial upwelling zone are opposite in sign to those associated with ENSO (re- call Fig. 8). The interdecadal and ENSO anomaly patterns of total cloud amount (Fig. 17, bottom) are also similar, with enhanced cloud cover over the western and central equa- torial Paciﬁc and diminished cloudiness over the far western Paciﬁc and the far eastern Indian Ocean. Re- duced cloud cover is also found in the stratus regions of the eastern tropical Paciﬁc in association with a warmer sea surface. We note that the stratus cloud signal is relatively more pronounced in the epoch difference than in the ENSO regression map; the reasons for this require further study. The ENSO cloudiness regression pattern is consistent with the results of Park and Leovy (2004). FIG. 15. Power spectra of (top) the NPI and (middle) the optimal tropical index based upon detrended data for the period 1900–97 (thick curves). The thin curves represent the power spectrum and its 95% conﬁdence limits for a ‘‘red noise’’ null hypothesis based upon a ﬁrst-order autoregressive process with the same autocorrelation as the observed time series (0.21 for the NPI and 0.32 for the tropical index). (bottom) Squared coherence between the NPI and the tropical index (thick curve). The 95% conﬁdence level is 0.6 (thin horizontal line). The short duration of the instrumental records limits our ability to assess whether the interdecadal variability sampled during the twentieth century is indicative of a preferred time scale or is part of a continuum of low- frequency variability. 4. Discussion As in Fig. 11 but for boreal winter SLP. The SLP color bar is in units of hPa. Rectangles outline regions used for the time series shown in Fig. 9. Given the evidence for a tropical Indo-Paciﬁc linkage to interdecadal climate variations over the North Paciﬁc, it is natural to inquire whether there are any distinctions between these connections and those that occur in as- FIG. 14. (top) The inverted NPI and (bottom) the optimal tropical index (see text for deﬁnition) during 1900–97. Both records have been normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents 1 std dev. The numeral to the right of the tropical index represents its correlation coefﬁcient with the inverted NPI. FIG. 14. (top) The inverted NPI and (bottom) the optimal tropical index (see text for deﬁnition) during 1900–97. Both records have been normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents 1 std dev. The numeral to the right of the tropical index represents its correlation coefﬁcient with the inverted NPI. FIG. 13. As in Fig. 11 but for boreal winter SLP. The SLP color bar is in units of hPa. Rectangles outline regions used for the time series shown in Fig. 9. 3121 E T A L . FIG. 16. (top) The inverted NPI, (middle) Darwin SLP, and (bottom) eastern equatorial Paciﬁc SST (the CTI) records during 1900–2002 (1900–97 for the CTI). All records are for boreal winter and have been normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents one std dev. 3121 T A L . D E S E R E T A L . 3121 15 AUGUST 2004 15 AUGUST 2004 D E FIG. 15. Power spectra of (top) the NPI and (middle) the op tropical index based upon detrended data for the period 190 (thick curves). The thin curves represent the power spectrum a 95% conﬁdence limits for a ‘‘red noise’’ null hypothesis based FIG. 16. (top) The inverted NPI, (middle) Darwin SLP, and (bottom) eastern equatorial Paciﬁc SST (the CTI) records during 1900–2002 (1900–97 for the CTI). All records are for boreal winter and have been normalized and smoothed with a three-point binomial ﬁlter. Each tick mark on the ordinate represents one std dev. 4. Discussion Paleoclimate proxies based upon climatically sensitive biological indicators are better sociation with the interannual ENSO cycle. Figure 17 shows a comparison of the ENSO and interdecadal pat- terns of SST and cloudiness anomalies during boreal winter. The ENSO patterns were obtained by regressing J O U R N A L O F C L I M A T E 3122 VOLUME 17 VOLUME 17 FIG. 17. Comparison between (left) ENSO and (right) epoch difference patterns of (top) SST and (bottom) cloudiness anomalies during boreal winter. The ENSO patterns are based upon linear regression of the anomaly ﬁelds upon the CTI during 1947–97, and the epoch difference maps are obtained by subtracting the period 1947–76 from the period 1977–97. The color bar scale for the SST (cloudiness) anomalies is given on the rhs of the ﬁgure in units of 8C (octas). Note that the ENSO-related anomalies are per unit std dev of the CTI. FIG. 17. Comparison between (left) ENSO and (right) epoch difference patterns of (top) SST and (bottom) cloudiness anomalies during boreal winter. The ENSO patterns are based upon linear regression of the anomaly ﬁelds upon the CTI during 1947–97, and the epoch difference maps are obtained by subtracting the period 1947–76 from the period 1977–97. The color bar scale for the SST (cloudiness) anomalies is given on the rhs of the ﬁgure in units of 8C (octas). Note that the ENSO-related anomalies are per unit std dev of the CTI. interdecadal variability. One such location suggested by our study is the tropical Indian and western Paciﬁc Oceans for both SST and precipitation-sensitive proxies. suited for addressing the issue of time scale due to their longer record lengths. Recently, Cole et al. (2000) pre- sented a 194-yr record (1804–1994) of skeletal d18O from a coral growing in the far western tropical Indian Ocean at Malindi Marine Park, Kenya (38S, 408E). Ac- cording to Cole et al. (2000), this record primarily re- ﬂects ﬂuctuations in SST as opposed to salinity. The detrended d18O time series is shown together with the leading tropical PC index in Fig. 18 (top). Interdecadal variations in d18O are present during both centuries, and those during the twentieth century are coherent with the instrumental record (the correlation coefﬁcient between the d18O and PC time series is 0.62 based upon data smoothed with a three-point binomial ﬁlter). The power spectrum of the d18O record (Fig. 4. Discussion 18, bottom) exhibits a large proportion of variance at periods longer than ;30 yr, with relatively little variance at higher fre- quencies. The predominantly low-frequency character of the d18O time series is reﬂected in the high year-to- year autocorrelation (0.64), a value considerably larger than that for nearby SST anomalies or the tropical PC record (;0.3). The higher persistence in the d18O record may be partly biological in nature and/or due to the way in which it was decimated to annual values (see Cole et al. 2000). Despite its duration, even the 194-yr d18O record is not sufﬁciently long to assess whether the en- hanced variance at low frequencies is indicative of a well-deﬁned (albeit broadband) spectral peak at inter- decadal periods or whether it reﬂects a red noise process with no preferred time scale. REFERENCES does not capture the full amplitude of the observed in- terdecadal variability evident in our ‘‘optimal tropical index.’’ Note that our interpretation does not preclude a feedback role for atmospherically induced SST anom- alies within the North Paciﬁc that may serve to amplify and/or ‘‘redden’’ the SLP response to tropical forcing, for example, through the large-scale dynamical atmo- spheric circulation response to North Paciﬁc SST anom- alies (Peng et al. 1997) or the mechanism of ‘‘reduced thermal damping’’ (Bretherton and Battisti 2000; Al- exander et al. 2002) associated with the thermal inertia of the deep winter ocean mixed layer (Alexander and Deser 1995; Deser et al. 2003). Nor does it preclude the existence of feedbacks from the North Paciﬁc to the Tropics via an atmospheric bridge (e.g., Barnett et al. 1999; Vimont et al. 2001) and/or an oceanic one (e.g., does not capture the full amplitude of the observed in- terdecadal variability evident in our ‘‘optimal tropical index.’’ Note that our interpretation does not preclude a feedback role for atmospherically induced SST anom- alies within the North Paciﬁc that may serve to amplify and/or ‘‘redden’’ the SLP response to tropical forcing, for example, through the large-scale dynamical atmo- spheric circulation response to North Paciﬁc SST anom- alies (Peng et al. 1997) or the mechanism of ‘‘reduced thermal damping’’ (Bretherton and Battisti 2000; Al- exander et al. 2002) associated with the thermal inertia of the deep winter ocean mixed layer (Alexander and Deser 1995; Deser et al. 2003). Nor does it preclude the existence of feedbacks from the North Paciﬁc to the Tropics via an atmospheric bridge (e.g., Barnett et al. 1999; Vimont et al. 2001) and/or an oceanic one (e.g., ——, M. A. Alexander, and M. S. Timlin, 1996: Upper-ocean thermal variations in the North Paciﬁc during 1970–1991. J. Climate, 9, 1840–1855. ——, ——, and ——, 2003: Understanding the persistence of sea surface temperature anomalies in midlatitudes. J. Climate, 16, 57–72. Dettinger, M. D., D. R. Cayan, G. M. McCabe, and J. A. Marengo, 2000: Multiscale streamﬂow variability associated with El Nin˜o/ Southern Oscillation. El Nin˜o and the Southern Oscillation— Multiscale Variability and Global and Regional Impacts, H. F. Diaz and V. Markgraf, Eds., Cambridge University Press, 113– 146. Folland, C. K., and D. E. Parker, 1995: Correction of instrumental biases in historical sea surface temperature data. Quart. J. Roy. Meteor. Soc., 121, 319–367. REFERENCES Alexander, M. A., and C. 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(top) Time series of the detrended inverted d18O coral record from the western Indian Ocean (thin black curve; Cole et al. 2000) and the instrumental tropical index (thick gray curve). Both time series have been normalized by their std dev, and the coral record (tropical index) was smoothed with a three-point (ﬁve-point) binomial ﬁlter to facilitate visual comparison. (bottom) Power spectrum of the d18O coral record based upon detrended data for the period 1801– 1994. The thin curves represent the power spectrum and its 95% conﬁdence limits for a red noise null hypothesis based upon a ﬁrst- order autoregressive process with the same autocorrelation as the observed time series (0.64). Chao, Y., M. Ghil, and J. C. McWilliams, 2000: Paciﬁc interdecadal variability in this century’s sea surface temperatures. Geophys. Res. Lett., 27, 2261–2264. Cole, J. E., R. B. Dunbar, T. R. McClanahan, and N. A. Muthiga, 2000: Tropical Paciﬁc forcing of decadal SST variability in the western Indian Ocean over the past two centuries. Science, 287, 617–619. Deser, C., and J. M. Wallace, 1990: Large-scale atmospheric circu- lation features of warm and cold episodes in the tropical Paciﬁc. J. Climate, 3, 1254–1281. ——, and M. S. Timlin, 1997: Atmosphere–ocean interaction on weekly timescales in the North Atlantic and Paciﬁc. J. Climate, 10, 393–408. 5. Summary and interpretation B time series have been normalized by their std dev, and the coral rec (tropical index) was smoothed with a three-point (ﬁve-point) binom ﬁlter to facilitate visual comparison. (bottom) Power spectrum of d18O l d b d d t d d d t f th i d 18 3123 15 AUGUST 2004 D D E S E R E T A L . 15 AUGUST 2004 Klecman et al. 1999; Zhang and McPhaden 2002). In- deed, these feedbacks may be essential for explaining the origin of the interdecadal climate variations within the tropical Indo-Paciﬁc, a question not addressed in the present study and one that remains outstanding. Acknowledgments. We wish to acknowledge helpful discussions with many colleagues, including Drs. M. Alexander, D. Battisti. M. Newman, K. Trenberth, and J. M. Wallace. The comments of the editor and two anonymous reviewers helped us to improve the presen- tation of the results. This work was supported by a grant from NOAA’s Ofﬁce of Global Programs to C. Deser and J. Hurrell. 5. Summary and interpretation The analyses presented in this study support the no- tion that interdecadal ﬂuctuations in the winter NPI dur- ing the twentieth century are associated with climate variations over the tropical Indo-Paciﬁc, in agreement with earlier studies (e.g., M97; Minobe 1997; Zhang et al. 1997; Garreaud and Battisti 1999). Speciﬁcally, SSTs in the tropical Indian and southeastern Paciﬁc Oceans, rainfall and cloudiness along the SPCZ and over the central equatorial Paciﬁc, cloudiness over the eastern tropical Paciﬁc, and the SLP difference between the tropical southeast Paciﬁc and Indian Oceans collectively exhibit variability that is coherent with the NPI on in- terannual and interdecadal time scales. This study has extended our knowledge of Paciﬁc interdecadal climate variability by considering a more comprehensive suite of climatic parameters in the tropical Indo-Paciﬁc than in previous studies and by focusing upon the relation- ships during boreal winter when the NPI is most active and the inﬂuence of the tropical Indo-Paciﬁc upon the atmospheric circulation over the North Paciﬁc is well established. In addition, our case study approach has allowed for an assessment of the similarities and dif- ferences among the three cases of interdecadal vari- ability sampled in the twentieth-century observational record. Although the Indian Ocean coral record shows a fa- vorable match to the instrumental time series and pro- vides evidence that the interdecadal ﬂuctuations contin- ue back at least 200 yr, other proxy records such as tree ring chronologies from North and South America are not as consistent in their behavior during the nineteenth century (Villalba et al. 1999). The lack of agreement among the proxy records suggests the need for addi- tional paleoclimate indicators at key locations of Paciﬁc Our interpretation of the results is that variations in the winter NPI at periods ranging from interannual to interdecadal represent, in part, a remote response to rain- fall anomalies over the tropical western and central Pa- ciﬁc via dynamical atmospheric teleconnection pro- cesses. 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https://openalex.org/W3198440950	https://www.frontiersin.org/articles/10.3389/fendo.2021.720769/pdf	English	null	Latent Adrenal Insufficiency: From Concept to Diagnosis	Frontiers in endocrinology	2,021	cc-by	10,006	Correspondence: Andre Lacroix Specialty section: This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Received: 04 June 2021 Accepted: 09 August 2021 Published: 27 August 2021 Specialty section: This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Latent Adrenal Insufﬁciency: From Concept to Diagnosis Nada Younes, Isabelle Bourdeau and Andre Lacroix Division of Endocrinology, Department of Medicine and Research Center, Centre Hospitalier de l’Universite´ de Montre´ al (CHUM), Montre´ al, QC, Canada Primary adrenal insufﬁciency (PAI) is a rare disease and potentially fatal if unrecognized. It is characterized by destruction of the adrenal cortex, most frequently of autoimmune origin, resulting in glucocorticoid, mineralocorticoid, and adrenal androgen deﬁciencies. Initial signs and symptoms can be nonspeciﬁc, contributing to late diagnosis. Loss of zona glomerulosa function may precede zona fasciculata and reticularis deﬁciencies. Patients present with hallmark manifestations including fatigue, weight loss, abdominal pain, melanoderma, hypotension, salt craving, hyponatremia, hyperkalemia, or acute adrenal crisis. Diagnosis is established by unequivocally low morning serum cortisol/aldosterone and elevated ACTH and renin concentrations. A standard dose (250 µg) Cosyntropin stimulation test may be needed to conﬁrm adrenal insufﬁciency (AI) in partial deﬁciencies. Glucocorticoid and mineralocorticoid substitution is the hallmark of treatment, alongside patient education regarding dose adjustments in periods of stress and prevention of acute adrenal crisis. Recent studies identiﬁed partial residual adrenocortical function in patients with AI and rare cases have recuperated normal hormonal function. Modulating therapies using rituximab or ACTH injections are in early stages of investigation hoping it could maintain glucocorticoid residual function and delay complete destruction of adrenal cortex. Keywords: cortisol, aldosterone, ACTH, renin, autoimmunity, hypocortisolism, adrenal insufﬁciency DEFINITION AND EPIDEMIOLOGY The adrenal cortex produces glucocorticoids, mineralocorticoids and androgens, under the inﬂuence of adrenocorticotropic hormone (ACTH) and the renin-angiotensin system (1, 2). In the event of adrenal cortex destruction, primary adrenal insufﬁciency (PAI) develops and is characterized by reduced serum concentrations of all three hormones: cortisol, aldosterone and adrenal androgens (3). However, since intra-adrenal cortisol is required for epinephrine production by the adrenal medulla, PAI is often associated with decreased phenylethanolamine N-methyltransferase (PNMT) activity, resulting in adrenomedullary dysfunction (1, 4). Central adrenal insufﬁciency (AI), encompasses both secondary and tertiary AI caused by low ACTH and low corticotropin releasing hormone (CRH), respectively (5). However, since aldosterone production is mainly controlled by renin, angiotensin II and potassium (1, 6), it is not affected in central AI. Received: 04 June 2021 Accepted: 09 August 2021 Published: 27 August 2021 REVIEW published: 27 August 2021 doi: 10.3389/fendo.2021.720769 Edited by: Edited by: Maria Fragoso, Institute of Cancer of Sao Paulo, Brazil Edited by: Maria Fragoso, Institute of Cancer of Sao Paulo, Brazil Reviewed by: Antonio Lerario, University of Michigan, United States Erick J.R. Silva, Reviewed by: Antonio Lerario, University of Michigan, United States Erick J.R. Silva, São Paulo State University, Brazil *Correspondence: Andre Lacroix andre.lacroix@umontreal.ca Citation: Younes N, Bourdeau I and Lacroix A (2021) Latent Adrenal Insufﬁciency: From Concept to Diagnosis. Front. Endocrinol. 12:720769. doi: 10.3389/fendo.2021.720769 August 2021 \| Volume 12 \| Article 720769 1 Frontiers in Endocrinology \| www.frontiersin.org Latent Adrenal Insufﬁciency Younes et al. PAI was ﬁrst described by Thomas Addison in 1855 in a case series of 11 patients and therefore it is often called Addison’s disease (7). Since then, prevalence has been on the rise especially in Europe, reaching 117/million in central Italy in the late 1990s (8) and 144/million in Norway in 2007 (9). More recently, an even higher prevalence was documented in a 2016 Icelandic nationwide study of patients over 18 years of age: 221/million population (10). Furthermore, an annual average increase in the prevalence of PAI of 1.8% per year was reported in Germany from 2008 to 2012 (11). The annual incidence of PAI is estimated to be around 0.44-0.62 per 100,000 (9, 12). PAI is more frequently found in women than men (11, 13) with a M:F ratio of 1: 3.5 (14). Age of onset is typically around 30-50 years old (1, 9, 10, 14). Autoimmune destruction of adrenal cortex has surpassed tuberculosis as the most common cause of PAI, in particular in high income countries (1, 15). Comorbid autoimmune disorders can often be found in patients with autoimmune AI (AAI), reported to be 46.5 and 66%, respectively in the German (11) and Norwegian studies (9). Most common comorbid conditions were thyroid disease, type 1 diabetes, vitiligo, vitamin B12 deﬁciency and primary ovarian insufﬁciency (POI) (9–11). When occurring with other autoimmune disorders, AAI may be part of an autoimmune polyglandular syndrome (APS). APS type 1 or autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is a rare monogenic disease, in which the autoimmune regulator gene (AIRE) is mutated and at least 2 of the following three occur: chronic mucocutaneous candidiasis, hypoparathyroidism and PAI (16). Generally, its transmission is autosomal recessive but may be dominant (16). Other conditions may include POI, enteropathy and rarely lymphomas (6, 16). More commonly found is APS type 2, a polygenic condition in which genes encoding cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), protein tyrosine phosphatase, nonreceptor type 22 (PTPN22), the transcriptional regulator protein BACH2, and the CD25–interleukin-2 receptor have been implicated (16), along with associations to certain haplotypes including DR3- DQ2, DR4-DQ8, DRB1-0301, and DRB1-0404 (15, 17). It commonly associates with type 1 diabetes and autoimmune thyroid disease (16). Citation: When AAI is conﬁrmed alongside other autoimmune diseases, excluding the cardinal ones needed for diagnosis of APS types 1 and 2, APS type 4 is diagnosed (5). cortisol deﬁciency are common to both primary and central AI. These include loss of appetite, weight loss, lethargy, gastrointestinal symptoms such as nausea, abdominal pain and vomiting (1, 6). Because aldosterone deﬁciency exists in PAI but not in central AI, symptoms and signs of mineralocorticoid deﬁciency including, dizziness and orthostatic hypotension, salt craving, hyponatremia, hyperkalemia and hyperchloremic acidosis, can be found (1, 19). Orthostatic hypotension may also be secondary to cortisol deﬁciency via reduced expression of catecholamine receptors on blood vessels and therefore may be found in central AI, albeit less pronounced than in PAI (1, 20). Although hyponatremia can be present in both primary and central AI, the underlying mechanisms differ. In PAI, hyponatremia is secondary to aldosterone deﬁciency resulting in renal salt wasting and hypovolemia with an increased risk of dehydration and acute kidney injury (19, 21). In central AI, it is a result of increased vasopressin secondary to cortisol deﬁciency, which in turn results in water retention and euvolemic hyponatremia (22). Other possible laboratory abnormalities are hypercalcemia, mild normocytic anemia, lymphocytosis, eosinophilia and hypoglycemia which occurred more frequently in children (1, 6). There is also a signiﬁcant decrease in natural killer cells cytotoxicity in patients with PAI, compromising innate immunity, hence contributing to increased viral infections (23). Androgen deﬁciency may result in low libido or reduced energy as well as thinning of axillary and pubic hair in post-menopausal women (1, 3). In pregnant women with unrecognized AI, establishing the diagnosis based on signs and symptoms is challenging, because cortisol deﬁciency symptoms may also be seen as part of a normal pregnancy. While only a few cases were diagnosed during pregnancy, the high maternal and fetal risks associated with unrecognized AI should prompt physicians to suspect the diagnosis in women with symptoms persisting into the second trimester or occurring secondary to illness or labor (24). A particularly distinctive feature of PAI is melanoderma which can be explained by elevated ACTH due to loss of negative feedback control usually exerted by cortisol on corticotropic cells in anterior pituitary gland (1). Citation: Elevated plasma ACTH activates melanocortin 1 receptors (M1R), resulting in excessive pigmentation, especially on areas exposed to sun and friction such as face, neck, knuckles, creases in the hand, elbows, areola of the nipple, scrotum, labia and newly acquired scars (6, 19). Mucosal pigmentation can also be noted and clinicians should look for brown patchy discoloration on lips, palate and gingiva (19). SIGNS AND SYMPTOMS OF AI- NATURAL HISTORY OF AUTOIMMUNE PAI-MORTALITY Although hyponatremia and hyperkalemia are very common features in adrenal crisis, clinicians should beware of patients with multiple episodes of vomiting and severe dehydration because they can present with normal levels of serum potassium and sodium (6). Adrenal crisis can be triggered by numerous precipitating events, namely gastrointestinal infections, fever, emotional stress, major surgery, pregnancy, strenuous physical activity and noncompliance (34–37, 39). In 7-14% of adrenal crises, no precipitating event could be identiﬁed (34, 36). Patients at higher risk of adrenal crisis were especially those who have a history of a previous adrenal crisis (threefold increase) (36), but also patients that are female (35), older (40) and have diabetes insipidus (35), type 1 diabetes mellitus (41) and cardiac, neurological or pulmonary comorbidities (35, 39). Conversely, the latter comorbidities were not shown to be associated with a higher risk of adrenal crises according to the new study by Quinkler et al. (38). However, they did have more infections associated with adrenal crisis occurrence, conﬁrming and elevated renin concentrations suggesting that zona glomerulosa may be more prone to autoimmune destruction than zona fasciculata (27–29). The latter may be initially spared owing to the high local concentrations of cortisol responsible for suppressing antigen presentation to immune cells and procuring local anti-inﬂammatory properties (30). The second stage is characterized by an impaired cortisol response to synthetic ACTH administered intravenously, while in third and fourth stages, ACTH increases and serum cortisol drops, respectively (27). Early stages of AAI are difﬁcult to diagnose because of the asymptomatic or pauci-symptomatic state since adrenal function is maintained by high plasma ACTH/renin (31). Stages 1 and 2 may be reversible in sporadic AAI. This was not true in APS types 1 and 2/4, where respectively stage 1 and stage 2 were deﬁned as the point of no return with a cumulative risk of 100% (28). Progression of AAI occurred in most patients within 11 years, while no progression was seen after 19 years offollow up in a large cohort of 143 antibody positive patients. Progression was higher in males, patients who had APS type 1 and impaired adrenal function (28). However, antibody titers did not affect risk of progression, probably because AAI is mediated by cellular immunity and antibodies are in fact markers of the disease (27, 28). SIGNS AND SYMPTOMS OF AI- NATURAL HISTORY OF AUTOIMMUNE PAI-MORTALITY PAI may be underrecognized because of the nonspeciﬁc symptoms of cortisol deﬁciency, especially in its early stages. This is often called latent AI and should be suspected in the presence of unexplained health complaints related to stress, such as gastrointestinal symptoms, fatigue and weight loss, in particular in patients with a history of autoimmune disease (25, 26). Betterle et al. suggested that autoimmune adrenocortical destruction goes through 4 stages (27) (Figure 1). The very ﬁrst stage of the disease is marked by aldosterone deﬁciency Onset of AI can be insidious, with many of the symptoms and signs being nonspeciﬁc and often leading to a delay in diagnosis (1). In a retrospective study including 216 patients with AI (18), more than half of patients consulted at least 3 physicians and were falsely diagnosed with either gastrointestinal or psychiatric disorders most frequently. A 5 year delay in diagnosis was reported in as much as 20% of patients (18). Symptoms of August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 2 Latent Adrenal Insufﬁciency Younes et al. FIGURE 1 \| Simpliﬁed schematic depiction of the 4 stages of autoimmune adrenocortical destruction. GURE 1 \| Simpliﬁed schematic depiction of the 4 stages of autoimmune adrenocortical destruction. on the deﬁnition used for adrenal crises and the characteristics of patients included in each of the studies. A prospective study by Hahner et al., evaluated the incidence of adrenal crisis in 423 patients followed up for 2 years. It was found to be persistently high even in adequately educated patients, reaching 8.3 crises/ 100 patient-years with a high mortality, estimated at 0.5 adrenal crisis related deaths/100 patient-years (36). A much lower incidence was reported in Switzerland despite insufﬁcient patient education regarding their disease (37). Recently, an analysis of the biggest cohort described to date, including 2694 patients from the European Adrenal Insufﬁciency Registry, revealed an adrenal crisis incidence of 6.53/100 patient-years, with more than one episode per year in 16% of patients with PAI (38). In the absence of a universal deﬁnition of adrenal crisis, its recognition can prove to be difﬁcult. The most used deﬁnition is an unstable hemodynamic state (absolute or relative hypovolemia/shock) recovering within 1 to 2 hours following parenteral glucocorticoid administration (4, 33). Frontiers in Endocrinology \| www.frontiersin.org PATHOGENESIS OF AUTOIMMUNE AI AAI is an autoimmune destructive process affecting all three zones of the adrenal cortex in which lymphocytes inﬁltrate the adrenal parenchyma leading to adrenal ﬁbrosis and atrophy. It is a slow process and AI may not be clinically relevant until most of adrenocortical cells are destroyed (48). Antibodies directed against 21-hydroxylase are speciﬁc to AAI and are rarely seen in the general population; hence their presence alongside positive adrenal cortex antibodies allow to accurately diagnose AAI in 99% of cases (19, 49, 50). The presence of antibodies targeting interferon-a2 and interferon-w should prompt genetic testing for APS type 1 (19, 49). While the detection of anti 21- hydroxylase antibodies is diagnostic of AAI, they have no direct role in pathogenesis and are only biologic markers of autoimmunity (48, 49). In fact, cytotoxic T-cells auto-reactive to steroidogenic enzymes, in particular 21-hydroxylase, inﬁltrate the adrenal cortex in response to proinﬂammatory chemokines, CXCL9 and CXCL10, released intrinsically by adrenocortical cells. Hence, adrenocortical cells contribute to their own destruction in the presence of both, a genetic predisposition and environmental triggers (48). Possible triggers are thought to be caused by local viral infections with increased tropism to adrenocortical cells, such as herpes simplex virus 1, cytomegalovirus and adenovirus, as well as interferon alfa treatments and the relatively new checkpoint inhibitors targeting CTLA-4, programmed cell death protein 1 (PD-1), and PD-1 ligand (PD-L1), used in melanoma and lung cancer treatment (48). Genetic variants implicated in AAI development include the autosomal recessive mutation in AIRE gene commonly responsible for APS type 1 (50) but was also implicated in AAI independently of APS type 1 in a recent genome wide association study (51), which reported 2 new alterations in AIRE, the strongest one being p.R471C. It also described nine independent risk loci implicated in central immunological tolerance (51), in particular PTPN22, CTLA4, and BACH2 loci whose role in pathogenesis is already established (50, 51) and SH2B3 and SIGLEC5 (51). HLA class II genes also play a central role in predisposition to isolated AAI or APS type 2/4. While HLA DRB1-0301 and DRB1-040 are associated with AAI, it seems that DRB1-0403 is protective against development of Addison’s disease (50). Other polymorphisms worth mentioning include the MHC class I chain-related gene A (MICA) allele 5.1, the CIITA (MHC class II transactivator), the master regulator of MHC class II expression, STAT4, PD-L1, and the vitamin D receptor (50). SIGNS AND SYMPTOMS OF AI- NATURAL HISTORY OF AUTOIMMUNE PAI-MORTALITY In 100 asymptomatic antibody positive patients followed up for 20 years, impairment of adrenal function occurred at an annual rate of 4.9% (29). Treatment with glucocorticoids in stressful conditions such as a major surgery or illness may be considered for patients at stages 1 and 2 and close monitoring of patients with higher risk of progression to clinical disease is warranted (children, APS type 1, high antibody titers and already at stage 1) (29, 32). Because of the insidious course of the disease, patients may not be diagnosed until they present with adrenal crisis. Adrenal crisis is an acute, life-threatening condition more frequently seen in PAI than central AI (33). The frequency of adrenal crisis varied from 4.4 to 17/100 patient-years (11, 34, 35), depending August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 3 Latent Adrenal Insufﬁciency Younes et al. their immunocompromised state and inability to defend against viral infections owing to a natural killer cell dysfunction (23, 38). Eight % of annual cases of adrenal crises will need in hospital treatment and admission rates were highest in patients aged more than 60 years old (41). In children, adrenal crisis frequency was reported to be 3.4/100 patient-years and children diagnosed with salt wasting congenital adrenal hyperplasia (CAH), adrenal hypoplasia congenital (AHC) and AAI were at higher risk (42). Compared to the general population, patients with AI often complain of reduced quality of life and work capacity with more sick day leaves potentially explained by the inability of present treatment modalities to replicate circadian rhythm of cortisol production (6, 13). Another signiﬁcant issue is the impact of PAI on fertility in women of reproductive age. Few cohort studies have shown that pregnancy rate decreases after diagnosis of PAI even in the absence of associated POI, and a higher risk of cesarean delivery, impaired fetal growth, preterm birth and low birth weight is found (43). Particularly, a German study including 39 women with CAH secondary to 21-hydroxylase deﬁciency and 54 with AAI, reported reduced fertility only in classic CAH and APS type 2, based on answers from a self-reporting questionnaire (44). This suggests that a more severe course of disease and/or the presence of other comorbid autoimmune disorders largely impact fertility outcomes. There are no clear explanations for the underlying mechanisms of reduced fertility in women with AAI, in the absence of POI. SIGNS AND SYMPTOMS OF AI- NATURAL HISTORY OF AUTOIMMUNE PAI-MORTALITY Perhaps their reduced quality of life and overall dissatisfaction with current treatment modalities may play a signiﬁcant role in hindering successful pregnancies. represents 83% of PAI diagnoses compared to 9.7, 6.1 and 1.2% for AHC, autoimmune AI and adrenoleukodystrophy, respectively (42). 97.2% of CAH were secondary to 21-hydroxylase deﬁciency in a Chinese pediatric cohort followed up for 29 years (47). A detailed discussion of these diagnoses is outside the scope of this review and will be discussed elsewhere. PATHOGENESIS OF AUTOIMMUNE AI All susceptibility loci described to date are in genes involved in adaptive or innate immunity (52), particularly affecting Frontiers in Endocrinology \| www.frontiersin.org Associated clinical/biological and radiological features Associated clinical/biological and radiological features Bilateral adrenal atrophy on computed tomography. Bilateral adrenal atrophy on computed tomography. Polygenic- positive 21-hydroxylase antibodies ± adrenal cortex antibodies/17-hydroxylase antibodies/steroid side chain cleavage enzyme autoantibodies- other AI diseases associated. Autosomal recessive AIRE mutation- positive interferon antibodies- chronic mucocutaneous candidiasis, hypoparathyroidism, ectodermal dystrophy. Polygenic- Type 1 diabetes and autoimmune thyroid disease AUTOIMMUNE ADRENALITIS ISOLATED APS TYPE 1 APS TYPE 2 INFECTIONS ACTIVE TUBERCULOUS INFECTION SYSTEMIC FUNGAL INFECTIONS WATERHOUSE-FRIDERICHSEN SYNDROME SEPTIC CHOC OPPORTUNISTIC INFECTIONS IN IMMUNOCOMPROMISED INFILTRATIVE DISEASES METASTASES FROM LUNG, BREAST, OR KIDNEY CARCINOMAS SARCOIDOSIS AMYLOIDOSIS HEMOCHROMATOSIS ADRENAL HEMORRHAGE COAGULATION DISORDERS ANTICOAGULANT TREATMENT ANTIPHOSPHOLIPID SYNDROME BILATERAL ADRENALECTOMY PRIMARY ADRENAL LYMPHOMA DRUGS INCREASE CORTISOL METABOLISM IMPAIRED STEROIDOGENESIS ANTAGONIZE GLUCOCORTICOID ACTION ON PERIPHERAL TISSUES ADRENOLYTIC TRIGGER AUTOIMMUNE REACTION ADRENOLEUKODYSTROPHY AUTOIMMUNE ADRENALITIS ISOLATED APS TYPE 1 APS TYPE 2 INFECTIONS ACTIVE TUBERCULOUS INFECTION SYSTEMIC FUNGAL INFECTIONS WATERHOUSE-FRIDERICHSEN SYNDROME SEPTIC CHOC OPPORTUNISTIC INFECTIONS IN IMMUNOCOMPROMISED INFILTRATIVE DISEASES METASTASES FROM LUNG, BREAST, OR KIDNEY CARCINOMAS SARCOIDOSIS AMYLOIDOSIS HEMOCHROMATOSIS ADRENAL HEMORRHAGE COAGULATION DISORDERS ANTICOAGULANT TREATMENT ANTIPHOSPHOLIPID SYNDROME BILATERAL ADRENALECTOMY PRIMARY ADRENAL LYMPHOMA DRUGS INCREASE CORTISOL METABOLISM IMPAIRED STEROIDOGENESIS ANTAGONIZE GLUCOCORTICOID ACTION ON PERIPHERAL TISSUES ADRENOLYTIC TRIGGER AUTOIMMUNE REACTION ADRENOLEUKODYSTROPHY Bilateral adrenal atrophy on computed tomography. Polygenic- positive 21-hydroxylase antibodies ± adrenal cortex antibodies/17-hydroxylase antibodies/steroid side chain cleavage enzyme autoantibodies- other AI diseases associated. Autosomal recessive AIRE mutation- positive interferon antibodies- chronic mucocutaneous candidiasis, hypoparathyroidism, ectodermal dystrophy. Polygenic- Type 1 diabetes and autoimmune thyroid disease Enlarged adrenal glands on computed tomography, calciﬁcations may be seen. Altered mental state, hypotension, fever, acute adrenal crisis. Other signs speciﬁc to inﬁltrative disease (mediastinal lymph nodes, chronic kidney disease, hypoparathyroidism, diabetes mellitus…) Hypophysitis may be associated with inﬁltrative diseases. Hypophysitis may be associated with inﬁltrative diseases. Sudden pain accompanied with acute adrenal crisis. Adrenal hemorrhage on computed tomography. Warfarin. Positive cardiolipin antibodies, lupus anticoagulant and anti-beta-2 glycoprotein 1. Enlarged adrenal glands on computed tomography. Induction of P450-cytochrome enzymes, CYP3A4, CYP2B1, CYP2B2: phenytoin, rifampicin, phenobarbital Ketoconazole, ﬂuconazole, mitotane, metyrapone, etomidate, aminoglutethimide, trilostane. Abiraterone acetate. by ﬁbrous tissue (48). In cases when conﬁrmatory test is not possible, a morning serum cortisol of less than 140 nmol/L paired with a morning plasma ACTH above 2-fold the upper limit of normal is consistent with PAI. Measuring plasma renin and aldosterone is also recommended to document mineralocorticoid deﬁciency (3). Associated clinical/biological and radiological features There are two types of synthetic corticotropin analogs (ACTH 1-24) that can be used in the corticotropin stimulation test: cosyntropin (Cortrosyn, Amphastar Pharmaceuticals. Inc) and tetracosactrin (Synacthen, Novartis Pharma, Switzerland) and since they both exist in 250 µg formulations, the standard dose test is more practical and is recommended by the Endocrine Society Clinical Practice guideline (3). Diagnostic cutoff values of serum cortisol depend on the assay used: in most immunoassays, 500 nmol/L is often used as the cutoff to establish diagnosis (6). However, newer monoclonal immunoassays such as the Elecsys®Cortisol II from regulatory T-cell function leading to local intra-adrenal self- reactive cytotoxic T-cells (53). However, novel therapies targeting the cellular immune response or speciﬁc genes implicated in AAI pathogenesis have yet to emerge. ETIOLOGIES SARCOIDOSIS Other signs speciﬁc to inﬁltrative disease (mediastinal lymph nodes, chronic kidney disease, hypoparathyroidism, diabetes mellitus…) AMYLOIDOSIS HEMOCHROMATOSIS Hypophysitis may be associated with inﬁltrative diseases. ADRENAL HEMORRHAGE Sudden pain accompanied with acute adrenal crisis. COAGULATION DISORDERS Adrenal hemorrhage on computed tomography. ANTICOAGULANT TREATMENT Warfarin. ANTIPHOSPHOLIPID SYNDROME Positive cardiolipin antibodies, lupus anticoagulant and anti-beta-2 glycoprotein 1. BILATERAL ADRENALECTOMY PRIMARY ADRENAL LYMPHOMA Enlarged adrenal glands on computed tomography. DRUGS INCREASE CORTISOL METABOLISM Induction of P450-cytochrome enzymes, CYP3A4, CYP2B1, CYP2B2: phenytoin, rifampicin, phenobarbital IMPAIRED STEROIDOGENESIS Ketoconazole, ﬂuconazole, mitotane, metyrapone, etomidate, aminoglutethimide, trilostane. Abiraterone acetate. ANTAGONIZE GLUCOCORTICOID ACTION ON PERIPHERAL TISSUES Mifepristone ADRENOLYTIC Mitotane TRIGGER AUTOIMMUNE REACTION Nivolumab and pembrolizumab ADRENOLEUKODYSTROPHY Young men X-linked recessive Defects in ABCD1 gene Negative antibodies to 21-hydroxylase Elevated serum very long chain fatty acids Progressive neurological deﬁcit, hypogonadism ETIOLOGIES The most common cause of PAI in the adult population is autoimmune destruction of the adrenal cortex (1). Other potential causes include infections, inﬁltrative diseases, adrenal hemorrhage, surgery, and drugs (1, 6). They are summarized in Table 1. AAI remains a diagnosis of exclusion and in the absence of positive antibodies directed against 21-hydroxylase, other diagnoses should be explored; an abdominal computed tomography should be performed. In the event of enlarged adrenal glands, differential diagnoses include an active tuberculous infection, systemic fungal infections in immunocompromised patients, metastases and lymphoma (1). In young men with negative antibodies to 21-hydroxylase, adrenoleukodystrophy should be suspected, even in the absence of neurological symptoms (1, 6). Adrenoleukodystrophy is an X-linked recessive disorder, hence affecting only boys, characterized by defects in ABCD1 gene causing elevated serum very long chain fatty acids. The clinical spectrum of adrenoleukodystrophy includes cerebral manifestations that may manifest in childhood such as behavior changes, school difﬁculties, cognitive deﬁcits up to dementia, psychoses and loss of vision and speech. It may be associated with adrenomyeloneuropathy presenting later in middle age with progressive lower body stiffness and weakness (45). The diagnosis of adrenoleukodystrophy should be conﬁrmed by molecular genetic testing (46). In children, however, CAH August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org Latent Adrenal Insufﬁciency Younes et al. regulatory T-cell function leading to local intra-adrenal self- reactive cytotoxic T-cells (53). However, novel therapies by ﬁbrous tissue (48). In cases when conﬁrmatory test is no possible, a morning serum cortisol of less than 140 nmol/ TABLE 1 \| Etiologies of primary adrenal insufﬁciency in adults (1, 5, 6, 45). Etiology Associated clinical/biological and radiological features AUTOIMMUNE ADRENALITIS Bilateral adrenal atrophy on computed tomography. ISOLATED Polygenic- positive 21-hydroxylase antibodies ± adrenal cortex antibodies/17-hydroxylase antibodies/steroid side chain cleavage enzyme autoantibodies- other AI diseases associated. APS TYPE 1 Autosomal recessive AIRE mutation- positive interferon antibodies- chronic mucocutaneous candidiasis, hypoparathyroidism, ectodermal dystrophy. APS TYPE 2 Polygenic- Type 1 diabetes and autoimmune thyroid disease INFECTIONS ACTIVE TUBERCULOUS INFECTION Enlarged adrenal glands on computed tomography, calciﬁcations may be seen. SYSTEMIC FUNGAL INFECTIONS WATERHOUSE-FRIDERICHSEN SYNDROME Altered mental state, hypotension, fever, acute adrenal crisis. SEPTIC CHOC OPPORTUNISTIC INFECTIONS IN IMMUNOCOMPROMISED INFILTRATIVE DISEASES Enlarged adrenal glands on computed tomography. METASTASES FROM LUNG, BREAST, OR KIDNEY CARCINOMAS Known primary cancer. ESTABLISHING THE DIAGNOSIS The Endocrine Society Clinical Practice Guideline published in 2016 recommend conﬁrming the diagnosis of PAI with a corticotropin stimulation test to assess adrenocortical function (3). An algorithm depicting the proposed diagnostic approach in PAI in adults is shown in Figure 2. In PAI, adrenal glands are unresponsive to corticotropin stimulation because zona fasciculata is already maximally stimulated by elevated endogenous ACTH (54) and because adrenal cortex is replaced August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 5 Younes et al. Latent Adrenal Insufﬁciency FIGURE 2 \| Proposed diagnostic approach for primary adrenal insufﬁciency in adults. FIGURE 2 \| Proposed diagnostic approach for primary adrenal insufﬁciency in adults. corticotropin test (1 µg) had better accuracy for diagnosing central AI compared to the standard dose (250 µg) (61). Even so, the low dose is less practical because it necessitates dilution from supplied ampules of 250 µg and is subject to human and technical errors (3, 61). Also, samples are best withdrawn 20 to 30 minutes following injection in order to avoid false positives (59, 61). Neither corticotropin stimulation test nor metyrapone test often used to diagnose central AI, can replace insulin tolerance test considered to be the gold standard for evaluation of hypothalamic pituitary axis. A study by Giordano et al. including 31 patients with central AI, failed to demonstrate superiority of either low dose or standard dose, when both achieved same diagnostic accuracy (62). Another potential role for low dose corticotropin test would be in establishing diagnosis of latent AAI. In fact, it was found abnormal in 88.4% of 33 patients with abnormal cortisol response compared to 66.6% for the standard dose, suggesting that the standard dose might miss some cases of latent PAI (63). More studies are needed to elucidate the diagnostic role of low dose corticotropin testing. Moreover, several factors may affect interpretation of cortisol response to stimulation tests. In particular, cortisol binding globulin (CBG) which binds the majority of circulating cortisol, leaving only 5-10% of plasma cortisol free (1, 6), can be responsible for pitfalls in diagnosis. Because assays measure total serum cortisol and not free cortisol, conditions increasing CBG such as pregnancy, oral contraceptive pills and mitotane, Roche Diagnostics, have lower cross reactivity with other steroids, thus giving values that are 20-30% lower than those with older assays (55, 56). Frontiers in Endocrinology \| www.frontiersin.org ESTABLISHING THE DIAGNOSIS Another emerging noninvasive diagnostic test is the measurement of salivary cortisol or cortisone at 60 minutes following administration of 500 µg nasal tetracosactide with mucoadhesive chitosan. This generated the same 60-minute plasma cortisol response as seen with 250 µg intravenous tetracosactide and slightly lower levels of salivary cortisol and cortisone with the nasal formulation (69). Granted, this noninvasive diagnostic method is safe and convenient for patients, it requires additional studies before wide application can be recommended. Finally, measurement of anti 21-hydroxylase antibodies is necessary to establish autoimmune etiology. Commercially available assays include immunoﬂuorescence and autoantibody assays. However, clinicians should keep in mind that they are not standardized and variations in between assays exist (3, 6). When AAI is diagnosed, screening for other comorbid autoimmune diseases should be undertaken; in particular autoimmune thyroid disease (70), type 1 diabetes (3) and POI, especially when steroid side chain cleavage enzyme autoantibodies are detected (29). glucocorticoid deﬁciency, without signs of under replacement, in 25 adults with AI, was signiﬁcantly less than that recommended by the Endocrine Society Clinical Practice Guideline (72). The mean replacement dose reported in their study was 7.6 ± 3.5 mg/m2, reﬂecting daily endogenous cortisol production (72). A recent systematic review of 47 studies reported that although prednisolone therapy increased risk of dyslipidemia and cardiovascular disease, it was as safe and efﬁcacious as hydrocortisone (73). It also suggested that lower doses of hydrocortisone (less than 20 mg/day) had better clinical outcomes, and failed to conclusively demonstrate an added beneﬁt of modiﬁed release hydrocortisone or continuous subcutaneous hydrocortisone infusion using insulin pumps (73). Current available regimens fail to replicate both circadian and ultradian rhythmicity of cortisol, potentially explaining the persistently low quality of life that patients with AI often complain about. In fact, non-pulsatile cortisol secretion was found to be associated with poor quality of sleep, poor working memory performance and mood disorders (74, 75). An in-depth discussion of novel forms of glucocorticoid substitution and their role in better mimicking physiologic cortisol secretion will be covered in other chapters of this special topic. Overreplacement of glucocorticoids predisposes to elevated blood pressure, diabetes, osteoporosis and obesity, and thus should be avoided (6). Monitoring of glucocorticoid therapy is based solely on clinical assessment using signs and symptoms of over and under replacement such as body weight, blood pressure, energy levels, hyperpigmentation and bone mineral density (3). ESTABLISHING THE DIAGNOSIS Two different studies suggested new cutoff thresholds when using the Elecsys®Cortisol II: 374 nmol/L (55) and 403 nmol/L at 30 minutes (56). Furthermore, because liquid chromatography tandem mass spectrometry (LC-MS/MS) measures cortisol more accurately than immunoassays, cutoff values were also redeﬁned to be lower, respectively 400-412 and 485 nmol/L, at 30 and 60 minutes following corticotropin analog injection (56, 57). This would potentially reduce overdiagnosis of AI. Additionally, a baseline cortisol less than 55 nmol/L successfully predicted abnormal response to stimulation test (56). A recent retrospective study conducted on 370 patients in Spain (58), conﬁrmed need for sex-speciﬁc and assay-speciﬁc cutoff values when interpreting corticotropin test to reduce false positives and increase speciﬁcity. It also allowed for a better diagnostic agreement between sampling times at 30 and 60 minutes compared to using general cutoff values. Sampling at 30 minutes following injection of corticotropin analog accurately diagnosed 95% of PAI. However, this was not true for central AI, where sampling at 60 minutes showed better diagnostic accuracy (58). Corticotropin stimulation test can be done anytime of the day because in PAI response to ACTH is independent of circadian rhythm, but morning testing might be more accurate to avoid overdiagnosis in healthy individuals (59, 60). A metanalysis including 13 studies showed that the low dose August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 6 Latent Adrenal Insufﬁciency Younes et al. lead to normal cortisol values, falsely reassuring clinicians (6, 64). Hence, trimester-speciﬁc cutoff values were suggested to eliminate false negatives: 700, 800 and 900 nmol/L, respectively in ﬁrst, second and third trimester (65). In situations where CBG is reduced (sepsis, cirrhosis, nephrotic syndrome, hyperthyroidism and SERPINA6 gene polymorphisms), low cortisol values must be interpreted with caution (6, 66). In such patients, measurement of salivary cortisol has the beneﬁt of being a direct, noninvasive measurement of free cortisol and correlates well with circadian variations of serum cortisol (67). While it is more often used in the diagnosis of hypercortisolism, a recent study demonstrated an added beneﬁt of measuring salivary cortisol in response to corticotropin stimulation in particular in patients taking oral estrogens and in cases of indeterminate serum cortisol at 60 minutes, deﬁned as values between 500 and 599 nmol/L (68). The diagnostic cutoff used in this study was 26 nmol/L (68). ESTABLISHING THE DIAGNOSIS Use of ACTH measurement is especially unreliable and will most deﬁnitely lead to overreplacement owing to the disrupted negative feedback of cortisol on ACTH (3). Although hair cortisol concentrations were found to be useful in identifying children overtreated with hydrocortisone, it requires a 1 cm thick hair sample and the technique is not yet widely available (76). Also, gene expression emerged recently as a potential tool for monitoring hydrocortisone therapy. In particular, expression of DSIPI, DDIT4 and FKBP5 increased 2 hours after hydrocortisone infusion and correlated well with normal serum cortisol levels (77). Frontiers in Endocrinology \| www.frontiersin.org Frontiers in Endocrinology \| www.frontiersin.org MANAGEMENT OF CHRONIC AND ACUTE PAI Cortisol release follows a circadian and ultradian rhythm. It peaks early in the morning then gradually declines to reach nadir around midnight (6). Pulsatile release of cortisol every 60-90 minutes seems to be intrinsically related to interactions between the pituitary and the adrenal glands and might be independent of supra-pituitary inﬂuences (71). The Endocrine Society Clinical Practice Guideline recommends treating all patients with PAI with glucocorticoids and mineralocorticoids when aldosterone deﬁciency is conﬁrmed (Figure 3). The authors agreed that hydrocortisone or cortisone acetate, given in two or three divided oral doses, should be the preferred therapeutic choices (3). Total daily dose should be the equivalent of 15-25 mg of hydrocortisone for adults, with the highest dose given in the morning, in an attempt to replicate the physiologic circadian rhythm (3). However, Caetano et al. recently showed that the daily hydrocortisone dose sufﬁcient to substitute for Mineralocorticoid deﬁciency is treated with ﬂudrocortisone, approximately 50-200 mcg in adults, in one single morning dose, and ad libitum salt consumption (3). Higher doses are often needed in speciﬁc circumstances such as in children because of early mineralocorticoid resistance, in athletic people and in very hot climates because of salt wasting due to excessive perspiration (3, 6). Clinicians should look for signs of overreplacement (hypertension, peripheral edema, hypokalemia) and those of under-replacement (orthostatic hypotension, salt craving and hyperkalemia). Adequate mineralocorticoid replacement can also be determined according to plasma renin concentration, which should be kept in the upper reference range (3). Until this day, no evidence exists to support dehydroepiandrosterone (DHEA) replacement therapy in all patients with PAI. A trial therapy of 25-50 mg of DHEA for August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 7 Latent Adrenal Insufﬁciency Younes et al. FIGURE 3 \| Summary of the three pillars of management in adults with primary adrenal insufﬁciency. HC, hydrocortisone; FC, ﬂudrocortisone; IM, intramuscularly; SC, subcutaneously. RESIDUAL GLUCOCORTICOID FUNCTION AND FUTURE PERSPECTIVES studies suggested that treatment with immunomodulators could be a step forward to reverse autoimmune destruction and allow for regeneration of adrenal cortex. Two of 13 patients who were given subcutaneous tetracosactide for a period of 20 weeks had urine glucocorticoid metabolite in the median range of healthy individuals and peak serum cortisol concentrations above 400 nmol/L, allowing for cessation of glucocorticoid therapy (86). A more recent study evaluated the effect of dual therapy with rituximab and depot tetracosactide in 13 patients with AAI (87). Although dual therapy did not allow full recovery of adrenal function, as deﬁned in this study by peak cortisol > 550 nmol/L at week 48 of treatment, it showed that endogenous cortisol production, quantiﬁed by urine metabolites, was increased in 62% of patients (87). Much remains to be explored in regenerative therapies and their role in recovering adrenal function. More studies are needed to allow for a better treatment approach that will mimic physiologic production of cortisol in order to ameliorate quality of life and reduce the morbidity related to PAI. studies suggested that treatment with immunomodulators could be a step forward to reverse autoimmune destruction and allow for regeneration of adrenal cortex. Two of 13 patients who were given subcutaneous tetracosactide for a period of 20 weeks had urine glucocorticoid metabolite in the median range of healthy individuals and peak serum cortisol concentrations above 400 nmol/L, allowing for cessation of glucocorticoid therapy (86). A more recent study evaluated the effect of dual therapy with rituximab and depot tetracosactide in 13 patients with AAI (87). Although dual therapy did not allow full recovery of adrenal function, as deﬁned in this study by peak cortisol > 550 nmol/L at week 48 of treatment, it showed that endogenous cortisol production, quantiﬁed by urine metabolites, was increased in 62% of patients (87). Much remains to be explored in regenerative therapies and their role in recovering adrenal function. More studies are needed to allow for a better treatment approach that will mimic physiologic production of cortisol in order to ameliorate quality of life and reduce the morbidity related to PAI. Adrenocortical plasticity is a well-established concept deﬁned by the ability of subcapsular adrenocortical stem cells to proliferate, then migrate into the three zones of the adrenal cortex where zone-speciﬁc differentiation occurs, and cells acquire steroidogenic function; all under the inﬂuence of ACTH (54). It was shown that cortisol August 2021 \| Volume 12 \| Article 720769 8 Latent Adrenal Insufﬁciency Younes et al. FIGURE 3 \| Summary of the three pillars of management in adults with primary adrenal insufﬁciency. HC, hydrocortisone; FC, ﬂudrocortisone; IM, intramuscularly; SC, subcutaneously. FIGURE 3 \| Summary of the three pillars of management in adults with primary adrenal insufﬁciency. HC, hydrocortisone; FC, ﬂudrocortisone; IM, intramuscularly; SC, subcutaneously. ee pillars of management in adults with primary adrenal insufﬁciency. HC, hydrocortisone; FC, ﬂudrocortisone; IM, intramuscularly; increased rapidly following subcutaneous hydrocortisone with a delay of only 11 minutes when compared to intramuscular hydrocortisone (79). A medical alert bracelet or an emergency card can help health care providers identify patients with PAI requiring lifesaving hydrocortisone administration in emergent situations (78). During adrenal crises, it is recommended to administer 100 mg of intravenous hydrocortisone followed by either continuous infusion of 200 mg of hydrocortisone per 24 hours or 50 mg intravenously every 6 hours alongside adequate isotonic saline infusion. Tapering of hydrocortisone can begin 24-48 hours following adrenal crisis, when patients can tolerate oral medication. Fludrocortisone can be re-introduced when hydrocortisone dose is less than 50 mg/day (3, 6). Stress dosing is also needed before dental and minor surgeries (25-75 mg/24h of hydrocortisone, depending on type of procedure). Major surgeries, trauma and delivery require same dosing regimen as in adrenal crises management (3). Management of PAI in special populations including children, patients with CAH, pregnant women and endurance athletes is not discussed in this review. 6 months can be initiated in premenopausal women complaining of reduced libido and depressive moods despite adequate glucocorticoid and mineralocorticoid therapy (3, 6). Unfortunately, well standardized and reliable DHEA formulations are lacking in many countries. Patient education on dose adaptation and sick day rules is an essential part of management (Figure 3). Because all patients with PAI are at risk of adrenal crisis, physicians should insist on increasing patient awareness to their disease by implementing treatment strategies, participating in group sessions, and prescribing at home hydrocortisone injection kits in case of emergencies (78). Hydrocortisone dose adjustments are required during sick days: patients should double or triple their daily oral dose for 48-72 hours until full recovery. If oral medication cannot be tolerated or absorbed, 100 mg of parenteral hydrocortisone should be promptly administered before seeking medical care (3). Subcutaneous hydrocortisone injection is an alternative in patients who cannot self-inject hydrocortisone intramuscularly. CONCLUSION AAI remains a life-threatening condition if not recognized early. Despite medical advances, adrenal crises still occur and quality of life of patients is largely impacted. Attention should be especially given to patients with latent AI, at early stages of adrenal destruction, to prevent adrenal crises from developing and going unnoticed. Impending novel therapies are being explored to determine the best approach to utilize residual adrenal cortisol production and to regenerate adrenocortical cells destroyed by the autoimmune process. Until this is a validated practice, the only effective treatment remains adequate glucocorticoid and mineralocorticoid replacement and patient and physician education. AUTHOR CONTRIBUTIONS NY, IB, and AL contributed to conception and design of this review. NY wrote the ﬁrst draft of the manuscript. IB and AL wrote sections of the manuscript and revised the ﬁnal draft. All authors contributed to the article and approved the submitted version. RESIDUAL GLUCOCORTICOID FUNCTION AND FUTURE PERSPECTIVES Adrenal mass is also inﬂuenced by ACTH: when deﬁcient, adrenal atrophy and hypofunction develop, whereas when increased, adrenal hyperplasia and hyperfunction occur (54). Adrenocortical stem cells may hold the key to understanding the mechanisms of a new emerging concept in autoimmune adrenalitis; that of residual glucocorticoid function. Indeed, residual glucocorticoid production was described in 30-50% of patients years after AAI was diagnosed (80, 81), more commonly in men and in those with a more recent diagnosis. In contrast, residual mineralocorticoid production was only found in 13.5% of patients (80). It was hypothesized that the lack of expression by adrenocortical stem cells of 21-hydroxylase and other enzymes implicated in steroidogenesis, may protect adrenal cortex from complete autoimmune destruction by retaining the possibility of repopulation by intact stem cells (54). Also, turnover of adrenocortical stem cells differs according to sex in rodents and could possibly explain why endogenous residual function is more commonly observed in men (54). Elevated ACTH in PAI may also contribute to the stimulation of proliferation and differentiation of adrenocortical stem cells. However, once hydrocortisone replacement therapy is introduced, endogenous cortisol production declines alongside the ACTH decline and it is not known whether this is responsible for a more rapid loss of residual function or it is but the natural history of autoimmune adrenalitis that is responsible for the loss of function over time (82). Glucocorticoid precursors such as 11- deoxycortisol, 11-deoxycorticosterone and corticosterone, are potential biomarkers of residual endogenous adrenal function because their concentrations correlated well with serum cortisol (81, 82). Although, some patients retained residual adrenal function, they were not protected from increased risk of adrenal crisis and quality of life was still signiﬁcantly altered (80). Spontaneous recovery of endogenous adrenal function in AAI is rarely described in the literature, 7-16 years following diagnosis, with most cases being partial recovery (83–85). Recent 6. Husebye ES, Pearce SH, Krone NP, Kämpe O. Adrenal Insufﬁciency. Lancet Lond Engl (2021) 397(10274):613–29. doi: 10.1016/S0140-6736(21) 00136-7 5. Charmandari E, Nicolaides NC, Chrousos GP. Adrenal Insufﬁciency. Lancet Lond Engl (2014) 383(9935):2152–67. doi: 10.1016/S0140-6736(13) 61684-0 7. On the Constitutional and Local Effects of Disease of the Supra-Renal Capsules/by Thomas Addison. 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Eur Thyroid J (2015) 4(3):201–6. doi: 10.1159/000433532 71. Lightman SL, Conway-Campbell BL. The Crucial Role of Pulsatile Activity of 53. Betterle C, Presotto F, Furmaniak J. Epidemiology, Pathogenesis, and Diagnosis of Addison’s Disease in Adults. J Endocrinol Invest (2019) 42 (12):1407–33. doi: 10.1007/s40618-019-01079-6 71. Lightman SL, Conway-Campbell BL. The Crucial Role of Pulsatile Activity of the HPA Axis for Continuous Dynamic Equilibration. Nat Rev Neurosci (2010) 11(10):710–8. doi: 10.1038/nrn2914 the HPA Axis for Continuous Dynamic Equilibration. Nat Rev Neurosci (2010) 11(10):710–8. doi: 10.1038/nrn2914 54. Pearce SHS, Gan EH, Napier C. Management of Endocrine Disease: Residual Adrenal Function in Addison’s Disease. Eur J Endocrinol (2021) 184(2):R61– 7. doi: 10.1530/EJE-20-0894 72. Caetano CM, Sliwinska A, Madhavan P, Grady J, Malchoff CD. Empiric Determination of the Daily Glucocorticoid Replacement Dose in Adrenal Insufﬁciency. J Endocr Soc (2020) 4(11):bvaa145. doi: 10.1210/ jendso/bvaa145 55. Raverot V, Richet C, Morel Y, Raverot G, Borson-Chazot F. Establishment of Revised Diagnostic Cut-Offs for Adrenal Laboratory Investigation Using the New Roche Diagnostics Elecsys® Cortisol II Assay. Ann Endocrinol (2016) 77 (5):620–2. doi: 10.1016/j.ando.2016.05.002 73. Kiko N, Kalhan A. Comparison of Various Glucocorticoid Replacement Regimens Used in Chronic Adrenal Insufﬁciency: A Systematic Review. Dubai Diabetes Endocrinol J (2020) 26(2):50–68. doi: 10.1159/000508321 56. Javorsky BR, Raff H, Carroll TB, Algeciras-Schimnich A, Singh RJ, Colón- Franco JM, et al. New Cutoffs for the Biochemical Diagnosis of Adrenal Insufﬁciency After ACTH Stimulation Using Speciﬁc Cortisol Assays. J Endocr Soc (2021) 5(4):bvab022. doi: 10.1210/jendso/bvab022 74. Kalafatakis K, Russell GM, Harmer CJ, Munafo MR, Marchant N, Wilson A, et al. Ultradian Rhythmicity of Plasma Cortisol Is Necessary for Normal Emotional and Cognitive Responses in Man. Proc Natl Acad Sci (2018) 115 (17):E4091–100. doi: 10.1073/pnas.1714239115 J Endocr Soc (2021) 5(4):bvab022. doi: 10.1210/jendso/bvab022 75. Kalafatakis K, Russell GM, Lightman SL. Mechanisms in Endocrinology: Does Circadian and Ultradian Glucocorticoid Exposure Affect the Brain? Eur J Endocrinol (2019) 180(2):R73–89. doi: 10.1530/EJE-18-0853 57. Ueland GÅ, Methlie P, Øksnes M, Thordarson HB, Sagen J, Kellmann R, et al. The Short Cosyntropin Test Revisited: New Normal Reference Range Using Lc-Ms/Ms. J Clin Endocrinol Metab (2018) 103(4):1696–703. doi: 10.1210/ jc.2017-02602 76. Noppe G, van Rossum EFC, Vliegenthart J, Koper JW, van den Akker ELT. REFERENCES Betterle C, Scalici C, Presotto F, Pedini B, Moro L, Rigon F, et al. The Natural History of Adrenal Function in Autoimmune Patients With Adrenal Autoantibodies. 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Ann Endocrinol (2018) 79(3):157–63. doi: 10.1016/ j.ando.2018.03.008 29. Betterle C, Coco G, Zanchetta R. Adrenal Cortex Autoantibodies in Subjects With Normal Adrenal Function. Best Pract Res Clin Endocrinol Metab (2005) 19(1):85–99. doi: 10.1016/j.beem.2004.11.008 August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org 10 Younes et al. Latent Adrenal Insufﬁciency 49. Husebye E, Løvås K. Pathogenesis of Primary Adrenal Insufﬁciency. Best Pract Res Clin Endocrinol Metab (2009) 23(2):147–57. doi: 10.1016/j.beem. 2008.09.004 67. Inder WJ, Dimeski G, Russell A. Measurement of Salivary Cortisol in 2012 - Laboratory Techniques and Clinical Indications. Clin Endocrinol (Oxf) (2012) 77(5):645–51. doi: 10.1111/j.1365-2265.2012.04508.x 50. Falorni A, Brozzetti A, Perniola R. From Genetic Predisposition to Molecular Mechanisms of Autoimmune Primary Adrenal Insufﬁciency. Front Horm Res (2016) 46:115–32. doi: 10.1159/000443871 68. Nolan BJ, Sorbello J, Brown N, Dimeski G, Inder WJ. Characterization of the Serum and Salivary Cortisol Response to the Intravenous 250 µg ACTH1-24 Stimulation Test. Endocrine (2018) 59(3):520–8. doi: 10.1007/s12020-017- 1505-0 51. Eriksson D, Røyrvik EC, Aranda-Guillén M, Berger AH, Landegren N, Artaza H, et al. GWAS for Autoimmune Addison’s Disease Identiﬁes Multiple Risk Loci and Highlights AIRE in Disease Susceptibility. Nat Commun (2021) 12 (1):959. doi: 10.1038/s41467-021-21015-8 69. Elder CJ, Vilela R, Johnson TN, Taylor RN, Kemp EH, Keevil BG, et al. Pharmacodynamic Studies of Nasal Tetracosactide With Salivary Glucocorticoids for a Noninvasive Short Synacthen Test. J Clin Endocrinol Metab (2020) 105(8):2692–703. REFERENCES Elevated Hair Cortisol Concentrations in Children With Adrenal Insufﬁciency on Hydrocortisone Replacement Therapy. Clin Endocrinol (Oxf) (2014) 81 (6):820–5. doi: 10.1111/cen.12551 58. Ortiz-Flores AE, Santacruz E, Jiménez-Mendiguchia L, Garcı́a-Cano A, Nattero-Chávez L, Escobar-Morreale HF, et al. Role of Sampling Times and Serum Cortisol Cut-Off Concentrations on the Routine Assessment of Adrenal Function Using the Standard Cosyntropin Test in an Academic Hospital From Spain: A Retrospective Chart Review. (2018). Available at: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC5942445/ (Accessed 2021 Apr 29). 77. Sævik ÅB, Wolff AB, Björnsdottir S, Simunkova K, Hynne MS, Dolan DWP, et al. Potential Transcriptional Biomarkers to Guide Glucocorticoid Replacement in Autoimmune Addison’s Disease. J Endocr Soc (2021) 5(3): bvaa202. doi: 10.1210/jendso/bvaa202 59. Park YJ, Park KS, Kim JH, Shin CS, Kim SY, Lee HK. Reproducibility of the Cortisol Response to Stimulation With the Low Dose (1 Microg) of ACTH. Clin Endocrinol (Oxf) (1999) 51(2):153–8. doi: 10.1046/j.1365-2265.1999. 00773.x 78. Guignat L. Therapeutic Patient Education in Adrenal Insufﬁciency. Ann Endocrinol (2018) 79(3):167–73. doi: 10.1016/j.ando.2018.03.002 79. Hahner S, Burger-Stritt S, Allolio B. Subcutaneous Hydrocortisone Administration for Emergency Use in Adrenal Insufﬁciency. Eur J Endocrinol (2013) 169(2):147–54. doi: 10.1530/EJE-12-1057 60. Dickstein G, Shechner C, Nicholson WE, Rosner I, Shen-Orr Z, Adawi F, et al. Adrenocorticotropin Stimulation Test: Effects of Basal Cortisol Level, Time of Day, and Suggested New Sensitive Low Dose Test. J Clin Endocrinol Metab (1991) 72(4):773–8. doi: 10.1210/jcem-72-4-773 80. Sævik ÅB, Åkerman A-K, Methlie P, Quinkler M, Jørgensen AP, Höybye C, et al. Residual Corticosteroid Production in Autoimmune Addison Disease. J Clin Endocrinol Metab (2020) 105(7):2430–41. doi: 10.1210/ clinem/dgaa256. 61. Kazlauskaite R, Evans AT, Villabona CV, Abdu TAM, Ambrosi B, Atkinson AB, et al. Corticotropin Tests for Hypothalamic-Pituitary- Adrenal Insufﬁciency: A Metaanalysis. J Clin Endocrinol Metab (2008) 93(11):4245– 53. doi: 10.1210/jc.2008-0710 81. Vulto A, Bergthorsdottir R, van Faassen M, Kema IP, Johannsson G, van Beek AP. Residual Endogenous Corticosteroid Production in Patients With Adrenal Insufﬁciency. Clin Endocrinol (Oxf) (2019) 91(3):383–90. doi: 10.1111/cen.14006 62. Giordano R, Picu A, Bonelli L, Balbo M, Berardelli R, Marinazzo E, et al. Hypothalamus-Pituitary-Adrenal Axis Evaluation in Patients With Hypothalamo-Pituitary Disorders: Comparison of Different Provocative Tests. Clin Endocrinol (Oxf) (2008) 68(6):935–41. doi: 10.1111/j.1365- 2265.2007.03141.x 82. Napier C, Allinson K, Gan EH, Mitchell AL, Gilligan LC, Taylor AE, et al. Natural History of Adrenal Steroidogenesis in Autoimmune Addison’s Disease Following Diagnosis and Treatment. J Clin Endocrinol Metab (2020) 105(7):2322–30. doi: 10.1210/clinem/dgaa187 63. 87. Napier C, Gan EH, Mitchell AL, Gilligan LC, Rees DA, Moran C, et al. Residual Adrenal Function in Autoimmune Addison’s Disease—Effect of Dual Therapy With Rituximab and Depot Tetracosactide. J Clin Endocrinol Metab (2020) 105(4):e1250–9. doi: 10.1210/clinem/dgz287 REFERENCES Dekkers OM, Timmermans JM, Smit JWA, Romijn JA, Pereira AM. Comparison of the Cortisol Responses to Testing With Two Doses of ACTH in Patients With Suspected Adrenal Insufﬁciency. Eur J Endocrinol (2011) 164(1):83–7. doi: 10.1530/EJE-10-0621 83. Smans LCCJ, Zelissen PMJ. Partial Recovery of Adrenal Function in a Patient With Autoimmune Addison’s Disease. J Endocrinol Invest (2008) 31(7):672–4. doi: 10.1007/BF03345623 84. Baxter M, Gorick S, Swords FM. Recovery of Adrenal Function in a Patient With Conﬁrmed Addison’s Disease. Endocrinol Diabetes Metab Case Rep (2013) 2013:130070. doi: 10.1530/EDM-13-0070 64. van Seters AP, Moolenaar AJ. Mitotane Increases the Blood Levels of Hormone-Binding Proteins. Acta Endocrinol (Copenh) (1991) 124(5):526– 33. doi: 10.1530/acta.0.1240526 85. Nordin BE. Addison’s Disease With Partial Recovery. Proc R Soc Med (1955) 48(12):1024–6. 65. Lebbe M, Arlt W. What Is the Best Diagnostic and Therapeutic Management Strategy for an Addison Patient During Pregnancy? Clin Endocrinol (Oxf) (2013) 78(4):497–502. doi: 10.1111/cen.12097 86. Gan EH, MacArthur K, Mitchell AL, Hughes BA, Perros P, Ball SG, et al. Residual Adrenal Function in Autoimmune Addison’s Disease: Improvement After Tetracosactide (ACTH1-24) Treatment. J Clin Endocrinol Metab (2014) 99(1):111–8. doi: 10.1210/jc.2013-2449 66. Wallace I, Cunningham S, Lindsay J. The Diagnosis and Investigation of Adrenal Insufﬁciency in Adults. Ann Clin Biochem (2009) 46(5):351–67. doi: 10.1258/acb.2009.009101 August 2021 \| Volume 12 \| Article 720769 Frontiers in Endocrinology \| www.frontiersin.org Younes et al. Latent Adrenal Insufﬁciency the publisher, the editors and the reviewers. 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https://openalex.org/W2772996212	https://pubs.rsc.org/en/content/articlepdf/2017/ra/c7ra11914c	English	null	Surface modification of carbon cloth anodes for microbial fuel cells using atmospheric-pressure plasma jet processed reduced graphene oxides	RSC advances	2,017	cc-by	6,216	aDepartment of Chemical and Materials Engineering, National I-Lan University, I-Lan 260, Taiwan. E-mail: shchang@niu.edu.tw bGraduate Institute of Applied Mechanics, National Taiwan University, Taipei City 10617, Taiwan. E-mail: jchen@ntu.edu.tw Introduction Graphene oxide (GO) is a derivative of graphene that possesses abundant epoxy and hydroxyl functional groups on the basal planes and carbonyl and carboxyl groups on the sheet edges. GO also exhibits hydrophilic properties and is useful for large-scale applications.16 However, the electrical properties of GO are generally poor, limiting its application in MFCs. Therefore, additional reducing processes, such as hydrazine vapor or thermal annealing treatments, are considered to remove the oxygen groups on GO and obtain reduced graphene oxide (rGO) with good electrical characteristics.17–19 Microbial fuel cells (MFCs) are promising for the generation of bioelectricity via the treatment of wastewater, because they can convert chemically bound energy into biomass-based electricity with the help of electrochemically active bacteria.1–4 It has been demonstrated that the surface characteristics of the anode electrodes are critical to the performance of MFCs.5 Among various types of electrodes, carbonaceous electrodes have gained much attention and shown considerable potential in MFC applications. Carbonaceous electrodes are commonly used in MFCs because of their advantages of high chemical stability, relatively low cost, good conductivity, and excellent biocompatibility.5,6 However, the undesirable hydrophobic property of the carbonaceous electrodes normally causes a poor electron transfer eﬃciency. Thus, surface modications of the carbonaceous electrodes are inevitably needed to enhance the power performance of MFCs.7–13 Several studies have demonstrated that MFCs congured with rGO-modied electrodes exhibit better electrochemical performance than those congured with unmodied elec- trodes.16,20–24 Gnana kumar et al.16 proposed a simple, environ- mentally benign, time- and cost-eﬃcient approach for the preparation of nanocomposites comprising polypyrrole (PPy) and rGO. The rGO/PPy nanocomposite signicantly increased the electron transfer eﬃciency and the intimate contact between the microorganisms and electrode. Liu et al.20 reported a novel MFC anode fabricated by electrochemically coating rGO rst and coating polyaniline (PANI) nanobers aerward on the surface of carbon cloth. Combining the advantages of PANI and rGO yields a maximum power density of 1390 mW m2, which is three times larger than that of the MFCs with the normal carbon cloth anode. Li et al.21 proposed a three-dimensional rGO–Ni foam as an anode for MFCs, which provided a large accessible surface area for microbial colonization and a uniform macro- porous scaﬀold for eﬀective mass diﬀusion of the culture medium. Open Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 This article is licensed under a Creative Commons Attribution 3.0 U In this study, we report on an easy, rapid, economical, and environmentally friendly method for surface modiﬁcation of carbon cloth anodes applicable in microbial fuel cells (MFCs) by screen-printing reduced graphene oxide (rGO) followed by calcining using an atmospheric-pressure plasma jet (APPJ). Screen printing of rGO and APPJ treatment signiﬁcantly increased the surface area of the eﬀective materials for bacterial adhesion. The combination of screen printing of rGO and APPJ treatment also made the carbon cloth highly hydrophilic, which beneﬁts the growth of bacteria on the surface of the carbon cloth. The rGO and APPJ-treated modiﬁed MFCs exhibited a maximum power density of 10.80 0.19 mW m2, whereas that of the unmodiﬁed MFCs was 6.02 0.01 mW m2. Both screen printing of rGO and APPJ treatment can be used for large-area surface modiﬁcation, which is promising for manufacturing large-scale MFC stacks. Received 30th October 2017 Accepted 8th December 2017 DOI: 10.1039/c7ra11914c rsc.li/rsc-advances Received 30th October 2017 Accepted 8th December 2017 RSC Advances RSC Advances Cite this: RSC Adv., 2017, 7, 56433 Received 30th October 2017 Accepted 8th December 2017 DOI: 10.1039/c7ra11914c rsc.li/rsc-advances PAPER n 15 December 2017. Downloaded on 10/24/2024 5:01:47 AM. ed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 5:01:47 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Surface modiﬁcation of carbon cloth anodes for microbial fuel cells using atmospheric-pressure plasma jet processed reduced graphene oxides Cite this: RSC Adv., 2017, 7, 56433 Introduction Tai et al.22 fabricated melamine sponges coated with rGO and carbon nanotubes, which provide a large electrically Recently, graphene has been widely used for the surface modication of carbonaceous electrodes because of its good biocompatibility, large specic surface area for accommodating microbes, high electronic conductivity, and eﬃcient electron transfer between bacteria and the electrode surface.14,15 However, monolayer graphene sheets normally agglomerate or form multilayer graphite owing to the strong p–p stacking.15 RSC Adv., 2017, 7, 56433–56439 \| 56433 This journal is © The Royal Society of Chemistry 2017 View Article Online View Article Online RSC Advances Paper kHz repetition frequency, and 30 standard liters per minute N2 ow rate. conductive surface for microorganism growth and electron transfer in MFCs. Mehdinia et al.23 and Garino et al.24 both re- ported that using microwave-assisted synthesized rGO/SnO2 nanocomposites as the anode material of MFCs could eﬀec- tively improve their power generation. In this study, we also proposed an environmentally friendly, time-saving, economic surface modication process to fabricate rGO-modied elec- trodes. Instead of synthesizing form chemical reactants, we used screen-printing process and atmospheric pressure plasma jet (APPJ) calcining to deposit rGO on the surface of carbon cloth anode. According to our previous studies,25–27 rGO paste can be easily screen-printed on the surface of substrates for applications in dye-sensitized solar cells and supercapacitors. However, the adhesion between the rGO paste and the substrate is generally poor, and, therefore, further thermal annealing is needed to calcine rGO on the substrate surface and burn the undesired organic residues. Compared to traditional annealing processes using a furnace, the APPJ process for surface modi- cation is a clean, nontoxic, environmentally friendly process, which is crucial for improving the eﬀectiveness of MFCs.25–27 Moreover, the APPJ surface modication technique can also introduce carboxyl and ammonium functional groups on the surface of carbon cloth. These promote the formation of anodic biolms and the adhesion of bacteria.28 Therefore, the aim of this study is to combine the advantages of screen-printing of rGO and the APPJ calcining process to enhance the power generation capabilities of MFCs. The surface morphology and the microbial colonization on the surface of the carbon cloths were examined using a 5136MM (Tescan) scanning electron microscope (SEM). The wettability properties of the surface of the carbon cloths were determined using an FTA125 (First Ten Angstroms) contact angle instru- ment. Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 5:01:47 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 5:01:47 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Results Fig. 1(a)–(d) show the top-view SEM images of the untreated, APPJ-treated, screen-printed rGO, and screen-printed rGO with APPJ calcining (rGO & APPJ-treated) carbon cloths, respectively. Fig. 1(a) shows that the carbon cloth comprises smooth carbon bers with a diameter of approximately 10 mm. Fig. 1(b) demonstrates that the carbon bers are not damaged aer the APPJ treatment. Fig. 1(c) shows that the carbon cloth is uniformly and densely covered by the screen-printed rGO akes; only a few carbon bers are visible. Besides, the surface morphology of the screen-printed rGO carbon cloth is rough with abundant porosity. Compared with the SEM image of the screen-printed rGO, Fig. 1(d) shows the presence of a larger number of carbon bers on the surface of the rGO & APPJ- treated carbon cloth, suggesting that some of the rGO akes and the organic binder in the rGO paste were burned out during the APPJ treatment. Meanwhile, the surface morphology of rGO 56434 \| RSC Adv., 2017, 7, 56433–56439 Introduction The surface chemical compositions of the carbon cloth electrodes were analyzed by a K-Alpha (Thermo Scientic) X-ray photoelectron spectroscopy (XPS) with a monochromatic Al Ka radiation source of 1468.6 eV. The power density and current density of the MFC were determined using an ECW-5600 (Jie- han) workstation. All the MFC experimental tests were carried out at ambient temperature. The internal resistance of the MFC was measured by electrochemical impedance spectroscopy (EIS) using a ZIVE SP1 (WonAtech) potentiostat. Experimental Membrane-free air-cathode single-chamber MFCs were con- structed in cylindrical tubes made of polymethyl methacrylate (cell sizing: ID ¼ 54 mm, L ¼ 96 mm) with an operating volume of 220 mL. A carbon cloth (CeTech Co. Ltd., Taiwan) with a projected area of approximately 22.9 cm2 on one side was used as the anode. The air cathode was nearly identical in size to the anode and consisted of a polytetrauorethylene diﬀusion layer (CeTech, Taiwan) on the air-facing side. The microbe used in this study was Aeromonas hydrophila NIU01. The culture medium in the MFCs used in this study was Luria–Bertani (LB) broth medium (tryptone: 10, yeast extract: 5, and sodium chloride: 10 (all in units of g L1)). The carbon cloth electrodes used in this study were purchased from CeTech, Taiwan. The procedure for the preparation of the rGO paste and the details of the screen-printing method are described in detail else- where.25–27 rGO amounting to 0.1 g (purity 99%, thickness < 5 nm, sheet diameter: 0.1–5 mm, Golden Innovation Business Co., Ltd.) was mixed with 3.245 g of terpineol (anhydrous, #86480, Fluka), 0.4698 g of ethanol, 1.4 g of 10 wt% ethyl cellulose (5–15 mPa s, #46070, Fluka) ethanolic solution, and 1.8 g of 10 wt% ethyl cellulose (30–50 mPa s, #46080, Fluka) ethanolic solution. The mixture was stirred at 450 rpm for 24 h using a magnetic stirrer. Finally, a 4 mL mixture was concen- trated for 5 min using a rotatory evaporator operated at 55 C to obtain the rGO pastes. Aer screen-printing the rGO paste, the carbon cloth was calcined by APPJ. The APPJ was carried out at a 4 mm s1 constant scanning rate, 275 V operation voltage, 25 Fig. 1 SEM micrographs of the (a) untreated, (b) APPJ-treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. Fig. 1 SEM micrographs of the (a) untreated, (b) APPJ-treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. 56434 \| RSC Adv., 2017, 7, 56433–56439 This journal is © The Royal Society of Chemistry 2017 This journal is © The Royal Society of Chemistry 2017 RSC Advances View Article Online RSC Advances View Article Online Paper changed from ake-like to sponge-like because of the APPJ treatment. Fig. 4 shows the results of water contact angle measurements of the untreated, APPJ-treated, screen-printed rGO, and rGO & APPJ-treated carbon cloths. As shown in Fig. Experimental 4, the untreated and screen-printed rGO carbon cloths exhibit high water contact angles of 127.9 11.4 and 123.5 11.8, respectively. On the contrary, the water contact angles of both the APPJ- treated and the rGO & APPJ-treated carbon cloths were approx- imately zero, indicating that the surfaces of the APPJ-treated carbon cloths are extremely hydrophilic. The high hydrophi- licity characteristic favors the biosorption of bacteria on the surface of electrodes.28 Fig. 2(a) and (b) show the magnied images of the screen- printed rGO (Fig. 1(c)) and the rGO & APPJ-treated (Fig. 1(d)) carbon cloths, respectively. Fig. 2(a) reveals that the screen- printed rGO akes merely adhere to the surface of the carbon bers. However, Fig. 2(b) shows that the rGO akes are sintered on the surface of the carbon bers. Although some of the screen-printed rGOs were burned during the APPJ treatment, Fig. 2 demonstrates that the adhesion between rGO and the carbon cloth improved aer the APPJ treatment. Open Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 5:01:47 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5(a)–(d) show the C 1s XPS spectra results of the untreated, APPJ-treated, screen-printed rGO, and rGO & APPJ- treated carbon cloths, respectively. Fig. 5(a) reveals that the C 1s characteristic peak of the untreated carbon cloth can be deconvoluted into a major sp3 C–C peak (284.8 eV) and a minor C–O peak (286.1 eV). The origin of the C–O peak is attributed to the contamination on the surface of the carbon cloth. Fig. 5(b) demonstrates that, except of the C–C and C–O peaks, the APPJ-treated carbon cloth exhibited additional C]O (287.3 eV) and COOH peaks (288.7 eV). Fig. 5(c) shows that the screen-printed rGO carbon cloth comprises sp3 C–C, sp2 C–C, C–O, C]O, and COOH peaks at approximately 284.8, 284.4, 286.1, 287.3, and 288.7 eV, respectively. The signicant sp2 C–C peak at approximately 284.4 eV is attributed to gra- phene in the screen-printed rGO. The signicant C–O and C]O peaks correspond to ethyl cellulose in the organic binder. The minor COOH peak is assigned to oxygen bonded to carbon at the edges of the rGO sheets.29–31 Fig. 5(d) shows that the rGO & APPJ-treated carbon cloth also consists of sp3 C–C, sp2 C–C, C–O, C]O, and COOH peaks at approximately 284.8, 284.4, 286.1, 287.3, and 288.7 eV, respectively. Experimental 6(b) shows that APPJ-treated carbon cloth exhibits a signicant N 1s peak, which can be deconvo- luted into a major C–N peak (399.3 eV) and a minor NR4 + peak (401.3 eV), which were introduced during the N2-APPJ treat- ment. Fig. 6(c) shows that the screen-printed rGO carbon cloth also does not exhibit the characteristic N 1s peak. However, aer the APPJ calcining, as shown in Fig. 6(d), the rGO & APPJ- Fig. 8(a) shows the power density response curves of the MFCs congured with the untreated, APPJ-treated, and rGO & APPJ-treated carbon cloth electrodes. According to Fig. 8(a), the highest power densities of the MFCs congured with the untreated, APPJ-treated, and rGO & APPJ-treated carbon cloth electrodes were approximately 6.02 0.01, 7.70 0.15, and 10.80 0.19 mW m2, respectively. Fig. 8(a) also shows the power density response curves of the identical MFCs congured with the APPJ-treated and rGO & APPJ-treated carbon cloth electrodes measured aer 145 days. The highest power densities of the MFCs congured with the APPJ-treated and rGO & APPJ- treated carbon cloth electrodes slightly decreased to approxi- mately 6.24 0.10 and 8.44 0.06 mW m2, respectively. This suggests that the MFCs congured with the APPJ-treated and rGO & APPJ-treated carbon cloth electrodes both exhibit good durability performance. Fig. 8(b) shows the results of the EIS measurements of the MFCs congured with the diﬀerent elec- trodes. The gure shows a single capacitive loop, which could be tted by the constant phase element (CPE) circuit model, in which the circuit comprises a CPE in parallel with a charge- transfer resistance (RCT), as demonstrated in Fig. 8(c). The impedance of the CPE can be calculated as ZCPE ¼ 1/T(ju)4.28,32 The Z-View® soware was used in this study to t the imped- ance of the CPE, in which 4 is dened as CPE-P and T is dened as CPE-T. The RCT values of the untreated, APPJ-treated, and rGO & APPJ-treated carbon cloth electrodes are 5827.9, 5323.3, and 972.1 U, respectively, indicating that the rGO & APPJ treatment can eﬀectively improve the charge transfer eﬃciency of the MFC. Fig. 8(d) shows the open-circuit voltage (OCV) Fig. 6 N 1s XPS spectra of the surfaces of (a) untreated, (b) APPJ- treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. Fig. Experimental RSC Adv., 2017, 7, 56433–56439 \| 56435 This journal is © The Royal Society of Chemistry 2017 View Article Online Paper RSC Advances Fig. 5 C 1s XPS spectra of the surfaces of (a) untreated, (b) APPJ- treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. treated carbon cloth showed the characteristic N 1s peak with a major C–N peak and a minor NR4 + peak. According to Fig. 5 and 6, we can conclude that the chemical bonding on the surface of the carbon cloth was eﬀectively modied from major sp3 C–C and minor C–O to dominant sp2 C–C with abundant carbonyl, carboxyl, and ammonium functional groups aer the rGO & APPJ treatment. Fig. 7(a)–(c) show the SEM images of the surfaces of the untreated, APPJ-treated, screen-printed rGO, and rGO & APPJ- treated carbon cloths, respectively, aer immersing in the chamber of the MFC for 24 h. The SEM results of the screen- printed rGO carbon cloth are not shown herein, because the screen-printed rGO akes peeled oﬀfrom the surface of the carbon cloth aer immersing in the chamber of the MFC. This is attributed to the poor adhesion between the screen-printed rGO and the carbon cloth before the APPJ calcining (Fig. 2(a)). Fig. 7(a) and (b) show that some microorganisms adhered on the surfaces of the untreated and the APPJ-treated carbon cloths. In addition, Fig. 7(b) shows that fragmentary biolms formed between the APPJ-treated carbon bers. Fig. 7(c) reveals that abundant microorganisms colonized on the surface of the rGO & APPJ-treated carbon cloth. Besides, the morphologies of rGO and the carbon bers cannot be clearly identied in Fig. 7(c), implying that the surfaces of rGO and the carbon bers are completely covered with the biolm. Thus, we demonstrated that rGO & APPJ treatment can eﬀectively facilitate the growth of microorganisms and the formation of biolms on the surface of carbon cloth. Fig. 5 C 1s XPS spectra of the surfaces of (a) untreated, (b) APPJ- treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. rGO & APPJ-treated carbon cloth were introduced during the APPJ treatment.28 Fig. 6(a)–(d) show the N 1s XPS spectra of the untreated, APPJ-treated, screen-printed rGO, and rGO & APPJ-treated carbon cloths, respectively. Fig. 6(a) shows that the characteristic N 1s peak was not observed on the surface of the untreated carbon cloth. Fig. Experimental However, compared with those of the peaks in the screen-printed rGO carbon cloth, the intensities of the C–O and the sp3 C–C peaks in the rGO & APPJ-treated carbon cloth are signicantly lower. Besides, it is clear from Fig. 5(d) that the intensity of the sp2 C–C peak is higher than that of the sp3 C–C peak. These results suggest that most of the organic binder in the rGO was burned out during the APPJ treatment. In addition, the carbonyl and the carboxyl functional groups observed on the surface of the Fig. 3(a) shows the cross-sectional SEM image of the rGO & APPJ-treated carbon cloth. Fig. 3(b), (c) and (d) present the magnied SEM images of Fig. 3(a) at the surface, center, and bottom regions, respectively. Fig. 3 shows that most of the rGO akes were deposited on the surface of the carbon cloth, as the rGO pastes were only screen-printed on one side of the carbon cloth. Nevertheless, as shown in Fig. 3(c) and (d), some rGO also percolated the center of the carbon cloth. This implies that the screen-printing of rGO not only provides contact areas on the surface of the carbon cloth, but also increases the surface area of the interior of the carbon cloth for the formation of biolms. Open Access Article. Published on 15 December 2017. Downloaded on 10/24/2024 5 This article is licensed under a Creative Commons Attribution 3.0 Unp Fig. 2 Magniﬁed SEM images of the (a) screen-printed rGO and the (b) rGO & APPJ-treated carbon cloths. Fig. 2 Magniﬁed SEM images of the (a) screen-printed rGO and the (b) rGO & APPJ-treated carbon cloths. Fig. 3 (a) Cross-sectional SEM micrographs of the rGO & APPJ- treated carbon cloth. Magniﬁed SEM micrographs of the (b) surface, (c) center, and (d) bottom regions of the cross-section of the rGO & APPJ-treated carbon cloth. Fig. 4 Water contact angles of the untreated, APPJ-treated, screen- printed rGO, and rGO & APPJ-treated carbon cloths. Fig. 3 (a) Cross-sectional SEM micrographs of the rGO & APPJ- treated carbon cloth. Magniﬁed SEM micrographs of the (b) surface, (c) center, and (d) bottom regions of the cross-section of the rGO & APPJ-treated carbon cloth. Fig. 4 Water contact angles of the untreated, APPJ-treated, screen- printed rGO, and rGO & APPJ-treated carbon cloths. This journal is © The Royal Society of Chemistry 2017 Experimental 6 N 1s XPS spectra of the surfaces of (a) untreated, (b) APPJ- treated, (c) screen-printed rGO, and (d) rGO & APPJ-treated carbon cloths. This journal is © The Royal Society of Chemistry 2017 56436 \| RSC Adv., 2017, 7, 56433–56439 View Article Online Paper RSC Advances Fig. 7 SEM micrographs of the (a) untreated, (b) APPJ-treated, and (c) rGO & APPJ-treated carbon cloths after immersing in the chamber of the MFC for 24 h. Fig. 7 SEM micrographs of the (a) untreated, (b) APPJ-treated, and (c) rGO & APPJ-treated carbon cloths after immersing in the chamber of the MFC for 24 h. Fig. 8 (a) Power density response curves and (b) EIS results of the MFCs conﬁgured with the untreated, APPJ-treated, and rGO & APPJ- treated carbon cloths. (c) The equivalent circuit model. (d) The open circuit voltage (OCV) results of the MFCs determined at an external resistance of 1000 U. improved by the rGO & APPJ treatment. The charge transfer resistance of the carbon cloth was reduced aer rGO & APPJ treatment because of the combined eﬀorts of good electrical conductivity and high surface area of the deposited rGO, improving the interfacial properties between the electrolyte and electrode and lowering the internal resistance and mass trans- fer losses.33–37 The improved electricity generation capability of the MFC congured with the rGO & APPJ-treated carbon cloth electrode can also be attributed to the increased surface area and the introduction of hydrophilic functional groups. As demonstrated in Fig. 1 to 3, the screen-printed rGO akes signicantly increase the surface area of the carbon cloth because of their high specic surface areas and porous struc- tures. Moreover, the surface morphology of the screen-printed rGO changed from ake-like to sponge-like aer the APPJ calcining, further increasing the surface area of rGO. The high surface area of the rGO & APPJ-treated carbon cloth electrode facilitates bacterial colonization and formation of biolms, as demonstrated in Fig. 7, escalating the extracellular electron transfer eﬃciency and the power generation capability of the MFC. Further, according to the XPS results shown in Fig. 5 and 6, the chemical bonds on the surface of the rGO & APPJ-treated carbon cloth consisted of sp2 C–C, carbonyl, carboxyl, and ammonium functional groups. The higher intensity of the sp2 C–C peak implies that the rGO & APPJ surface modications improved the electron transfer in the carbon cloth. Experimental The carbonyl, carboxyl, and ammonium functional groups contribute to the hydrophilic property of the rGO & APPJ-treated carbon cloth, as shown in Fig. 4, which promotes biocompati- bility for the growth of microorganisms.38 Additionally, the carboxyl functional group promotes the transfer of electrons from the bacteria to the electrodes, because of the hydrogen bonding with the membrane-bound peptide bonds in bacterial cytochromes associated with the intracellular electron transfer chain.39 The positive charge of the ammonium functional groups also helps in the formation of biolms and the adhesion of bacteria on the surface of the carbon cloth.40 Fig. 8 (a) Power density response curves and (b) EIS results of the MFCs conﬁgured with the untreated, APPJ-treated, and rGO & APPJ- treated carbon cloths. (c) The equivalent circuit model. (d) The open circuit voltage (OCV) results of the MFCs determined at an external resistance of 1000 U. results of the MFCs congured with the untreated, APPJ-treated, and rGO & APPJ-treated carbon cloth electrodes determined at an external resistance of 1000 U. Fig. 8(d) reveals that the MFC congured with the rGO & APPJ-treated carbon cloth electrode possessed a good durability performance for more than 700 h. This journal is © The Royal Society of Chemistry 2017 Acknowledgements The authors gratefully acknowledge the nancial support provided by the Ministry of Science and Technology (MOST), Taiwan, under Grant No. MOST 104-2221-E-197-004-MY3 (S. H. Chang), 105-2221-E-002-047-MY3 (J. Z. Chen) and 106-2221-E- 197-202-MY3 (B. Y. Chen). Discussion In our previous paper,42 we estimated that this ultrashort APPJ pro- cessing time (11 s) can render an estimated energy consump- tion per unit area of 1.1 kJ cm2, which is only one-third of that consumed in a conventional furnace calcination process. This is, therefore, a time- and energy-saving process benecial for future mass production. Moreover, APPJ treatment can easily introduce abundant hydrophilic functional groups on the surface of the carbon cloth, which are formed from the nitrogen and oxygen atoms in the atmosphere instead of exogenous chemicals.28 This indicates that APPJ is an environmentally friendly process, which is important for the growth of micro- organisms in MFCs. Compared with other graphene-based surface modication techniques, the screen-printing of rGO & APPJ treatment is time saving and cost eﬀective, consumes low energy, and is suitable for large-area processing. Therefore, the screen printing of the rGO & APPJ treatment process is prom- ising for the manufacture of large-scale MFC stacks for practical engineering applications. observe the reduction in C–O bonding contents that is contributed by ECs (as evidenced by XPS), in a shorter pro- cessing time. This reduction in C–O bonding content aer nitrogen APPJ processing is also observed in this study (Fig. 5). C–C bonding content is mainly contributed by rGOs and carbon bers of the carbon cloth. As the APPJ processing time is further increased, the rGOs are damaged and oxidized/evaporated.25,26 In this case, with rGOs on the carbon cloth, the C–C peak remains at a high level aer APPJ processing because of the presence of carbon bers (which are typically approximately 10 mm in diameter) on the carbon cloth. Using optical emission spectroscopy (OES) to control the processing time properly, we can burn ECs while retaining much of the rGOs.43 The optimal APPJ processing time is typically within 15 s. Furthermore, this APP process and screen-printing of rGO can be performed on substrates with various types of materials, including porous and solid ones. This provides high exibility in material deposition and surface modication processes. This nitrogen APPJ pro- cessing of screen-printed rGO pastes is economically favorable in terms of the thermal budget in materials processing. In our previous paper,42 we estimated that this ultrashort APPJ pro- cessing time (11 s) can render an estimated energy consump- tion per unit area of 1.1 kJ cm2, which is only one-third of that consumed in a conventional furnace calcination process. Discussion This is, therefore, a time- and energy-saving process benecial for future mass production. Moreover, APPJ treatment can easily introduce abundant hydrophilic functional groups on the surface of the carbon cloth, which are formed from the nitrogen and oxygen atoms in the atmosphere instead of exogenous chemicals.28 This indicates that APPJ is an environmentally friendly process, which is important for the growth of micro- organisms in MFCs. Compared with other graphene-based surface modication techniques, the screen-printing of rGO & APPJ treatment is time saving and cost eﬀective, consumes low energy, and is suitable for large-area processing. Therefore, the screen printing of the rGO & APPJ treatment process is prom- ising for the manufacture of large-scale MFC stacks for practical engineering applications. formation of biolms on the surface of the carbon cloth. The electrochemical measurements demonstrated that the highest power density of the MFC signicantly increased from 6.02 0.01 to 10.80 0.19 mW m2 aer the rGO & APPJ treatment. The EIS analysis showed that the total internal resistance of the MFC was signicantly reduced aer the rGO & APPJ treatment. The screen printing of rGO followed by the APPJ calcining is a clean, time-saving, and economical surface modication process. The possibility of easy large-area surface modication using both screen printing of rGO and APPJ calcining is ideal for the manufacture of large-scale MFC stacks with maximized power generation. Notes and references 1 B. Logan, B. Hamelers, R. Rozendal, U. Schr¨oder, J. Keller, 1 B. Logan, B. Hamelers, R. Rozendal, U. Schr¨oder, J. Keller, S. Freguia, P. Aelterman, W. Verstraete and K. Rabaey, Environ. Sci. Technol., 2006, 40, 5181–5192. S. Freguia, P. Aelterman, W. Verstraete and K. Rabaey, Environ. Sci. Technol., 2006, 40, 5181–5192. 2 B. E. Logan and M. Elimelech, Nature, 2012, 488, 313–319. 3 B. E. Logan and K. Rabaey, Science, 2012, 337, 686–690. 4 C. Santoro, C. Arbizzani, B. Erable and I. Ieropoulos, J. Power Sources, 2017, 356, 225–244. 5 M. Zhou, M. Chi, J. Luo, H. He and T. Jin, J. Power Sources, 2011, 196, 4427–4435. 6 P. Y. You and S. K. Kamarudin, Chem. Eng. J., 2017, 309, 489– 502. 7 Y. C. Yong, X. C. Dong, M. B. Chan-Park, H. Song and P. Chen, ACS Nano, 2012, 6, 2394–2400. 56438 \| RSC Adv., 2017, 7, 56433–56439 Discussion In this study, we combined the advantages of screen-printing of rGO and APPJ treatment to enhance the power generation of MFCs. According to the electrochemical measurements shown in Fig. 8(a), the power density generated from the MFC is approximately 80% higher aer the rGO & APPJ treatment. Further, the EIS results shown in Fig. 8(b) reveal that the total internal resistance of the MFC congured with the rGO & APPJ- treated carbon cloth electrode (972.1 U) is much lower than that of the unmodied MFC (5827.9 U). This implies that the eﬃ- ciency of the transfer of electrons from bacteria to electrodes is APPJ has been found to be highly reactive with carbonaceous materials.25,26,41–43 Reactive plasma species in nitrogen APPJ react vigorously with ethyl cellulose (EC) and rGOs. The reac- tivity is even higher with EC than with rGOs. In this reaction of nitrogen APPJ with rGO pastes that contain ECs, we can typically This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 7, 56433–56439 \| 56437 View Article Online RSC Advances Paper observe the reduction in C–O bonding contents that is contributed by ECs (as evidenced by XPS), in a shorter pro- cessing time. This reduction in C–O bonding content aer nitrogen APPJ processing is also observed in this study (Fig. 5). C–C bonding content is mainly contributed by rGOs and carbon bers of the carbon cloth. As the APPJ processing time is further increased, the rGOs are damaged and oxidized/evaporated.25,26 In this case, with rGOs on the carbon cloth, the C–C peak remains at a high level aer APPJ processing because of the presence of carbon bers (which are typically approximately 10 mm in diameter) on the carbon cloth. Using optical emission spectroscopy (OES) to control the processing time properly, we can burn ECs while retaining much of the rGOs.43 The optimal APPJ processing time is typically within 15 s. Furthermore, this APP process and screen-printing of rGO can be performed on substrates with various types of materials, including porous and solid ones. This provides high exibility in material deposition and surface modication processes. This nitrogen APPJ pro- cessing of screen-printed rGO pastes is economically favorable in terms of the thermal budget in materials processing. Conclusions 8 V. S. Sarathi and K. S. Nahm, Biosens. Bioelectron., 2013, 43, 461–475. This study investigated the surface and electrochemical prop- erties of carbon cloth electrodes surface-modied by screen printing of rGO and APPJ treatment for applications in MFCs. The SEM results showed that the screen-printed rGO was deposited on the surface of the carbon cloth as well as perco- lated into the central region, providing more surface area for the adhesion of bacteria. The subsequent APPJ calcining not only burned the organic binder in the rGO paste, but also enhanced the adhesion between rGO and the carbon cloth. The wettability measurements revealed that the carbon cloth was highly hydrophilic aer rGO & APPJ treatment. The XPS results showed the presence of abundant carbonyl, carboxyl, and ammonium hydrophilic functional groups on the surface of the rGO & APPJ- treated carbon cloth. These hydrophilic functional groups improve the biocompatibility, facilitate the transfer of electrons from the attached bacteria to the electrodes, and assist in the 9 Y. Wang, B. 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https://openalex.org/W4387403503	https://www.e3s-conferences.org/articles/e3sconf/pdf/2023/67/e3sconf_icmpc2023_01011.pdf	English	null	Power Quality Improvement and Sustainable operation in A Standalone Micro Grid by Regulating Frequency in A Deregulated Market	E3S web of conferences	2,023	cc-by	6,014	Power Quality Improvement and Sustainable operation in A Standalone Micro Grid by Regulating Frequency in A Deregulated Market Phani Kumar K.S.V.1, Vinay Kumar Awaar2,Venkateshwarlu Sonnati3, Mahammad Rafi K4, Gopal Krishna5 1,3CVR College of Engineering, Ibrahimpatnam, Hyderabad, Telangana, INDIA. 2Gokaraju Rangaraju Institute of Engineering & Technology, Hyderabad, Telangana, INDIA. 4Muffakham Jah College of Engineering and Technology, Hyderabad, Telangana, INDIA. 5Uttaranchal Institute of Technology, Uttaranchal University, Dehradun, INDIA. Abstract. Cooperative decision-making has always benefited from achieving objectives subjected to system constraints in a microgrid. This paper considers the power balance between generation and load as a significant power quality issue. Hence, frequency regulation is regarded as a primary challenge in the system. When the system operator works towards profit for the power producer, an integrated operation provides a solution. The sources considered in this system are Solar PhotoVoltaics (SPV), FuelCells (FC), Diesel Generators (DG) and Battery driven Electric Vehicles (BEV), where the BEVs operate in Vehicle microgrid mode. A central controller and local controllers are present to operate the generators at desired levels. A cascaded fuzzy controller is designed that chooses the best suitable BEV to be connected to the microgrid. The system is implemented in a MATLAB Simulation environment, and various scenarios and cases have been considered for evaluating the system response and its sustainability. Vinay Kumar Awaar: vinaykumaar.a@gmail.com , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). 1 Introduction Integrating distributed resources in the microgrid system introduces many power quality issues. System parameters like the quality of voltage supplied to the loads and the frequency response of the supply power delivery management issues must be addressed by the system operator. Various researchers have suggested an approach involving optimised control of the resources in the system and compensation techniques to overcome the issues of operation of renewable energy in the system that can be reduced. The collective operation of these resources is analysed under different economic considerations too. The participation of electric vehicles as a power source in the system has been considered in great scenarios. However, significant research is yet to be available in the context of microgrids and its sustainability. The contribution of our exchange between batteries of electric vehicles and microgrids for frequency regulation in the system is a significant consideration given the , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 increasing availability of battery-driven electric vehicles in the system. A typical 100- kilowatt pic power management system is needed in which the source of power generation includes distribution like solar wind fuel sales, diesel generators and electric vehicles. Battery-driven electric vehicles are available in the market, ready to participate in the power exchange. The microgrid scenario limits the usage of locally available vehicles. The coordination between these vehicles is implemented using a synchronous mode of operation. The coordinator approach of the vehicles during a contingency has been discussed in [6] and [7] but without integrating other resources. The load considerations in the literature are limited to the step inputs to study the study state response of the system. The real-time changes in the lords of our system are considered to be included in the study. During the load changes, the system's frequency should be regulated between 49.9 Hertz to 50.1 Hertz according to the grid code standards. In the literature, more importance should be given to these regulations and many results violate them. Electric vehicles' participation in frequency regulation in a grade scenario has long been discussed, but introducing microgrids is a new area of research. This paper must consider the scope of our transfer from the microgrid to the vehicles since it is assumed that the vehicles get charged off the grid from a source such as solar photovoltaics. 1 Introduction Many private players are coming into the market in this sector to charge electric vehicles with the solar power available. The vehicle owner can schedule the availability of the vehicle at the parking station, which is interconnected to the microgrid. Hence, the microgrid operator has information about a BEV's incoming and outgoing time [8]. The vehicles in the pool are selected based on different approaches, such as (a) equal division of the discharge rates irrespective of their battery capacity. (b) proportional division of discharge rates based on battery capacities. y p These methods are available for distributing the required power demand among electric vehicles. The state of health of the batteries shall not be considered in this case and may be considered in the future. The benefit of transferring power to the micro grade is also not considered in 9. In the first approach, the control operation of the power flow from the vehicles needs to be checked. If all the vehicles available in the microgrid deliver power, then control over the tower generation is not obtained. This can be achieved through a mechanism widely used in the literature, using a fuzzy logic controller [10]. Some authors have considered optimising operation costs to the BEV owners using a mathematical approach. Nontraditional methods like black hole algorithms are implemented in [12] and [13] for a microgrid scenario. The reserve power availability in the system needs to be discussed. The coordination between the sources needs to be considered. An aggregator-based communication is set up to use the power at BEV parking stations. The rest of the paper is organised by presenting how BEVs are included in the microgrid system in the next section. It is followed by the implementation methodology of a cascaded fuzzy controller to decide which BEV to be connected to the microgrid optimally. The following section showcases the simulation results and analysis of different scenarios and cases considered in the microgrid operation. The conclusion is presented at the end of the paper with the cited references. 2 BEV-based Frequency Regulation in a Microgrid without Storage System Hence the change in frequency signal obtained from the power system transfer function block is taken as a feedback signal and given to the local controllers through the central controller. This feedback signal obtained is scrutinised further and sent to the central controller only if a certain threshold of frequency violation is observed. This value is considered as ±0.02 Hz in this work to obtain a smooth generating unit operation. The system parameters are presented in Table 1, where M1, M2, M3, and M2G are the vehicle categories [18]-[21]. A PI controller is designed to convert the power generation signal (scheduled) to the battery discharge rate equivalent. The tuning of proportional and integral gains is done to obtain a faster response and avoid overshoots. Each power source has a controller to convert the power schedule signal to the power generation command and send it to the respective generating unit. In the case of SPV, a neuro-fuzzy-based approach is used to decide the voltage level at which the DC-DC controller should operate so that the reserve can be changed based on the requirement. FC's control unit changes the amount of fuel input, i.e. controlling the volume flow rate of hydrogen and oxygen into the chambers. For DG, a PI controller decides the amount of diesel input into the IC engine coupled with a synchronous generator. In this way, the local controllers will coordinate with the power-scheduled signal operating in an integrated way. There may be a change in load and steady-state error in the system's frequency despite the unit commitment and scheduling of the power from the central controller. Hence the change in frequency signal obtained from the power system transfer function block is taken as a feedback signal and given to the local controllers through the central controller. This feedback signal obtained is scrutinised further and sent to the central controller only if a certain threshold of frequency violation is observed. This value is considered as ±0.02 Hz in this work to obtain a smooth generating unit operation. The system parameters are presented in Table 1, where M1, M2, M3, and M2G are the vehicle categories [18]-[21]. Table 1. Distributed Generation Source Parameters considered. Table 1. Distributed Generation Source Parameters considered. 2 BEV-based Frequency Regulation in a Microgrid without Storage System A distributed generation system needs an efficient management scheme so that the interlinked parameters can be optimally controlled under the given constraints. The primary concern of 2 2 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 the system operator is to increase profits while effectively utilising available resources. A storage system (usually a battery) increases the system's reliability. A few drawbacks have been identified in the system considered. the system operator is to increase profits while effectively utilising available resources. A storage system (usually a battery) increases the system's reliability. A few drawbacks have been identified in the system considered. They are: They are: y • The solar power reserve shall only be available during the daytime, while this facility is unavailable at night. • The solar power reserve shall only be available during the daytime, while this facility is unavailable at night. • The reserve power obtained from solar is not used during the off-peak load period, which brings loss to the operator or a burden to other generators. • The reserve power obtained from solar is not used during the off-peak load period, which brings loss to the operator or a burden to other generators. • The electric vehicles in the system, which can exchange power with the microgrids, are not considered here. • The electric vehicles in the system, which can exchange power with the microgrids, are not considered here. The microgrid considered in the paper consists of SPV, FC, DG and BEVs. The system includes a central controller to do the power scheduling operation. Depending on the load changes and the frequency deviations occurring in the system, each distributed power generator must adjust its power output. A local controller placed near the generator site does this power output adjustment. A typical PI controller converts the power change signal to respective parameter changes depending on the source. The modelling of different sources and loads is mentioned in previous works [18]. This paper contributes by addressing issues like managing the fleet of electric vehicles and coordinating with other generating sources in the microgrid. A group of BEVs send information such as SoC and the battery's discharge rate to the BEVs' aggregator. This aggregator analyses the information and sends data to the central controller regarding the current power supply and the total capacity of the fleet of BEVs connected to the microgrid. 2 BEV-based Frequency Regulation in a Microgrid without Storage System Depending on the load condition, the central controller sends a signal to all the resources regarding the power schedule for that time. The microgrid considered in the paper consists of SPV, FC, DG and BEVs. The system includes a central controller to do the power scheduling operation. Depending on the load changes and the frequency deviations occurring in the system, each distributed power generator must adjust its power output. A local controller placed near the generator site does this power output adjustment. A typical PI controller converts the power change signal to respective parameter changes depending on the source. The modelling of different sources and loads is mentioned in previous works [18]. This paper contributes by addressing issues like managing the fleet of electric vehicles and coordinating with other generating sources in the microgrid. A group of BEVs send information such as SoC and the battery's discharge rate to the BEVs' aggregator. This aggregator analyses the information and sends data to the central controller regarding the current power supply and the total capacity of the fleet of BEVs connected to the microgrid. Depending on the load condition, the central controller sends a signal to all the resources regarding the power schedule for that time. A PI controller is designed to convert the power generation signal (scheduled) to the battery discharge rate equivalent. The tuning of proportional and integral gains is done to obtain a faster response and avoid overshoots. Each power source has a controller to convert the power schedule signal to the power generation command and send it to the respective generating unit. In the case of SPV, a neuro-fuzzy-based approach is used to decide the voltage level at which the DC-DC controller should operate so that the reserve can be changed based on the requirement. FC's control unit changes the amount of fuel input, i.e. controlling the volume flow rate of hydrogen and oxygen into the chambers. For DG, a PI controller decides the amount of diesel input into the IC engine coupled with a synchronous generator. In this way, the local controllers will coordinate with the power-scheduled signal operating in an integrated way. There may be a change in load and steady-state error in the system's frequency despite the unit commitment and scheduling of the power from the central controller. 2 BEV-based Frequency Regulation in a Microgrid without Storage System DG FC SPV M1 M2 M2G M3 M3 Total Minimum power (pu) 0.1 0.1 0 0 0 0 0 0 0.2 Maximum power (pu) 0.3 0.3 0.29 0.1 0.125 0.1625 0.3 0.3125 1.89 3 3 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 Table 2. Battery Electric Vehicle Parameters considered. VT NS NP SOCm in (%) NC (Ah) TC (ms) SOCIn (%) DT(s) Power (W) Power @ 0.9C (pu) M1 15 15 20 5 25 66 20 800 0.1 M2 19 19 20 5 25 89 60 1000 0.125 M2 G 22 15 20 4 25 74 0 1300 0.1625 M3 40 40 20 5 25 78 0 2400 0.3 M3 42 42 20 5 25 42 70 2500 0.3125 Total 8000 1 Distributed Generation Source Parameters considered are shown in Table 2, with the keywords mentioned as Vehicle Type (VT), No of cells in series (NS), No of cells in Parallel (NP), SoC Minimum value set (SOCmin) in %, Nominal Capacity (NC) in Ah, Time Constant (TC) in ms, InitialSOC (SOCIn) in %, Dead Time (DT) in s, Power in Watts, Power @ 0.9C in pu. Based on the inp t signal recei ed from the central controller local controllers shall VT NS NP SOCm in (%) NC (Ah) TC (ms) SOCIn (%) DT(s) Power (W) Power @ 0.9C (pu) M1 15 15 20 5 25 66 20 800 0.1 M2 19 19 20 5 25 89 60 1000 0.125 M2 G 22 15 20 4 25 74 0 1300 0.1625 M3 40 40 20 5 25 78 0 2400 0.3 M3 42 42 20 5 25 42 70 2500 0.3125 Total 8000 1 Distributed Generation Source Parameters considered are shown in Table 2, with the keywords mentioned as Vehicle Type (VT), No of cells in series (NS), No of cells in Parallel (NP), SoC Minimum value set (SOCmin) in %, Nominal Capacity (NC) in Ah, Time Constant (TC) in ms, InitialSOC (SOCIn) in %, Dead Time (DT) in s, Power in Watts, Power @ 0.9C in pu. 2 BEV-based Frequency Regulation in a Microgrid without Storage System Distributed Generation Source Parameters considered are shown in Table 2, with the keywords mentioned as Vehicle Type (VT), No of cells in series (NS), No of cells in Parallel (NP), SoC Minimum value set (SOCmin) in %, Nominal Capacity (NC) in Ah, Time Constant (TC) in ms, InitialSOC (SOCIn) in %, Dead Time (DT) in s, Power in Watts, Power @ 0.9C in pu. @ p Based on the input signal received from the central controller, local controllers shall ensure power generation from the units committed. The SPV, FC and DGs used in the simulation work are lumped units; hence, power obtained as an output is the aggregated value, and the central controller does not have individual access to the internal units. This is considered a drawback in the work done by [18]. Hence in the proposed work, the additionally included power source of BEV is designed so that individual units, i.e. each vehicle battery, can be connected or disconnected from the microgrid based on the power schedule requirement. However, this scheme is introduced in later stages of the work. Initially, the power from BEVs is lumped for ease of control. Every vehicle owner can decide whether to connect their battery to the microgrid. This decision introduces various scenarios of simulation that are possible. Every possible scenario should be considered to prove the feasibility of the study. Hence in this paper, there are three scenarios considered. The following aspects are included in the scenarios—load variations for every 60 seconds. Vehicle SoC variations are considered due to the different driving cycle patterns of the owners—extreme loading conditions. g The main challenge here is maintaining the system’s frequency within the operating limits. The additive adaptive algorithm needs to continuously check the frequency change in the system and use the priority-based technique to commit the units and schedule the power. Different from traditional generators, there is little choice in the selection of generators since the availability of the source decides the priority. The load is categorised as critical and non- critical. The critical loads include computers, medical equipment, street lighting, etc., accounting for 0.1pu of the total load. The maximum load on the system is expected to be 150kW. The base value of power generation is 100kW, and in this study, it is assumed that the maximum load is 1.55pu levied on the system. 2 BEV-based Frequency Regulation in a Microgrid without Storage System In contrast, the total generation capacity of the system is 1.79pu, which includes BEVs. The maximum generation capabilities of SPV are 0.29pu, while the reserve considered here is 10% and hence the usual operating point would be 0.26pu. Figure 1 shows the simulation environment considered. Three blocks highlighted with the star mark correspond to the contributions done in this work, while the others are taken from various references. Four scenarios have been considered with the following parameters. The main challenge here is maintaining the system’s frequency within the operating limits. The additive adaptive algorithm needs to continuously check the frequency change in the system and use the priority-based technique to commit the units and schedule the power. Different from traditional generators, there is little choice in the selection of generators since the availability of the source decides the priority. The load is categorised as critical and non- critical. The critical loads include computers, medical equipment, street lighting, etc., accounting for 0.1pu of the total load. The maximum load on the system is expected to be 150kW. The base value of power generation is 100kW, and in this study, it is assumed that the maximum load is 1.55pu levied on the system. In contrast, the total generation capacity of the system is 1.79pu, which includes BEVs. The maximum generation capabilities of SPV are 0.29pu, while the reserve considered here is 10% and hence the usual operating point would be 0.26pu. Figure 1 shows the simulation environment considered. Three blocks highlighted with the star mark correspond to the contributions done in this work, while the others are taken from various references. Four scenarios have been considered with the following parameters. 4 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 • Scenario-1 Step load change • Scenario-2 Load considered varies from 1.2pu to 1.35 pu and EV power output var i h l d • Scenario-2 Load considered varies from 1.2pu to 1.35 pu and EV power output varies with load • Scenario-3 Extreme load conditions are considered and EVs taken out based on SoC • Scenario-3 Extreme load conditions are considered and EVs taken out base • Scenario–4 SPV Power variation • Scenario–4 SPV Power variation In this [18] work carried out; certain aspects need to be considered, affecting the real- time implementation of the proposed concept. 3 Simulation Results The variable parameters identified in the simulation are related to the distributed sources considered for power generation and the load. As the load changes insteps, the generation tries to adjust to maintain the power balance. These BEVs are assumed to be parked in two parking lots and ready to supply power to the microgrid. The amount of power that can be pumped into the microgrid from a source is limited based on the current ratings of the infrastructure available. The variable parameters identified with the BEVs are listed below. 1. The availability of the vehicle during the simulation period 1. The availability of the vehicle during the simulation period 2. SoC of the vehicle battery 3. Cost of power exchange Different scenarios are considered in the paper while increasing the complexity of variable changes in the system. The central controller continuously monitors the frequency in the system, and the frequency deviation is communicated to the sources if the deviation is beyond a threshold value of ±0.02Hz. The limits considered in the paper for maximum frequency deviation are ±0.05 Hz, and the fundamental frequency considered is 50Hz. The system is considered to have low inertia since there is only one synchronous generator, i.e., DG. The communication between the local controllers at the generation, central controller and load is assumed to be based on fibre optic communication since it is the most reliable, fastest means of data transfer available and that is currently being used. During the result analysis, the focus shall be on the allotment of an available BEV to supply power to the microgrid and the coordinated approach by all the generators to respond to the load changes in the system. Other simulation aspects are mentioned in [19], and this paper extends the previous works. The scenario is considered to check whether the fuzzy logic controller designed can select the best suitable BEV to be connected to the microgrid. This is based on the current SoC of the battery, the discharge rate demand by the central controller and the selling cost chosen by the owner of BEV. For simplicity, the load assumed is constant throughout the simulation time. Two cases are considered in the scenario. 2 BEV-based Frequency Regulation in a Microgrid without Storage System The daily power consumption/load curves provided by the load dispatch centres show that the variation in the load values is continuous. p y p The average load demand curve is found by removing the higher order frequency terms to get a smooth waveform. Inspired by this, the traditional step change signal applied for the small signal stability analysis of the power system is mixed with another high-frequency signal to resemble the daily load curve variations. Private people own the BEVs, and if the owner wills to participate in the V2mG energy transfer, there should be a common place where this connection facility (infrastructure) is available. Hence in this study, two parking stations are considered, where the vehicle owners can park their vehicles and connect to the microgrid, as shown in Figure 2. Figure 3 showcases the cascaded fuzzy logic controller proposed and used in the Electric vehicle selection. Based on scheduling, the parking lots get a signal to generate power, which is equally distributed to BEVs. Suppose total vehicles are connected to the microgrid. In that case, the infrastructure shall fail since the power transfer capability of parking lots has a maximum value, limitation of the number of vehicles that can simultaneously pump power can be regulated to five. Fig. 1. Simulation model developed. 5 Fig 1 Simulation model developed Fig. 1. Simulation model developed. Fig. 1. Simulation model developed. 5 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 Fig. 2. Simulation model developed. Fig. 3. Cascaded fuzzy-logic block diagram representation. Fig. 2. Simulation model developed. Fig. 2. Simulation model developed. Fig. 3. Cascaded fuzzy-logic block diagram representation. 3.1.1 Case-1 In this case, the randomly chosen SoC levels of the BEVs and different selling costs of the BEV energy are given in Table 3. The load considered in this case is only 0.75pu. The system's frequency response is shown in Fig. 4. Since the system starts from an initial condition of zero, the initial frequency response of the system can be neglected. It can be observed that the frequency is within the limits of ±0.05 Hz and the percentage deviation 6 6 6 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 during the overall simulation time of 480s is less than 1%. The overall share of each generator during the simulation time is given in Fig. 5. At time t=180s, the load of 0.75pu is divided by the central controller among the sources, as shown in Table 4, as per the priority-based algorithm. The load to be shared by the BEVs together is 0.315 pu divided among the BEVs, as shown in Fig. 6. The first fuzzy logic controller gives a priority number based on the current SoC of the battery and the current discharge rate demand. This priority number is sent as input to the second fuzzy controller, which decides the final ranking of the BEVs based on the cost. Ranking of BEVs is done on a scale of 0 to 10. The minor scale value corresponds to a higher ranking. The local controller then performs a sorting operation to list the BEVs in ascending order. Based on the total power to be shared by all the BEVs in parking lots, vehicles having a higher ranking will be connected to the microgrid. The BEV selection for the constant load considered is given in Fig.7 and the ranking of BEVs is shown in Fig.8. Fig. 4. Frequency error obtained for case 1. Fig. 5. Power generation done by each generator. Fig. 4. Frequency error obtained for case 1. Fig. 4. Frequency error obtained for case 1. Fig. 5. Power generation done by each generator. 7 7 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 Fig. 6. Load sharing by BEVs in parking lots for case-1. Fig. 7. Selection of BEVs in parking lots based on fuzzy controllers for case-1. Fig. 6. Load sharing by BEVs in parking lots for case-1. Fig. 7. 3.1.1 Case-1 Load sharing by all the generators for case-2. 2. Fig. 10. Load sharing by BEVs in parking lots for case-2. Fig. 10. Load sharing by BEVs in parking lots for case-2. 3.1.1 Case-1 Selection of BEVs in parking lots based on fuzzy controllers for case-1. Table 3. Selling prices considered for the vehicle to microgrid energy transfer. Parking Lot No. 1 Parking Lot No. 2 EV No: SoC Price (Rs.) EV No: SoC Price (Rs.) 1 50 5.025 6 50 5.04 2 50 1.675 7 50 5.574 3 50 3.35 8 50 4.884 4 50 4.623 9 50 1.524 Fig. 6. Load sharing by BEVs in parking lots for case-1. Fig. 6. Load sharing by BEVs in parking lots for case-1. Fig. 7. Selection of BEVs in parking lots based on fuzzy controllers for case-1. ase 1. Fig. 7. Selection of BEVs in parking lots based on fuzzy controllers for case-1. Table 3. Selling prices considered for the vehicle to microgrid energy transfer. Table 3. Selling prices considered for the vehicle to microgrid energy transfer. Table 3. Selling prices considered for the vehicle to microgrid energy transfer. Table 3. Selling prices considered for the vehicle to microgrid energy transfer. Parking Lot No. 1 Parking Lot No. 2 EV No: SoC Price (Rs.) EV No: SoC Price (Rs.) 1 50 5.025 6 50 5.04 2 50 1.675 7 50 5.574 3 50 3.35 8 50 4.884 4 50 4.623 9 50 1.524 8 , 010 (2023) E3S Web of Conferences 11 430 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 5 50 5.963 10 50 1.458 3.1.2 Case-2 3.1.2 Case-2 The load considered in this case is 0.95 pu which is close to the base value of the power demand expected. This value is chosen to test the ability of fuzzy controllers. The overall power-sharing by all the generators is given in Fig. 9. The BEVs generate enough power to keep the frequency within limits, as shown in Fig. 10. Here, the load share of BEVs is now 0.335pu. The sorted ranking order for this load share is shown in Fig. 11. The discharge rate requirement, in this case, is higher than the previous one. Hence the priority has changed. The frequency regulation, in this case, is shown in Fig. 12. g Fig. 9. Load sharing by all the generators for case-2. Fig. 10. Load sharing by BEVs in parking lots for case-2. Fig. 9. Load sharing by all the generators for case-2. Fig. 9. Load sharing by all the generators for case-2. Fig. 9. 9 9 , 010 (2023) E3S Web of Conferences ICMPC 2023 11 430 https://doi.org/10.1051/e3sconf/202343001011 Fig. 12. Selection of BEVs in parking lots based on fuzzy controllers for case-2. Fig. 13. Frequency error obtained for case-2. Fig. 12. Selection of BEVs in parking lots based on fuzzy controllers for case-2. Fig. 12. Selection of BEVs in parking lots based on fuzzy controllers for case-2. g p g y Fig. 13. Frequency error obtained for case-2. Fig. 13. Frequency error obtained for case-2. Conclusions The variable parameters in a deregulated power system where renewable sources are used for power generation, the system operation and power quality of supply in an autonomously operating microgrid with sustainability are maintained. Distributed resources are operated together to maintain a balance between generated powers to maintain power quality (frequency regulation) and satisfy dynamic requests of loads (active) to improve reliability (for sensitive/critical loads) and sustainability. A control strategy that can address the needs of every generating source and coordinate well with other parts of the system has been developed. 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https://openalex.org/W4394759575	https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1367734/pdf	English	null	Role of aryl hydrocarbon receptors in infection and inflammation	Frontiers in immunology	2,024	cc-by	15,487	OPEN ACCESS OPEN ACCESS EDITED BY Esaki M. Shankar, Central University of Tamil Nadu, India REVIEWED BY Debora Decote-Ricardo, Federal Rural University of Rio de Janeiro, Brazil Danielle Oliveira Nascimento, Federal Rural University of Rio de Janeiro, Brazil CORRESPONDENCE Weihua Nong nongweihua666@163.com Ranhui Li ranhui81@163.com †These authors have contributed equally to this work RECEIVED 09 January 2024 ACCEPTED 02 April 2024 PUBLISHED 12 April 2024 CITATION Xu L, Lin L, Xie N, Chen W, Nong W and Li R (2024) Role of aryl hydrocarbon receptors in infection and inﬂammation. Front. Immunol. 15:1367734. doi: 10.3389/fimmu.2024.1367734 COPYRIGHT © 2024 Xu, Lin, Xie, Chen, Nong and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. EDITED BY Esaki M. Shankar, Central University of Tamil Nadu, India REVIEWED BY Debora Decote-Ricardo, Federal Rural University of Rio de Janeiro, Brazil Danielle Oliveira Nascimento, Federal Rural University of Rio de Janeiro, Brazil CORRESPONDENCE Weihua Nong nongweihua666@163.com Ranhui Li ranhui81@163.com †These authors have contributed equally to this work RECEIVED 09 January 2024 ACCEPTED 02 April 2024 PUBLISHED 12 April 2024 CITATION Xu L, Lin L, Xie N, Chen W, Nong W and Li R (2024) Role of aryl hydrocarbon receptors in infection and inﬂammation. Front. Immunol. 15:1367734. doi: 10.3389/fimmu.2024.1367734 COPYRIGHT © 2024 Xu, Lin, Xie, Chen, Nong and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. EDITED BY Esaki M. TYPE Review PUBLISHED 12 April 2024 DOI 10.3389/fimmu.2024.1367734 TYPE Review PUBLISHED 12 April 2024 DOI 10.3389/fimmu.2024.1367734 KEYWORDS AhR, viruses, bacteria, parasites, fungus AhR, viruses, bacteria, parasites, fungus AhR, viruses, bacteria, parasites, fungus Introduction modulate the vasoactive apelin-APJ peptide system4, thereby providing prophylactic protection against dysplastic and apoptotic responses in respiratory epithelial cells, ultimately safeguarding the integrity of the lung barrier (16). The role of AhR in toxicology has been recognized throughout history, but it has been increasingly recognized as an important disease modulator in recent decades. AhR is reported to be associated with many diseases driven by immune and inﬂammatory processes. This review mainly discusses the role of AhR in inﬂammation and infection. We will also discuss the mechanisms of widespread involvement of AhR during infection of pathogens, including viruses, bacteria, fungi, and parasites, which can be retrieved until now. The aryl hydrocarbon receptor (AhR) belongs to the basic helix- loop-helix (bHLH) family and functions as a transcription factor that is activated through binding with its cognate ligand. This activation is involved in regulating host immune homeostasis and metabolism (1). Since its discovery by Alan Poland in the mid- 1970s, the AhR signaling pathway has been extensively studied in various ﬁelds, including biology, immunology, and pathophysiology (2). The AhR protein is able to interact with numerous endogenous and exogenous ligands. Exogenous ligands, such as 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) and various viruses, can activate the AhR signaling pathway in host cells (3–5). Endogenous AhR ligands, such as Kynurenine (Kyn) and arachidonic acid, induce the translocation of AhR from the cytoplasmic matrix to the nucleus, thereby regulating the transcription of various target genes (6). Activation of AhR leads to the upregulation of cytochrome P450 enzymes, including CYP1A1 and CYP1B1, which play a crucial role in facilitating detoxiﬁcation and drug metabolism (2). In the context of host- microbe interactions, AhR has the ability to collaborate with various signaling pathways, including signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-kb), epidermal growth factor receptor (EGFR), and hypoxia-inducible factor-1a (HIF-1a) signaling pathways (7). In parallel with the signiﬁcant advancement of the AhR signaling pathway, there has been a gradual improvement in several hypotheses concerning microbial infection. The role of AhR extends beyond its involvement in pathogen infection, encompassing immune system regulation such as the promotion of Treg cell generation (8), mediation of autophagy-related neurotoxicity (9), regulation of circadian rhythm, and initiation of carcinogenesis processes (7, 10). However, the precise mechanisms underlying these phenomena require further investigation in future research. OPEN ACCESS Shankar, Central University of Tamil Nadu, India Linglan Xu 1,2†, Luping Lin 1†, Nan Xie 2, Weiwei Chen 2, Weihua Nong 1* and Ranhui Li 2,3* REVIEWED BY Debora Decote-Ricardo, Federal Rural University of Rio de Janeiro, Brazil Danielle Oliveira Nascimento, Federal Rural University of Rio de Janeiro, Brazil 1Key Laboratory of Research on Clinical Molecular Diagnosis for High Incidence Diseases in Western Guangxi, Department of Obstetrics and Gynecology, Afﬁliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China, 2Hunan Provincial Key Laboratory for Special Pathogens Prevention and Control, Institute of Pathogenic Biology, Hengyang Medical School, University of South China, Hengyang, China, 3Hunan Prevention and Treatment Institute for Occupational Diseases and Afﬁliated Prevention and Treatment Institute for Occupational Diseases, University of South China, Changsha, China *CORRESPONDENCE Weihua Nong nongweihua666@163.com Ranhui Li ranhui81@163.com The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by various ligands, including pollutants, microorganisms, and metabolic substances. It is expressed extensively in pulmonary and intestinal epithelial cells, where it contributes to barrier defense. The expression of AhR is pivotal in regulating the inﬂammatory response to microorganisms. However, dysregulated AhR expression can result in endocrine disorders, leading to immunotoxicity and potentially promoting the development of carcinoma. This review focuses on the crucial role of the AhR in facilitating and limiting the proliferation of pathogens, speciﬁcally in relation to the host cell type and the species of etiological agents involved in microbial pathogen infections. The activation of AhR is enhanced through the IDO1-AhR-IDO1 positive feedback loop, which is manipulated by viruses. AhR primarily promotes the infection of SARS-CoV-2 by inducing the expression of angiotensin-converting enzyme 2 (ACE2) and the secretion of pro- inﬂammatory cytokines. AhR also plays a signiﬁcant role in regulating various types of T-cells, including CD4+ T cells and CD8+ T cells, in the context of pulmonary infections. The AhR pathway plays a crucial role in regulating immune responses within the respiratory and intestinal barriers when they are invaded by viruses, bacteria, parasites, and fungi. Additionally, we propose that targeting the agonist and antagonist of AhR signaling pathways could serve as a promising therapeutic approach for combating pathogen infections, especially in light of the growing prevalence of drug resistance to multiple antibiotics. 01 Frontiers in Immunology frontiersin.org 10.3389/fimmu.2024.1367734 Xu et al. Introduction Additionally, existing research indicates that the molecular chaperone heat- shock protein 90 (HSP90) plays a role in the activation of the AhR by forming a complex with co-chaperones HSP90, which then translocates to the nucleus (11). When AhR is inactive, HSP90, AhR, co-chaperone p23 (p23), and the hepatitis B virus X- associated protein (XAP2) bind together to form a stable cytoplasmic complex (12). This multiprotein complex is believed to enhance the structural stability of AhR and contribute to its subcellular localization. Furthermore, the examination of the antagonist and agonist of AhR has brought about promising opportunities for development, leading to an accumulation of research in this area. Numerous prevalent nutraceuticals contain AhR inhibitor components, including folate, vitamin B12, and curcumin, which have the potential to regulate viral pathophysiology (17). The effective utilization of AhR antagonists could signiﬁcantly address the issue of multi-drug resistance in viral infections in the foreseeable future. This review aims to summarize the signiﬁcant role of AhR in invasive agent infections and its potential as a novel therapeutic target for microbial infections. Frontiers in Immunology AhR and coronavirus infections that further investigation into the role of AhR is warranted in order to identify potential therapeutic strategies for managing SARS- CoV-2 infection. The disease known as Post-Acute Sequelae of SARS-CoV-2 infection (PAS) arises from the viral infection of SARS-CoV-2 and is characterized by persistent and severe acute respiratory symptoms lasting for a duration of three months or longer (26). Recent research ﬁndings suggest that the brain tissue of patients with PASC exhibits a signiﬁcant expression of indoleamine 2, 3- dioxygenase-2 (IDO2), which plays a role in modulating SARS- CoV-2 infection through autophagy and apoptosis (27). This modulation could potentially be alleviated by the administration of an AhR antagonist. It is worth noting that the activation and expression of IDO-2 are triggered by the metabolites of tryptophan, speciﬁcally the Kynurenine generated by IDO-1. The Kynurenine/ AhR/IDO-2 axis is implicated in the early initiation of severe SARS- CoV-2 viral infection, whereby the activation of IDO-2 and AHR leads to cellular responses such as apoptosis and autophagy in individuals with severe COVID-19 (28). Additionally, the SARS- CoV-2 viral infection has the ability to activate AhR through various mechanisms, including the induction of indole 3 pyruvate (I3P) by interleukin (IL) 4-inducible1 (IL41), resulting in a shift in the immunological mechanisms involved in SARS-CoV-2 infection (Figure 1). The increase in Kynurenine leads to a decrease in serotonin, which is synthesized from tryptophan and plays a crucial role in the melatonergic pathway in COVID-19. The main mechanism of SARS-CoV-2 infection involves the AhR-induced The SARS-CoV2 virus gains entry into host cells by means of the interaction between spike glycoprotein and angiotensin- converting enzyme 2 (ACE2), resulting in various respiratory symptoms (21). The activation of AhR can facilitate the infection of SARS-CoV-2 by upregulating ACE2 expression, in conjunction with an increase in viral nuclear protein (NP) expression (22). The administration of 6-formylindolo (3,2-b) carbazole (FICZ) and omeprazole (OMP), which are agonists of AhR, reveals that AhR activation can decrease ACE2 expression, thereby inhibiting SARS- CoV-2 infection (23). According to research ﬁndings, pelargonidin, a natural ﬂavonoid, is capable of reducing the expression of ACE2 in colon inﬂammation through an AhR-dependent mechanism. Through its binding to a fatty acid binding pocket in the receptor binding region of the SARS-CoV-2 spike protein, pelargonidin hinders the interaction between SARS-CoV-2 and ACE2, thereby inhibiting virus proliferation. These conclusions are based on molecular docking studies (24). AhR and coronavirus infections Coronaviruses (CoVs) are a group of positive sense single- stranded RNA viruses capable of infecting a wide range of hosts. Among them, Coronavirus 2 (SARS-CoV-2) is the causative agent of Corona Virus Disease (COVID) 19, a severe acute respiratory syndrome that emerged in China in 2019 (18). Inhibition of the AhR has been found to reduce the proliferation of both HCoV-229E (human Corona Virus 229E, a type of Corona Virus that is less pathogenic and usually causes respiratory symptoms) and SARS- CoV-2, suggesting that AhR activation serves as a mechanism for coronaviruses to evade the immune response and promote viral replication (3). The bioﬂavonoid dihydroxyﬂavone pinostrobin (PSB) functions as a negative modulator in the AhR/CYP1A1 signaling pathway by inﬂuencing the catabolic pathways of linoleic and arachidonic acid, thereby exhibiting antiviral effects (19). It has been observed that various coronaviruses can activate AhR signaling in hosts, leading to an increase in the expression of AhR-pathway genes in patients infected with SARS-CoV-122 (4). The activation of the AhR induced by coronaviruses leads to the up- regulation of downstream AhR elements, resulting in the manifestation of “Systemic AhR Activation Syndrome” (SAAS), which includes symptoms such as inﬂammation, ﬁbrosis, thromboembolism, and potentially multiple organ dysfunction syndrome or mortality (20). While many mechanisms of coronavirus infection have been extensively studied, this discussion primarily focuses on the interplay between SARS-CoV- 2 infection and the AhR signaling pathway. By characterizing the Increasing evidence suggests that the AhR plays a crucial role in the invasion of microorganisms and the regulation of both innate and acquired immune responses to various microbial infections. AhR can regulate the expression of pro-inﬂammatory cytokines, including IFN-g, TNF-a, and various interleukins. The enzyme indoleamine 2,3-dioxygenase-1 (IDO-1), involved in tryptophan metabolism, can be stimulated by inﬂammatory cytokines such as IFN-g, leading to the production of kynurenine (Kyn) and promoting the antiviral activity of AhR (13, 14). A previous study conducted by Drozdzik et al. has demonstrated that IL-1 and TNF- a have the ability to regulate the functional expression of AhR in a human salivary cell line (15). Furthermore, AhR has been found to Frontiers in Immunology 02 frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 mechanisms underlying SARS-CoV-2 pathogenesis and the interaction between the host immune system and the pathogen, we can facilitate future advancements in therapeutic approaches. AhR and coronavirus infections The SARS-CoV-2-induced “cytokine storm” leads to an upregulation of pro-inﬂammatory cytokines, which in turn promotes Kyn production through the enzyme indoleamine 2,3- dioxygenase (IDO). Consequently, this indirect effect results in a reduction in melatonin production. Activation of the AhR enhances the induction of cytochrome P450 1B1 (CYP1B1), which in turn facilitates the conversion of melatonin to N-acetylserotonin (NAS). However, during SARS-CoV-2 infection, the levels of butyrate are suppressed due to the inhibition of butyrate’s histone deacetylase (HDAC) activity. Butyrate exerts its effects by reducing the activity of the pyruvate dehydrogenase complex (PDC), leading to the conversion of pyruvate to acetyl-CoA, which serves as a co- factor in the melatonergic pathway. According to research ﬁndings, it has been observed that vitamin D has the ability to up-regulate the expression of tryptophan hydroxylase (TPH) 2, resulting in an increased production of tryptophan (Try) and subsequently leading to an elevation in serotonin synthesis. Additionally, melatonin has the capacity to bind to the vitamin D receptor and subsequently enhance the transcriptional activity mediated by vitamin D. It is important to note that the red arrow signiﬁes a positive effect, whereas the green arrow represents a negative effect. FIGURE 1 The present study shows the interaction between tryptophan metabolites and the AhR in the context of SARS-CoV-2 infection. The accompanying illustration illustrates the mechanism by which the SARS-CoV-2 virus modulates tryptophan metabolism to enhance the production of AhR ligands, namely kynurenine (Kyn), indole 3 pyruvate (I3P), and serotonin. Notably, Kyn and I3P serve as ligands for AhR. The SARS-CoV-2-induced “cytokine storm” leads to an upregulation of pro-inﬂammatory cytokines, which in turn promotes Kyn production through the enzyme indoleamine 2,3- dioxygenase (IDO). Consequently, this indirect effect results in a reduction in melatonin production. Activation of the AhR enhances the induction of cytochrome P450 1B1 (CYP1B1), which in turn facilitates the conversion of melatonin to N-acetylserotonin (NAS). However, during SARS-CoV-2 infection, the levels of butyrate are suppressed due to the inhibition of butyrate’s histone deacetylase (HDAC) activity. Butyrate exerts its effects by reducing the activity of the pyruvate dehydrogenase complex (PDC), leading to the conversion of pyruvate to acetyl-CoA, which serves as a co- factor in the melatonergic pathway. AhR and coronavirus infections Additionally, the study suggests that cigarette smoke condensates (CSC) may play a role in the regulation of ACE2 and transmembrane serine protease 2 (TMPRSS2) expression, potentially leading to SARS-CoV-2 infection by activating the AhR signaling pathway in gingival epithelial cells. It appears that CSC treatment may enhance the internalization of SARS-CoV-2 pseudovirus, as ACE2 is known to inﬂuence this process (25). In conclusion, these ﬁndings indicate FIGURE 1 The present study shows the interaction between tryptophan metabolites and the AhR in the context of SARS-CoV-2 infection. The accompanying illustration illustrates the mechanism by which the SARS-CoV-2 virus modulates tryptophan metabolism to enhance the production of AhR ligands, namely kynurenine (Kyn), indole 3 pyruvate (I3P), and serotonin. Notably, Kyn and I3P serve as ligands for AhR. The SARS-CoV-2-induced “cytokine storm” leads to an upregulation of pro-inﬂammatory cytokines, which in turn promotes Kyn production through the enzyme indoleamine 2,3- dioxygenase (IDO). Consequently, this indirect effect results in a reduction in melatonin production. Activation of the AhR enhances the induction of cytochrome P450 1B1 (CYP1B1), which in turn facilitates the conversion of melatonin to N-acetylserotonin (NAS). However, during SARS-CoV-2 infection, the levels of butyrate are suppressed due to the inhibition of butyrate’s histone deacetylase (HDAC) activity. Butyrate exerts its effects by reducing the activity of the pyruvate dehydrogenase complex (PDC), leading to the conversion of pyruvate to acetyl-CoA, which serves as a co- factor in the melatonergic pathway. According to research ﬁndings, it has been observed that vitamin D has the ability to up-regulate the expression of tryptophan hydroxylase (TPH) 2, resulting in an increased production of tryptophan (Try) and subsequently leading to an elevation in serotonin synthesis. Additionally, melatonin has the capacity to bind to the vitamin D receptor and subsequently enhance the transcriptional activity mediated by vitamin D. It is important to note that the red arrow signiﬁes a positive effect, whereas the green arrow represents a negative effect. FIGURE 1 The present study shows the interaction between tryptophan metabolites and the AhR in the context of SARS-CoV-2 infection. The accompanying illustration illustrates the mechanism by which the SARS-CoV-2 virus modulates tryptophan metabolism to enhance the production of AhR ligands, namely kynurenine (Kyn), indole 3 pyruvate (I3P), and serotonin. Notably, Kyn and I3P serve as ligands for AhR. FIGURE 1 The present study shows the interaction between tryptophan metabolites and the AhR in the context of SARS-CoV-2 infection. The accompanying illustration illustrates the mechanism by which the SARS-CoV-2 virus modulates tryptophan metabolism to enhance the production of AhR ligands, namely kynurenine (Kyn), indole 3 pyruvate (I3P), and serotonin. Notably, Kyn and I3P serve as ligands for AhR. The SARS-CoV-2-induced “cytokine storm” leads to an upregulation of pro-inﬂammatory cytokines, which in turn promotes Kyn production through the enzyme indoleamine 2,3- dioxygenase (IDO). Consequently, this indirect effect results in a reduction in melatonin production. Activation of the AhR enhances the induction of cytochrome P450 1B1 (CYP1B1), which in turn facilitates the conversion of melatonin to N-acetylserotonin (NAS). However, during SARS-CoV-2 infection, the levels of butyrate are suppressed due to the inhibition of butyrate’s histone deacetylase (HDAC) activity. Butyrate exerts its effects by reducing the activity of the pyruvate dehydrogenase complex (PDC), leading to the conversion of pyruvate to acetyl-CoA, which serves as a co- factor in the melatonergic pathway. According to research ﬁndings, it has been observed that vitamin D has the ability to up-regulate the expression of tryptophan hydroxylase (TPH) 2, resulting in an increased production of tryptophan (Try) and subsequently leading to an elevation in serotonin synthesis. Additionally, melatonin has the capacity to bind to the vitamin D receptor and subsequently enhance the transcriptional activity mediated by vitamin D. It is important to note that the red arrow signiﬁes a positive effect, whereas the green arrow represents a negative effect. AhR and coronavirus infections According to research ﬁndings, it has been observed that vitamin D has the ability to up-regulate the expression of tryptophan hydroxylase (TPH) 2, resulting in an increased production of tryptophan (Try) and subsequently leading to an elevation in serotonin synthesis. Additionally, melatonin has the capacity to bind to the vitamin D receptor and subsequently enhance the transcriptional activity mediated by vitamin D. It is important to note that the red arrow signiﬁes a positive effect, whereas the green arrow represents a negative effect. 03 Frontiers in Immunology frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 demonstrated that EBV possesses the capability to induce human lymphomas and various carcinomas, including gastric cancer (GC) (34). In EBV-positive gastric carcinoma (GC) cells, it has been observed that latent membrane protein 2A (LMP2A) activates the phosphorylation of extracellular signal-regulated kinase (ERK), thereby inhibiting the expression of AhR through the MAPK/ ERK pathway. LMP2A is considered one of the most crucial molecules involved in the carcinogenic transformation of EBV, as it plays a role in maintaining the viral latent state and modulating intracellular signaling pathways. The downregulation of AhR expression can be observed in EBV-positive GC tissues, thereby attenuating the pro-proliferative effect and nuclear translocation (35). The examination of the interactions between the Epstein-Barr virus (EBV) and the AhR signaling pathway warrants comprehensive exploration, as it holds signiﬁcant potential for advancing therapeutic approaches in the treatment of EBV- associated cancers. suppression of pineal melatonin, resulting in melatonin-insensitive modulation of mitochondrial metabolism. This suggests that melatonin and AhR antagonists could potentially serve as prophylactic therapies for severe SARS-CoV-2 infection. Additionally, vitamin D and butyrate also contribute to the disruption of gut microbiota caused by COVID-19, as they can increase the toxicity and number of NK cells (17). There are several additional mechanisms mediated by AhR in the context of SARS-CoV-2 infection. Notably, one study shows that Fc signaling were enriched in AHR-dependent transcriptional modules, as revealed by pathway enrichment analysis of AhR- dependent and AhR-independent components in transcriptional responses to coronavirus infection. The AhR signaling pathway primarily inhibits the regenerative activity of lung epithelial basal cells, which play a crucial role in tissue repair (29). This inhibition leads to the accumulation of stem cells and facilitates the establishment of coronavirus infection. Coronaviruses have been observed to induce the activation of AhR through a pathway that is not dependent on IDO1. AhR and inﬂuenza A virus infection The inﬂuenza virus, particularly inﬂuenza A virus (IAV), is a pathogen that frequently leads to global pandemics and mortality. Numerous epidemiological studies have demonstrated a correlation between AhR activation and the severity of respiratory infections (36). The activation of AhR has the potential to inﬂuence inﬂuenza virus infection through the responses of various lymphocytes, including CD4+ T cells, dendritic cells, T follicular helper cells, and CD8+ T cells. The forthcoming discussion will provide a detailed description of the interactions between these lymphocytes and AhR. The persistence of CD4+ T cell responses to inﬂuenza virus infection is attributed to the binding of AhR ligands, which directly affect CD4+ T cells (37). Research has demonstrated that AhR not only inﬂuences CD4+ T cells during infection but also impacts the patterns of DNA methylation in these cells (38). Analysis of gene and protein expression reveals that the expansion and function of CD4+ T cells are suppressed during viral infection due to increased AhR activation. Furthermore, the treatment of mice with an AhR agonist during IAV infection results in differential methylation of seven out of eleven genes in CD4+ T cells. Following treatment with drugs that alter DNA methylation, various functions of CD4+ T cell responsiveness were restored. These ﬁndings suggest that DNA methylation may serve as a mechanism regulated by AhR, capable of consistently modifying T cell functions in mice (38). Previous research has elucidated that a similar mechanism can be observed in CD8+ T cells, where increased AhR activation leads to genomic variations in DNA methylation patterns (39). Notably, developmental AhR activation visibly diminishes the polyfunctionality of cytotoxic T lymphocytes (CTL) and alters the transcriptional progression of CD8+ T cells (40). S-adenosylmethionine (SAM) has been found to enhance polyfunctionality and augment the population of speciﬁc virus CD8+ T cells by promoting DNA methylation. It is postulated that the reduction in methylation induced by AhR-binding ligands may lead to persistent alterations in antiviral CD8+ CTL functions (40). In the context of inﬂuenza A virus (IAV) infection, AhR AhR and coronavirus infections This activation leads to an increase in the expression of TCDD-inducible poly (ADP-ribose) polymerase (TiPARP), which serves as a downstream effector and modulates the expression of cytokine genes (4). Additionally, SARS-CoV-2 infection has been found to stimulate the AhR signaling pathway, thereby promoting viral replication by upregulating the transcription of ACE2 and dampening the antiviral immune response mediated by IFN-I. The activation of AhR in the IFN/ IDO/Kyn axis by the viral infection leads to lung inﬂammation, which subsequently promotes the expression of mucin production, intercellular adhesion, and cytokine release. Ultimately, this process contributes to the increased replication of SARS-CoV-2 (30). Consequently, AhR emerges as a signiﬁcant host factor for SARS- CoV-2-infected cells and a potential target for antiviral therapies against SARS-CoV-2. Therefore, it is crucial to further investigate and comprehend the underlying mechanisms that govern SARS- CoV-2 infection for future research endeavors. Frontiers in Immunology The role of AhR in EB virus infection The Epstein-Barr virus (EBV), a prevalent type of herpes virus, is capable of persisting in memory B cells without causing symptoms (31). However, there is still a potential risk for the development of autoimmune diseases, such as sicca syndrome (SS), due to EBV-associated cellular transformation and subsequent viral invasion. The activation of the EBV infection from a latent to a lytic state is facilitated by TCDD, which induces the transcription of BZLF1. The reactivation of EBV, triggered by AhR ligands, plays a role in the development of SS in EBV-positive salivary epithelial cells and B cells. Inoue et al. postulate that the induction of EBV reactivation by TCDD may enhance the immune response in the salivary glands of patients with SS (32). This is achieved through the involvement of latent membrane protein 1 (LMP1), which binds to TRAF1-3 and facilitates the interaction between the NF-kB pathway and AhR signaling pathway during the B-cell transformation process initiated by EBV invasion (33). Further investigations have Frontiers in Immunology 04 frontiersin.org 10.3389/fimmu.2024.1367734 Xu et al. negatively modulates host immune responses by inhibiting dendritic cell (DC) activity in priming naive CD8+ T cells, thereby diminishing the generation of CD8+ CTL (41). Moreover, the AhR presents a promising avenue for manipulating T follicular helper (Tfh) cell reactions, thereby reducing the development of Tfh cells and T cell-dependent B cell responses during inﬂuenza virus infection (42). DNA domain, facilitating infection-associated accession in iNOS expression and neutrophil recruitment. Notably, the increase in these two factors is not correlated, indicating that AhR activation yields independent outcomes (48). These ﬁndings demonstrate the role of AhR in regulating the equilibrium between moderate and excessive inﬂammation in the context of inﬂuenza A virus infection. The Aryl hydrocarbon receptor nuclear translocator (ARNT) is known to bind to AhR and facilitate its nuclear transportation (49). Recent studies have indicated interaction between ARNT and the polymerase acidic (PA) protein of the H5N1 virus (50). The PA subunit is a complex protein that plays a crucial role in viral pathogenicity by regulating the replication and transcription of the IAV (51). Through a yeast two-hybrid screen, ARNT has been identiﬁed as a novel host factor that interacts with the PA protein. Speciﬁcally, the bHLH/PAS region of ARNT primarily interacts with the C-terminal domain of the PA subunit. The role of AhR in EB virus infection During this interaction, the overexpression of ARNT was found to have a signiﬁcant impact on the downregulation of polymerase activity, thereby limiting the propagation and transcription of the inﬂuenza viral genome. This ultimately led to the accumulation of the PA protein in the nucleus (50). In conclusion, the activation of AhR has the potential to inﬂuence inﬂuenza virus infection through various mechanisms (Figure 2). AhR plays a regulatory role in the functions and responses of CD4+ T cells, dendritic cells, CD8+ T cells, and neutrophils during inﬂuenza virus infection. ARNT binds to AhR and interacts with the C-terminal domain in the PA subunit of the H5N1 virus, ultimately resulting in the nuclear accumulation of the PA protein. In future studies, we intend to investigate additional Furthermore, the activation of the AhR not only affects lymphocytes but also leads to neutrophilia in peripheral tissues (43). Among the various agonists for AhR, the 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) is considered one of the most potent, exacerbating inﬂammatory pulmonary diseases upon exposure to AhR ligands. Different AhR ligands, such as TCDD and 6-formylindolo (3, 2-b) carbazole (FICZ), can induce immunomodulatory variations in response to inﬂuenza A virus infection, depending on the duration of AhR activation and the speciﬁc types of activated cell receptors (44). The activation of the AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in an increased number of neutrophils in the lung during infection with inﬂuenza A virus (IAV) (45). Recent studies indicate that extrinsic bone marrow-derived cells, mediated by AhR, play a role in regulating the directed migration of neutrophils, which speciﬁcally increase at the site of antigen challenge in response to pulmonary infection (46). Additionally, our ﬁndings demonstrate that AhR activation enhances the accumulation of neutrophils in the lung and upregulates the expression of inducible nitric oxide synthase (iNOS) within the same context (47). The nuclear translocation of AhR is dependent on its binding to the intrinsic FIGURE 2 The involvement of the AhR in inﬂuenza A virus (IAV) infection is of signiﬁcant interest. Activation of AhR has been shown to inﬂuence the response of various cell types, potentially impacting the course of IAV infection. Notably, AhR not only affects the function and differentiation of CD4+ T cells during IAV infection, but also inﬂuences DNA methylation patterns in these cells. Similar mechanisms may be observed in CD8+ T cells. The role of AhR in EB virus infection Furthermore, AhR presents a promising target for modulating T follicular helper (Tfh) cell responses, offering a potential avenue for therapeutic intervention. During infection with IVA, the AhR exerts a suppressive effect on the host’s immune responses by negatively regulating the function of dendritic cells (DCs) in their ability to prime naive CD8+ T cells. Consequently, this may lead to a reduction in the number of cytotoxic T lymphocytes (CTLs). Additionally, the AhR agonist, TCDD, enhances the expression of inducible nitric oxide synthase (iNOS) and promotes the recruitment of neutrophils in endothelial and respiratory epithelial cells, respectively. However, it is important to note that there exists a reciprocal inhibition between iNOS and neutrophils. ARNT interacts with the polymerase acidic (PA) protein, thereby exerting a signiﬁcant inﬂuence on the viral pathogenicity of the inﬂuenza virus. The red arrow symbolizes a positive effect, whereas the green arrow signiﬁes a negative effect. FIGURE 2 The involvement of the AhR in inﬂuenza A virus (IAV) infection is of signiﬁcant interest. Activation of AhR has been shown to inﬂuence the response of various cell types, potentially impacting the course of IAV infection. Notably, AhR not only affects the function and differentiation of CD4+ T cells during IAV infection, but also inﬂuences DNA methylation patterns in these cells. Similar mechanisms may be observed in CD8+ T cells. Furthermore, AhR presents a promising target for modulating T follicular helper (Tfh) cell responses, offering a potential avenue for therapeutic intervention. During infection with IVA, the AhR exerts a suppressive effect on the host’s immune responses by negatively regulating the function of dendritic cells (DCs) in their ability to prime naive CD8+ T cells. Consequently, this may lead to a reduction in the number of cytotoxic T lymphocytes (CTLs). Additionally, the AhR agonist, TCDD, enhances the expression of inducible nitric oxide synthase (iNOS) and promotes the recruitment of neutrophils in endothelial and respiratory epithelial cells, respectively. However, it is important to note that there exists a reciprocal inhibition between iNOS and neutrophils. ARNT interacts with the polymerase acidic (PA) protein, thereby exerting a signiﬁcant inﬂuence on the viral pathogenicity of the inﬂuenza virus. The red arrow symbolizes a positive effect, whereas the green arrow signiﬁes a negative effect. FIGURE 2 The involvement of the AhR in inﬂuenza A virus (IAV) infection is of signiﬁcant interest. The role of AhR in respiratory syncytial virus infection Respiratory syncytial virus (RSV) is a prominent causative agent of lower respiratory tract infection, with the potential to precipitate early and recurrent pulmonary disease. Prior research has demonstrated that the AhR plays a role in modulating immune responses to respiratory viral infections by regulating the interplay among different cell types, such as dendritic cells (DCs) (52). In vivo, DCs serve as direct recipients of AhR ligands, and their accumulation is associated with heightened allergen sensitization and exacerbated pathology in the context of RSV infection. The impacts of respiratory syncytial virus (RSV) infection encompass the monitoring of pathophysiological AhR activity, resulting in excessive mucus production and heightened Th2 cytokine responses (53). Additionally, there exists an interplay between bacteria and viruses, which synergistically contributes to the development of severe disease and clinical symptoms (54). Lipopolysaccharide (LPS), acting as the ligand for Toll-like receptor 4 (TLR4), interacts with RSV infection to amplify inﬂammatory reactions, modulate AhR signaling, and alter cytokine proﬁles. TLR4 activation can be induced by LPS, leading to enhanced viral clearance. Consequently, the level of TLR4 activation can serve as an indicator of disease severity in RSV infection. The interaction between RSV and LPS triggers AhR signaling and respiratory tract inﬂammation through the TRIF- MMP-9-neutrophil-MMP-9 signaling pathway (55). Consequently, inhibiting this co-infection interaction may prove beneﬁcial in the development of vaccines and therapies for RSV infection in the future. The AhR transcription factor is highly conserved and has been found to play a signiﬁcant role in promoting tumorigenesis and carcinogenicity in transformed host cells. This occurs through the suppression of apoptosis and the disregarding of AhR-dependent cell growth arrest when activated by its endogenous ligand, Kynurenine (60). Tian et al. have provided a description of AhR’s ability to modulate the toxicities of dioxins and similar compounds (61). Additionally, Opitz et al. have demonstrated that the endogenous ligand of AhR, derived from TDO, Kyn, can down-regulate the anti- tumor effects and enhance the survival rate of tumor cells through the AhR pathway (62). It is hypothesized that HCV infection may expedite the development of hepatocellular carcinoma (HCC) by amplifying the tryptophan (Trp)-TDO-Kyn-AhR pathway, thereby impeding immune responses against viral and tumor antigens, ultimately leading to tumorigenesis (63). The persistence of HCV infection induces chronic inﬂammation, attracting dendritic cells and macrophages, which in turn elevate the levels of IDO in the liver. The role of AhR in EB virus infection This process ultimately plays a crucial role in HCV assembly. Therefore, targeting this pathway could serve as an effective strategy for combating HCV infection and hepatic LDs overproduction. The role of AhR in respiratory syncytial virus infection Consequently, the upregulation of IDO contributes to the metabolic conversion of Trp into Kyn through TDD, facilitating the activation of AhR. The activation of AhR signaling, in conjunction with cytokine signaling, induces the differentiation of naive CD4+ T cells into Tregs. Tregs possess the capability to suppress immune responses, thereby preventing the formation of hepatocellular carcinoma (HCC) (63). During the progression of HCC establishment, it has been observed that AhR signaling is heightened in hepatocytes during HCV infection. However, the precise mechanisms governing pathogen immune evasion through the TDO-Kyn-AhR pathway require further elucidation. Frontiers in Immunology The role of AhR in EB virus infection Activation of AhR has been shown to inﬂuence the response of various cell types, potentially impacting the course of IAV infection. Notably, AhR not only affects the function and differentiation of CD4+ T cells during IAV infection, but also inﬂuences DNA methylation patterns in these cells. Similar mechanisms may be observed in CD8+ T cells. Furthermore, AhR presents a promising target for modulating T follicular helper (Tfh) cell responses, offering a potential avenue for therapeutic intervention. During infection with IVA, the AhR exerts a suppressive effect on the host’s immune responses by negatively regulating the function of dendritic cells (DCs) in their ability to prime naive CD8+ T cells. Consequently, this may lead to a reduction in the number of cytotoxic T lymphocytes (CTLs). Additionally, the AhR agonist, TCDD, enhances the expression of inducible nitric oxide synthase (iNOS) and promotes the recruitment of neutrophils in endothelial and respiratory epithelial cells, respectively. However, it is important to note that there exists a reciprocal inhibition between iNOS and neutrophils. ARNT interacts with the polymerase acidic (PA) protein, thereby exerting a signiﬁcant inﬂuence on the viral pathogenicity of the inﬂuenza virus. The red arrow symbolizes a positive effect, whereas the green arrow signiﬁes a negative effect. 05 Frontiers in Immunology frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 Conversely, increasing the levels of CYP1A1 in AhR-inactivated cells can restore lipid and triglyceride accumulation. Additionally, the antagonist Flutamide, which targets the AhR, impairs the host cell’s capacity to generate HCV by disrupting lipid accumulation. In conclusion, the ﬁndings of Ohashi et al. provide evidence that HCV promotes the activation of the AhR/CYP1A1 pathway, leading to the reorganization of hepatic cell functions and subsequent accumulation of lipid droplets (58). This process ultimately plays a crucial role in HCV assembly. Therefore, targeting this pathway could serve as an effective strategy for combating HCV infection and hepatic LDs overproduction. pathways that are speciﬁc to these cell lineages and their interaction with AhR-dependent regulatory signals. Conversely, increasing the levels of CYP1A1 in AhR-inactivated cells can restore lipid and triglyceride accumulation. Additionally, the antagonist Flutamide, which targets the AhR, impairs the host cell’s capacity to generate HCV by disrupting lipid accumulation. In conclusion, the ﬁndings of Ohashi et al. provide evidence that HCV promotes the activation of the AhR/CYP1A1 pathway, leading to the reorganization of hepatic cell functions and subsequent accumulation of lipid droplets (58). The role of AhR in hepatitis C virus infection The hepatitis C virus (HCV) pathogen is known to cause hepatitis C and contribute to the development of hepatocellular carcinoma and other severe liver diseases. Recent analysis suggests that HCV-induced hepatocellular carcinoma tumors exhibit an increased expression of genes involved in the AhR signaling pathway (56). Consequently, AhR emerges as a pivotal factor in HCV infection. A previous study has indicated that MicroRNA (MiRNA) plays a signiﬁcant role in HCV proliferation and lipid metabolism, thereby limiting the progression of liver ﬁbrosis and exhibiting potential antitumor effects (64). Additionally, the expression of miR-10a follows a circadian pattern and inﬂuences the expression of the brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1), which is involved in circadian gene regulation (65). This modulation occurs through the downregulation of the RA receptor-related gene, which serves as a target gene for miR-10a. The interaction between miR-10a and Bmal1 is crucial for the regulation of circadian rhythm. MiR-10a plays a signiﬁcant role in the catabolism of lipids, protein, glucose, and bile acid through the down-regulation of Bmal1 genes. It also regulates fatty acid metabolism by inhibiting the expression of sterol regulatory element binding protein (SREBP1), SREBP2, and fatty acid synthase (FASN), as well as other lipid synthesis genes. Additionally, MiR-10a modulates gluconeogenesis through Hepatitis C virus (HCV) exerts control over the metabolic processes of host cells, leading to the formation of speciﬁc cytomembrane structures, namely lipid droplets (LDs). This manipulation facilitates the replication of the virus and the efﬁcient assembly of viral particles (57). The AhR serves as a target for ﬂutamide and plays a pivotal role in regulating the accumulation of LDs and the generation of HCV in hepatic cells (58). Additionally, the cytochrome P450 (CYP) family of genes, downstream targets of AhR, actively participate in the metabolism of xenobiotics, with CYP1A1 being a notable example (59). The ﬁndings of the study suggest that inhibiting the AhR-induced CYP1A1 enzyme can effectively reduce the excessive production of lipid droplets. Frontiers in Immunology 06 frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 cells (69). Notably, Kyn levels, an endogenous ligand of AhR, are signiﬁcantly increased in ﬁbroblasts infected with HCMV (70). The presence of unidentiﬁed metabolites such as Kyn may potentially impact HCMV replication and provide novel insights into the intricate interplay between HCMV and host cell metabolism. AhR and herpesvirus infection Herpes simplex virus 1 (HSV-1) is a pathogen capable of infecting epithelial cells and invading neurons (73). The reactivation of latent herpesvirus infections is commonly observed in individuals with compromised immune systems. Recent research indicates that the AhR signaling pathway plays a role in suppressing the production of IL-17 in mice infected with gamma-herpesvirus 68, resulting in a weakened immune response (74). The levels of indolamine 2-3 dioxygenase (IDO1) and tryptophan dioxygenase (TDO2), which are key components of the Kyn pathway and AhR ligands, are essential for the replication of herpesvirus in ﬁbroblasts. Lung dendritic cells (DCs) expressing AhR and CD103 are observed to accumulate in response to TDO2-expressing ﬁbroblasts, replacing AhR+ lymphocytes. This suggests that the ﬁbroblast (TDO2)/DC (AHR) axis plays a crucial role in herpesvirus infection (74). Additionally, we have observed an elevated level of interferon (IFN) signaling upon genetic ablation of AhR during HSV-1 infection. HSV-1 has the ability to exploit host mechanisms to evade antiviral defenses and enhance its own replication. The impact of AhR agonist treatment on interferon regulatory factor 3 (IRF-3) rather than IFN-b is responsible for the observed enhanced resistance to HSV-1 infection in treated cells (75). The speciﬁc mechanism by which IRF-3 inﬂuences HSV-1 infection is currently unknown, but studies indicate a similar mechanism exists in human cytomegalovirus (76). Consequently, the use of AhR antagonists at the site of invasive infection may serve as a potential therapeutic approach for combating viral infections, as AhR plays a negative role in restricting cell-autonomous antiviral resistance. The role of AhR in hepatitis C virus infection The reduction of AhR has the potential to decrease the replication of HCMV, whereas the activation of AhR using an exogenous ligand can enhance virus proliferation. Wise et al. propose that Hypoxia- inducible factor 1a plays a signiﬁcant role in the antiviral response by inhibiting the synthesis of Kyn and activating AhR during HCMV infection (71). Additionally, HCMV infection regulates the activation of AhR, which in turn modulates the transcription of infected cells. In a precise manner, it can be stated that the AhR plays a crucial role in facilitating the HCMV-induced G1/S block, which effectively hinders cellular DNA replication and consequently impedes the progression of infected ﬁbroblasts into the S phase. By employing two distinct locked nucleic acids to suppress AhR expression, it becomes evident that the accumulation of viral DNA is signiﬁcantly reduced. Hence, it can be inferred that the activation of AhR by Kyn during HCMV infection serves as a mediator for essential functions in the interaction between the virus and the host (72). These investigations present a unique perspective on a previously unexplored aspect of the mechanism underlying the interaction between the virus and the host cell over an extended period of time. peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a), protein synthesis through mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K), and bile acid synthesis through liver receptor homolog 1 (LRH1). The downstream genes of Bmal1, which are regulated by miR-10a, exhibit a strong association with the exacerbation of pathological liver catabolism in individuals with chronic hepatitis C (65). Importantly, miR-10a holds promise as a potential effective biomarker for assessing the prognosis of liver cirrhosis in forthcoming studies. The role of AhR in human immunodeﬁciency virus infection Human immunodeﬁciency virus (HIV), the causative agent of acquired immunodeﬁciency syndrome (AIDS), disrupts the equilibrium of the immune system and facilitates the occurrence of opportunistic infections. HIV induces chronic inﬂammation and immune dysfunction, resulting in diverse modiﬁcations to cellular catabolic pathways, including glucose, lipids, and amino acids (66). To impede the advancement of HIV-1 infection, the regulation of cellular metabolic pathways presents a viable approach. Studies indicate that the Kynurenine tryptophan metabolite, acting as an endogenous ligand of AhR, contributes to the accelerated progression of HIV-1. AhR facilitates HIV-1 transcription by binding to the 5ˊ-long terminal repeat domain of the virus and ampliﬁes the accumulation of the positive transcription elongation factor complex, thereby phosphorylating RNA Pol II (67). It is postulated that targeting the downstream effects of AhR may be pivotal in controlling HIV-1 infection. Conversely, AhR can also serve as a hindrance to HIV-1 infection by means of CD4+ T cells. The observed reduction in IL-22, IL-17A, and IL-10 production in AhR knockout cells suggests that AhR actively facilitates HIV-1 replication. This analogous mechanism is observed in the pharmacological group of AhR, wherein antagonists are employed to impede the binding of AhR and its ligands. The subsequent cohort additionally impedes the replication of HIV-1 during the stages of integration and reverse transcription within memory CCR6+ CD4+ T cells. Furthermore, we have identiﬁed through chromatin immunoprecipitation that HIV-1 is a target of AhR. These ﬁndings manifest as the facilitation of HIV-1 replication in CD4+ T cells of individuals living with HIV-1 who are undergoing antiretroviral therapy (ART). Consequently, AhR exerts control over T cell transcription via tissue circulation and outgrowth, thereby representing a signiﬁcant therapeutic approach in the management of individuals living with HIV-1 (68). Frontiers in Immunology The role of AhR in other viral infections recognition that AhR can enhance the integrity of the intestinal immune and physical barriers by regulating the expression of tight junction proteins and reducing cecal inﬂammation (83). Moreover, AhR activation exerts a multifaceted inﬂuence on gut microbiota homeostasis through intricate mechanisms (Figure 3). The Stimulator of interferon genes (STING1) is a protein that interacts with guanosine mono-phosphate-adenosine monophosphate (cGAMP) and speciﬁcally interacts with the N- terminal region of AhR. When located in the nucleus, STING1 modulates AhR activation. The formation of the STING1-AhR complex requires the presence of nuclear partners such as RNF20 (ring ﬁnger protein 20), XRCC6 (X-ray repair cross complementing 6), DHX9 (DExH-box helicase 9), and PML (the only positive regulators). STING1 activation triggers the activation of AhR in the nucleus, leading to the improvement of intestinal homeostasis and the promotion of the expression of CYP1A1, IL22, and other related factors (84). Indole-3-carboxylic acid (ICA), a catabolic derivative of Lactobacillus gallinarum, competes with Kyn for binding sites on AhR and also suppresses IDO1 expression. The antagonistic effect of ICA on Kyn function results in the down-regulation of Foxp3+ transcription, indicating that ICA has the potential to regulate the IDO1/Kyn/AhR axis, thereby reducing the differentiation of CD4+Treg cells and enhancing the function of CD8+T cells (85). In addition to the extensive survey conducted, numerous other viral infections are linked to the AhR signaling pathway, including BK polyomavirus, Zika virus, Junı́n virus, and others. BK polyomavirus infection is frequently observed following kidney transplantation, leading to the development of BK polyoma virus allograft nephropathy (BKPyVN) (77). Given AhR’s role in drug metabolism, this transcription factor regulates the polarization of macrophages by inﬂuencing viral tolerance and inﬂammatory response. Based on the ﬁndings of the study on renal immune response, it has been observed that the expression of AhR in the BKPyVN group is signiﬁcantly elevated compared to the control group with T-cell mediated rejection (TCMR). Furthermore, analysis of the inﬁltrating graft biopsies reveals an increased presence of AhR in the nuclei of CD45+ and CD68+ cells in BKPyVN cases. These ﬁndings suggest a potential interplay between adaptive and innate immunity, which may contribute to a deeper comprehension of BKPyVN (78). Zika virus (ZIKV) infection is associated with mild symptoms but can result in various birth defects, notably microcephaly, which has garnered signiﬁcant attention regarding the treatment of ZIKV (79). The role of AhR in other viral infections Recent research indicates that ZIKV infection induces the activation of the aryl hydrocarbon receptor (AhR) through the production of Kyn, as observed in genome-wide transcriptional studies. This activated AhR subsequently hampers ZIKV replication by constraining the intrinsic immune response mediated by the promyelocytic leukemia (PML) protein. Persistent study of AhR inhibition is warranted due to its potential to reduce the replication of both ZIKV strains in vitro and other related ﬂavivirus dengue (29). Additionally, AhR plays crucial roles in Human T-cell leukemia virus type 1 (HTLV-1) plus-strand transcription by directly binding to the HTLV-1 LTR dioxin response element (DRE) site (CACGCATAT). Furthermore, the manipulation of AhR overexpression in HTLV-1 infected T- cells through the activation of the nuclear factor kappa B (NF-kB) pathway, as well as the level of AhR ligands Kyn, determines HTLV- 1 latency-reactivation-latency (80). The adenovirus-dioxin- responsive bioassay system proves to be a valuable tool for the detection of dioxins, as it relies on the AhR responsiveness of adenovirus (81). Additionally, it has been observed that the AhR signaling pathway can exert pharmacological control over the replication of Junı́n virus (JUNV). In JUNV-infected hepatic cells, an upregulation of AhR has been identiﬁed, and through immunoﬂuorescence detection, it has been demonstrated that the replication of JUNV strains can be dose-dependently inhibited by an AhR antagonist (82). Future research could focus on the exploration of unidentiﬁed modulating AhR ligands as a means of mitigating harmful immune responses. The modiﬁcation of the extracellular matrix, regulated in part by AhR-associated genes, may have notable implications for the integrity of the intestinal cell barrier and the immune response during salmonella infection in the host (86). Comparable functions can also be observed in distant organs, as AhR agonists derived from the photooxidation of Trp possess the capacity to ameliorate Eschrichia coli-induced endometritis by restoring functional barriers and mitigating inﬂammatory responses (87). The activation of AhR through Lactobacillus reuteri tryptophan metabolism plays a role in mitigating Escherichia coli-induced mastitis and maintaining host homeostasis by limiting NF-kB activation and improving barrier function (88). Additionally, Streptococcus gallolyticus interacts with AhR-mediated pathways involved in cellular cytochrome P450 (CYP) 1 biotransformation, potentially leading to DNA damage and effectively eliminating intestinal pathogens, thus protecting the epithelial barrier (89). The AhR molecule is involved in the cooperative inﬂuence of VD3 and butyrate on the integrity of the intestinal barrier against Salmonella infection. The role of AhR in other viral infections This is achieved by augmenting antibacterial responses and reducing the expression of tight junction proteins, thereby impeding pathogen invasion (90). However, it is important to note that AhR activation can have both beneﬁcial and detrimental effects. Speciﬁcally, the activation of AhR by Benzo(a) pyrene has the potential to mitigate or eradicate septic peritonitis resulting from systemic Salmonella enterica infection (91). Furthermore, Zhu et al. have reported that Helicobacter pylori (H. pylori) infection hinders the expression of both AhR and aryl- hydrocarbon receptor repressor (AHRR) in the mucosa of the digestive tract, potentially leading to the progression of chronic inﬂammation and prolonged bacterial sustenance (92). In light of this, Soyocak et al. have postulated that AhR and AHRR may play a role in the gastric pathogenesis associated with H. pylori (93). Subsequently, they have discovered an increased expression of The role of AhR ligand in human cytomegalovirus infection Human cytomegalovirus (HCMV), a member of the herpesvirus family, modulates the activation of cellular pathways involved in metabolism in order to facilitate its own replication. Speciﬁcally, HCMV enhances glycolysis and lipid synthesis in target Frontiers in Immunology 07 frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 Xu et al. Frontiers in Immunology The role of AhR in bacterial infection Increasing evidence suggests that AhR plays a signiﬁcant role in the pathogenesis of various bacteria, contributing to the development of inﬂammatory bowel disease. There is a growing Frontiers in Immunology 08 frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 FIGURE 3 Multiple mechanisms are involved in the regulation of AhR for maintaining gut microbiota homeostasis. One such mechanism involves the translocation of STING1 from the endoplasmic reticulum (ER) to the Golgi apparatus, followed by its subsequent movement into the nucleus through AhR and AhR ligands. The formation of the STING1-AhR complex requires the presence of positive regulators such as PML, which facilitate the expression of CYP1A1 and IL22. Indole-3-carboxaldehyde (IAld) and ICA, which are synthesized by L. gallinarum, have been found to suppress the expression of IDO1 and counteract the function of Kyn, consequently leading to a reduction in Foxp3+ transcription. This reduction in transcription may potentially hinder the differentiation of CD4+ Treg cells. FIGURE 3 Multiple mechanisms are involved in the regulation of AhR for maintaining gut microbiota homeostasis. One such mechanism involves the translocation of STING1 from the endoplasmic reticulum (ER) to the Golgi apparatus, followed by its subsequent movement into the nucleus through AhR and AhR ligands. The formation of the STING1-AhR complex requires the presence of positive regulators such as PML, which facilitate the expression of CYP1A1 and IL22. Indole-3-carboxaldehyde (IAld) and ICA, which are synthesized by L. gallinarum, have been found to suppress the expression of IDO1 and counteract the function of Kyn, consequently leading to a reduction in Foxp3+ transcription. This reduction in transcription may potentially hinder the differentiation of CD4+ Treg cells. The role of AhR in parasites infection AhR in H. pylori-positive patients with chronic gastritis. The infection of macrophages by Mycobacterium tuberculosis triggers AhR signaling through the regulation of IL23A transcription, offering potential avenues for the treatment of pulmonary disease due to the high expression of AhR in the lungs (94). In terms of cutaneous defense, both Staphylococcus epidermidis and Staphylococcus aureus possess the capability to activate AhR in Human Keratinocytes, thereby inducing the gene expression of CYP1A1 and CY-P1B1 for cutaneous innate defense (95, 96). When the AhR signaling pathway is activated, S. epidermidis exhibits contrasting effects on skin cells derived from atopic and healthy skin (97). Additionally, AhR serves as a component of the host defense response in both Pseudomonas aeruginosa and Pseudomonas plecoglossicida, by binding to pyocyanin produced by P. aeruginosa and up-regulating the expression levels of cyp1a, respectively (98, 99). Porphyromonas gingivalis possesses the capacity to induce inﬂammatory responses through the inhibition of the AhR signaling pathway in periodontitis (100). The AhR pathway plays a role in maintaining cellular homeostasis by regulating tubulin dynamics. The obstruction of AhR by an excess of Trp-derived indole leads to a reduction in the expression of detyrosinated tubulin, thereby facilitating the growth of Chlamydia trachomatis (101). Collectively, these ﬁndings collectively offer substantiation for innovative therapeutic approaches concerning bacterial infection. However, a comprehensive investigation into the involvement of AhR in the pathogenic mechanisms of bacteria remains unpublished. Consequently, there is still a considerable distance to traverse in order to advance the intricacies of research efforts focused on AhR-targeted therapeutic interventions for inﬂammatory diseases. AhR in H. pylori-positive patients with chronic gastritis. The infection of macrophages by Mycobacterium tuberculosis triggers AhR signaling through the regulation of IL23A transcription, offering potential avenues for the treatment of pulmonary disease due to the high expression of AhR in the lungs (94). In terms of cutaneous defense, both Staphylococcus epidermidis and Staphylococcus aureus possess the capability to activate AhR in Human Keratinocytes, thereby inducing the gene expression of CYP1A1 and CY-P1B1 for cutaneous innate defense (95, 96). When the AhR signaling pathway is activated, S. epidermidis exhibits contrasting effects on skin cells derived from atopic and healthy skin (97). Additionally, AhR serves as a component of the host defense response in both Pseudomonas aeruginosa and Pseudomonas plecoglossicida, by binding to pyocyanin produced by P. aeruginosa and up-regulating the expression levels of cyp1a, respectively (98, 99). Conclusions and perspectives AhR has been shown to promote tumorigenesis, carcinogenicity, and clonal expansion of transformed liver cells, thereby accelerating the development of hepatocellular carcinoma in individuals with HCV infection. The involvement of LMP1, LMP2A, and the MAPK/ERK pathway in the carcinogenic transformation of EBV is known to attenuate the pro-proliferation effect and nuclear translocation upon AhR inhibition. However, the relationship between AhR pathway and lung carcinoma remains unclear, warranting further investigation in this direction. Additionally, the role of AhR in pathogenic bacterial infections is complex and can have both beneﬁcial and detrimental effects. The positive aspects encompass the protection of the epithelial barrier and cutaneous defense, whereas the negative aspect entails the promotion of chronic inﬂammation and the consequent long-term bacterial persistence. The overall functionality is contingent upon the bacterial species, cell types, and other yet-to-be-explored factors. AhR primarily governs T cell levels and the secretion of certain cytokines, such as IFN and interleukin, in order to alleviate inﬂammation during parasitic and fungal infections. Furthermore, the majority of the aforementioned studies primarily focus on singular infections, necessitating a comprehensive exploration of AhR activation in various microbial co-infections. Ultimately, AhR presents a promising therapeutic target for immunomodulation, thereby mitigating the emergence of multi-drug resistance. Enhanced comprehension of the pathogenic mechanisms underlying viral modulation of AhR will facilitate the identiﬁcation of more effective targets for antiviral therapy. Conclusions and perspectives Additionally, the role of AhR in pathogenic bacterial infections is complex and can have both beneﬁcial and detrimental effects. The positive aspects encompass the protection of the epithelial barrier and cutaneous defense, whereas the negative aspect entails the promotion of chronic inﬂammation and the consequent long-term bacterial persistence. The overall functionality is contingent upon the bacterial species, cell types, and other yet-to-be-explored factors. AhR primarily governs T cell levels and the secretion of certain cytokines, such as IFN and interleukin, in order to alleviate inﬂammation during parasitic and fungal infections. Furthermore, the majority of the aforementioned studies primarily focus on singular infections, necessitating a comprehensive exploration of AhR activation in various microbial co-infections. Ultimately, AhR presents a promising therapeutic target for immunomodulation, thereby mitigating the emergence of multi-drug resistance. Enhanced comprehension of the pathogenic mechanisms underlying viral modulation of AhR will facilitate the identiﬁcation of more effective targets for antiviral therapy. There have been numerous decades of research dedicated to AhR since its discovery in the previous century, and signiﬁcant progress has been made in understanding the role of AhR in the infection caused by etiological agents. In this review, we have provided an overview of the current knowledge regarding the interaction between AhR’s structural conformations and infections caused by causative agents, as well as the mechanisms of AhR agonists and antagonists in guiding the development of innovative antiviral therapeutic strategies. The AhR receptor primarily mediates the inﬂammatory response of the respiratory and intestinal barriers when invaded by viruses, bacteria, parasites, and fungi. By upregulating ACE2 expression and regulating tryptophan-associated metabolites like Kyn and IDO, AhR serves as a negative regulator in SARS-CoV-2 infection. The mechanisms by which AhR inﬂuences the lung barrier involve the modulation of lymphocytes and neutrophils, leading to the induction of inﬂammatory cytokines and immune responses during infections caused by inﬂuenza virus and other respiratory viruses. Further investigation is required to understand the precise interaction between AhR and immune components. In terms of circadian rhythm regulation, the induction of Bmal1 by miR-10a has been found to decrease the expression of genes related to RA receptors. In addition to the already known circadian genes, further exploration is needed to elucidate the mechanism of rhythm involving additional aryl hydrocarbon receptor nuclear translocators. The role of AhR in parasites infection Porphyromonas gingivalis possesses the capacity to induce inﬂammatory responses through the inhibition of the AhR signaling pathway in periodontitis (100). The AhR pathway plays a role in maintaining cellular homeostasis by regulating tubulin dynamics. The obstruction of AhR by an excess of Trp-derived indole leads to a reduction in the expression of detyrosinated tubulin, thereby facilitating the growth of Chlamydia trachomatis (101). Collectively, these ﬁndings collectively offer substantiation for innovative therapeutic approaches concerning bacterial infection. However, a comprehensive investigation into the involvement of AhR in the pathogenic mechanisms of bacteria remains unpublished. Consequently, there is still a considerable distance to traverse in order to advance the intricacies of research efforts focused on AhR-targeted therapeutic interventions for inﬂammatory diseases. Previous research indicates that AhR may limit parasite replication and infection outcome by functioning as a ligand- activated transcription factor in various regulatory pathways (102). However, the speciﬁc mechanisms through which AhR exerts its antiparasitic effect remain unclear. Plasmodium falciparum, the causative agent of malaria, is responsible for signiﬁcant morbidity and mortality rates, particularly among the South African population. AhR signaling plays a critical role in malaria by maintaining the equilibrium between infection cell proliferation, immune response, and inﬂammation (103). Acute kidney injury (AKI) is a signiﬁcant complication of malaria that is linked to heme metabolism and the generation of AhR agonists. The potential therapeutic approach of targeting AhR and heme metabolism shows promise in managing AKI by controlling parasitemia and mitigating tissue damage (104). Furthermore, based on research investigating the pathological progression of P. berghei Anka, it appears that AhR may also play a role in regulating parasite replication and inﬂammation (105). Infections caused by Leishmania major exhibit a higher prevalence and mortality rate among human infections, as indicated by the Neglected Tropical Diseases listed by the World Health Organization (106). The absence of AhR in mice infected with L. major resulted in reduced levels of Treg cells, elevated levels of Interferon (IFN)-g and IL-12, and a decline in the secretion of IL-10 and IL-4 (107). Additionally, Münck et al. conducted a study that examined genome variations in granuloma macrophages, providing further evidence of the association between the AhR signaling pathway and early skin micromilieu in L. major infection. Furthermore, it has Frontiers in Immunology 09 frontiersin.org Xu et al. 10.3389/fimmu.2024.1367734 Conclusions and perspectives been demonstrated that treatment with AhR antagonists resulted in a decrease in tumor necrosis factor (TNF)-a mRNA expression induced by L. major in monocytes (108). These aforementioned studies have provided evidence of the diverse roles played by AhR in various parasites. Additionally, research has indicated that AhR is implicated in the inﬂammatory response to Trypanosoma cruzi, Toxoplasma gondii, schistosomiasis japonica, and coccidium through various mechanisms (102, 107, 109–111). After acquiring comprehension of the regulatory function of AhR in inﬂammation, it aids in the advancement of antiparasitic medications. There have been numerous decades of research dedicated to AhR since its discovery in the previous century, and signiﬁcant progress has been made in understanding the role of AhR in the infection caused by etiological agents. In this review, we have provided an overview of the current knowledge regarding the interaction between AhR’s structural conformations and infections caused by causative agents, as well as the mechanisms of AhR agonists and antagonists in guiding the development of innovative antiviral therapeutic strategies. The AhR receptor primarily mediates the inﬂammatory response of the respiratory and intestinal barriers when invaded by viruses, bacteria, parasites, and fungi. By upregulating ACE2 expression and regulating tryptophan-associated metabolites like Kyn and IDO, AhR serves as a negative regulator in SARS-CoV-2 infection. The mechanisms by which AhR inﬂuences the lung barrier involve the modulation of lymphocytes and neutrophils, leading to the induction of inﬂammatory cytokines and immune responses during infections caused by inﬂuenza virus and other respiratory viruses. Further investigation is required to understand the precise interaction between AhR and immune components. In terms of circadian rhythm regulation, the induction of Bmal1 by miR-10a has been found to decrease the expression of genes related to RA receptors. In addition to the already known circadian genes, further exploration is needed to elucidate the mechanism of rhythm involving additional aryl hydrocarbon receptor nuclear translocators. AhR has been shown to promote tumorigenesis, carcinogenicity, and clonal expansion of transformed liver cells, thereby accelerating the development of hepatocellular carcinoma in individuals with HCV infection. The involvement of LMP1, LMP2A, and the MAPK/ERK pathway in the carcinogenic transformation of EBV is known to attenuate the pro-proliferation effect and nuclear translocation upon AhR inhibition. However, the relationship between AhR pathway and lung carcinoma remains unclear, warranting further investigation in this direction. References 1. Shinde R, McGaha TL. The aryl hydrocarbon receptor: connecting immunity to the microenvironment. Trends Immunol. (2018) 39:1005–20. doi: 10.1016/ j.it.2018.10.010 1. Shinde R, McGaha TL. The aryl hydrocarbon receptor: connecting immunity to the microenvironment. Trends Immunol. (2018) 39:1005–20. doi: 10.1016/ j.it.2018.10.010 2. Sondermann NC, Faßbender S, Hartung F, Hätälä AM, Rolfes KM, Vogel CFA, et al. Functions of the aryl hydrocarbon receptor (AHR) beyond the canonical AHR/ ARNT signaling pathway. Biochem Pharmacol. (2023) 208:115371. doi: 10.1016/ j.bcp.2022.115371 14. Neavin D, Liu D, Ray B, Weinshilboum R. 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NX: Writing – review & editing, Data curation. WC: Writing – review & editing, Data curation. WN: Writing – review & editing, Funding acquisition, Supervision. RL: Writing – review & editing, Funding acquisition, Supervision. We would like to thank Dr. Aihua Lei’s valuable suggestions. The role of AhR in fungus infection The precise mechanisms by which the AhR signaling pathway regulates the pathogenicity of fungal infections remain poorly understood. Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is recognized as the most prevalent mycosis in Latin America (112). The immunological equilibrium mechanism, modulated by the IDO/AhR axis, plays a crucial role in maintaining the balance between Th17/Treg cells and the severity of lung PCM (113). The lung, acting as a barrier organ, expresses high levels of AhR. Research indicates that the involvement of IDO and AhR is essential for the control of pulmonary fungal infection and the tolerance of mycosis (114). The aryl hydrocarbon receptor (AhR) plays a crucial role in regulating the delicate equilibrium between the fungal-host relationship, commonly referred to as “commensalism vs infection” (115). Through its involvement in tryptophan metabolism in both the host and microbial communities, AhR effectively coordinates the activities of T cells (115). In this context, indoleamine 2,3-dioxygenase 1 (IDO1) assumes a signiﬁcant role in orchestrating T-cell immune homeostasis at mucosal surfaces, which is contingent upon AhR. AhR and IDO1 may contribute to the development of a novel therapeutic approach that supports the survival of the host and the establishment of fungal commensalism. This is achieved by facilitating the coevolution of commensal fungal groups with human immunity and the microbiota (115). Various studies have suggested that AhR could potentially mediate the toxicity of ochratoxin A and aﬂatoxin B1. This is accomplished by promoting the expression of P450 and inducing oxidative stress in kidney cells, respectively (116, 117). Solis et al. reported that the AhR functions through Src family kinases to phosphorylate the epidermal growth factor receptor, thereby inducing the endocytosis of Candida albicans (118). Subsequent investigations have demonstrated that AhR can also be activated by Kynurenic acid, leading to the inhibition of the myosin light chain kinase-phospho- myosin light chain signaling pathway, which alleviates inﬂammation in the lower digestive tract caused by Candida albicans (119). Additionally, AhR triggers the IL-22/IL-18 pathway, providing protection against intestinal damage resulting from vulvovaginal Candidiasis (120). All of the aforementioned statistics suggest that AhR has the potential to pave the way for antifungal therapy in future medical advancements. Frontiers in Immunology frontiersin.org 10 Xu et al. 10.3389/fimmu.2024.1367734 Funding The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. This work was supported by funds from the Natural Science Foundation of Hunan Province (NO.2023JJ0921), the Hunan Provincial Department of Education Project (NO.22A0318), the National Innovation and Entrepreneurship Training program for college students (No. 202110555103), the 2022 Scientiﬁc Research and Technology Development Program of Baise City (No. 20224124), Health Research Project of Hunan Provincial Health Commission (No. 202202074464). Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 13. Mao R, Zhang J, Jiang D, Cai D, Levy JM, Cuconati A, et al. 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https://openalex.org/W4353031526	https://mau.diva-portal.org/smash/get/diva2:1746366/FULLTEXT01	English	null	Ion kinetic energy- and ion flux-dependent mechanical properties and thermal stability of (Ti,Al)N thin films	Acta materialia	2,023	cc-by	16,036	A R T I C L E I N F O Keywords: Ion kinetic energy TiAlN Stress state Point defects Frenkel pairs Hard coatings Elastic modulus Thermal stability Density functional theory Ion-irradiation-induced changes in structure, elastic properties, and thermal stability of metastable c-(Ti,Al)N thin films synthesized by high-power pulsed magnetron sputtering (HPPMS) and cathodic arc deposition (CAD) are systematically investigated by experiments and density functional theory (DFT) simulations. While films deposited by HPPMS show a random orientation at ion kinetic energies (Ek)>105 eV, an evolution towards (111) orientation is observed in CAD films for Ek>144 eV. The measured ion energy flux at the growing film surface is 3.3 times larger for CAD compared to HPPMS. Hence, it is inferred that formation of the strong (111) texture in CAD films is caused by the ion flux- and ion energy-induced strain energy minimization in defective c-(Ti,Al)N. The ion energy-dependent elastic modulus can be rationalized by considering the ion energy- and orientation- dependent formation of point defects from DFT predictions: The balancing effects of bombardment-induced Frenkel defects formation and the concurrent evolution of compressive intrinsic stress result in the apparent independence of the elastic modulus from Ek for HPPMS films without preferential orientation. However, an ion energy-dependent elastic modulus reduction of ~18% for the CAD films can be understood by considering the 34% higher Frenkel pair concentration formed at Ek=182 eV upon irradiation of the experimentally observed (111)-oriented (Ti,Al)N in comparison to the (200)-configuration at similar Ek. Moreover, the effect of Frenkel pair concentration on the thermal stability of metastable c-(Ti,Al)N is investigated by differential scanning calorimetry: Ion-irradiation-induced increase in Frenkel pairs concentration retards the wurtzite formation temperature by up to 206 ◦C. Ion kinetic energy- and ion flux-dependent mechanical properties and thermal stability of (Ti,Al)N thin films Soheil Karimi Aghda a,, Damian M. Holzapfel a, Denis Music b, Yeliz Unutulmazsoy c, Stanislav Mr´az a, Dimitri Bogdanovski a, G¨onenç Fidanboy a, Marcus Hans a, Daniel Primetzhofer d, Alba San Jos´e M´endez e, Andr´e Anders c,f, Jochen M. Schneider a a Materials Chemistry, RWTH Aachen University, Kopernikusstr. 10, Aachen 52074, Germany b Department of Materials Science and Applied Mathematics, Malm¨o University, Malm¨o 20506, Sweden c Leibniz Institute of Surface Engineering (IOM), Permoserstr. 15, Leipzig 04318, Germany d Department of Physics and Astronomy, Uppsala University, L¨agerhyddsv¨agen 1, Uppsala 75120, Sweden e Deutsches Elektronen-Synchrotron DESY, Notkestr. 85, Hamburg 22607, Germany f Felix Bloch Institute, Leipzig University, Linn´estr. 5, Leipzig 04103, Germany Corresponding author. E-mail address: karimi@mch.rwth-aachen.de (S. Karimi Aghda). Acta Materialia 250 (2023) 118864 Acta Materialia 250 (2023) 118864 Contents lists available at ScienceDirect Available online 22 March 2023 1359-6454/© 2023 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.actamat.2023.118864 Received 6 November 2022; Received in revised form 24 January 2023; Accepted 15 March 2023 * Corresponding author. E-mail address: karimi@ 2. Experimental details (Ti,Al)N thin films were deposited by two physical vapor deposition (PVD) processing routes – high power pulsed magnetron sputtering (HPPMS) and cathodic arc deposition (CAD) – at comparable ion kinetic energy (Ek) ranges to explore the effect thereof on the evolution of structure, mechanical properties, and thermal stability. ii p p y For the first series of (Ti,Al)N thin films, a HPPMS process was used in the industrial deposition system CemeCon CC 800/9 (Würselen, Germany) employing a Melec SIPP2000USB-10-500-S pulser in combi nation with a 10 kW ADL GX 100/1000 DC power supply. A rectangular magnetron with a Ti0.5Al0.5 composite target (> 99.7% purity, Plansee Composite Materials GmbH, Germany) with the dimensions 8.8 × 50 cm2 was used for the depositions in a stationary geometry at a target-to- substrate distance of 10 cm. α-Al2O3(0001) substrates (1 × 1 cm2) and Fe foils (99.5% purity) were mounted on a Cu holder facing the target. Prior to depositions, the substrates were heated to the deposition temperature (Ts) of ~450 ◦C and etched in Ar atmosphere at 1.0 Pa for 30 min, using a mid-frequency Advanced Energy Pinnacle Plus generator operating at a voltage of -650 V with a frequency of 250 kHz and pulse off-time of 1600 ns. The chamber base pressure at the deposition temperature was below 0.75 mPa. A total pressure of 0.43 Pa was employed for sputtering with a constant N2/(Ar+N2) flow ratio of 0.13. The HPPMS power supply was operated with a pulse on-time of τHPPMS = 50 μs at a duty cycle of 2.5% corresponding to a frequency of f = 500 Hz. A time- averaged power of 2500 W was used, which resulted in the peak power density of 630 W/cm2. A pulsed substrate bias potential Vs was varied in the range from -20 V (floating) to -200 V with a pulse on-time of τs = 100 μs, synchronized to the target HPPMS pulse with a +30 μs phase shift, see Fig. S1 of the supplementary materials. The substrate bias phase shift was calculated considering the formation time of metal ions within each HPPMS pulse and the time-of-flight of the ions from the target to the substrate [45]. Therefore, majority of the ions accelerated towards substrate are expected to be film-forming species rather than Ar+. The substrates were kept electrically floating between the HPPMS pulses. 2. Experimental details The deposition time was 90 min resulting in films with a thick ness of ~2.2 μm. i In recent years, formation and stability of different types of point defects such as vacancies [8,24–28], interstitials [24,26], Schottky de fects [26], and Frenkel pairs [25,27–29] in c-TMN and c-(TM,Al)N have been investigated. Point defects cause significant alterations in the structural and mechanical properties of nitrides. For example, vacancies as the lowest-energy defects [26] stabilize the mechanically unstable c-MoN [30,31] and WN [32], reduce the elastic modulus of Ti0.5Al0.5N [24], and increase the elastic modulus of NbN [33]. In addition to va cancies, Frenkel pair defect types have also been shown to energetically stabilize the cubic structure of binary MoN [34]. By correlating exper iments and ab initio simulations for c-(Cr,Al)N [29], c-(V,Al)N [27], and c-(Ti,Al)N [28], it has been demonstrated that Frenkel pairs and va cancies form during high-energy ion irradiation. The kinetic energy Ek determines the rate of point defect formation and annihilation. The concentration of Frenkel defects and vacancies also directly impacts the physical properties of ternary nitrides such as stress state and elastic modulous, as it has systematically been investigated for c-(V,Al)N [27]. modulous, as it has systematically been investigated for c (V,Al)N [27]. At elevated temperatures, metastable c-(Ti,Al)N has been reported to exhibit age hardening [7] within the temperature range of 600–1000 ◦C, which can be explained by spinodal decomposition of the metastable solid solution into isostructural c-TiN- and c-AlN-rich domains [35]. Further increase in annealing temperature results in the transformation of metastable c-AlN-rich domains into the thermodynamically stable wurtzite (w-)AlN phase [7], which is accompanied by an undesirable volume increase of 22% [36] and hardness reduction up to 35% [37]. While many parameters affect the thermal stability of c-(Ti,Al)N such as the Al [37] and N [8,38] content, microstructure [39,40], stress state [41,42], and synthesis processing route [8,38,43], the effect of point defects on thermal stability has been just recently investigated [8,38, 44]. The focus in these studies was mainly on the presence of vacancies which accommodate the thin film off-stoichiometry. For magnetron sputtered (Ti,Al)Nx [8], minimization of the vacancy concentration resulted in enhanced thermal stability. In contrast, for cathodic arc-deposited (Ti,Al)Nx thin films [44], the highest thermal stability was obtained for thin films with N vacancies. Recently, Holzapfel et al. 2. Experimental details [28] have investigated the effect of ion irradiation-induced Frenkel pair de fects, generated within the Ek range from 64 to 91 eV, on the thermal stability of c-(Ti,Al)N deposited by CAD. It has been demonstrated that the presence of Frenkel pairs formed by ion irradiation enhances the thermal stability by retarding the formation of the wurtzite solid solu tion. This enhancement was attributed to the much larger activation energy for bulk diffusion of Al in the presence of Frenkel pairs, based on ab initio predictions [28]. The second series of (Ti,Al)N thin films was deposited by CAD using the industrial deposition plant Ingenia P3eTM (Oerlikon Surface Solu tions, Liechtenstein). α-Al2O3 (0001) substrates (1 × 1 cm2) and Fe foils (99.5% purity) were mounted onto the sample holder. When a base pressure < 3 × 10−4 Pa was reached, the substrates were heated to Ts = 450◦C and etched at a pressure of 0.5 Pa in an Ar/H2 atmosphere for 20 min. The thin films were deposited in a stationary geometry with a cathode-to-substrate distance of 16 cm. A Ti0.5Al0.5 composite cathode was used. The N2 pressure in the deposition chamber was kept constant at 8.0 Pa. A DC substrate bias potential was varied between -11 V (floating) and -200 V. The deposition time was 20 min resulting in films with a thickness of ~3.5 μm. The venting temperature was always < 100 ◦C for both processing routes to avoid surface chemistry modifica tion due to air exposure [46]. The Fe foil substrates were etched in nitric acid solution with HNO3/ deionized H2O volume ratio of 1/5 and subsequently dried. Then, the thin film flakes were milled into a powder using a mortar, and powder samples corresponding to thin film samples (on the Al2O3 substrates) were produced. Here, we systematically investigate the effect of ion irradiation on the structure evolution, mechanical properties, and thermal stability of c-(Ti,Al)N thin films deposited by different processing routes, HPPMS and CAD, within the ion kinetic energy range of 19–236 eV. The experimental findings are correlated with ion irradiation simulations of c-(Ti,Al)N using the thermal spike model [27–29]. It is demonstrated that the ion irradiation-induced changes in elastic modulus and thermal An energy-resolving mass spectrometer (PPM 422, Pfeiffer Vacuum, Asslar, Germany) was employed to characterize the plasma chemistry through a mass-to-charge scan at a fixed energy. 1. Introduction kinetically limited low-temperature growth conditions [10]. The state-of-the-art physical vapor deposition techniques such as high power pulsed magnetron sputtering (HPPMS) [11,12] and cathodic arc depo sition (CAD) [13,14] are frequently employed to synthesize c-(Ti,Al)N. kinetically limited low-temperature growth conditions [10]. The state-of-the-art physical vapor deposition techniques such as high power pulsed magnetron sputtering (HPPMS) [11,12] and cathodic arc depo sition (CAD) [13,14] are frequently employed to synthesize c-(Ti,Al)N. Due to the high power density in HPPMS [15] and CAD processes [14], high-energy ionized particle fluxes are directed towards the growing film surface. Additionally, a substrate bias potential ranging from tens to hundreds of volts is often applied to control the ion kinetic energy (Ek) during film growth. Therefore, structural defects are ex pected to form and annihilate, of which some are preserved during Metastable cubic titanium aluminum nitride (c-(Ti,Al)N, space group Fm3m, NaCl prototype structure) [1,2] is the current benchmark coating material system in cutting and forming [3,4] as well as in oxidation protection applications [1,5,6]. The importance of (Ti,Al)N for such applications originates from the combination of high hardness and elasticity [2], low wear [1], large thermal stability [7,8], and superior oxidation resistance [9]. These unique properties are enabled by the formation of metastable cubic (Ti,Al)N solid solutions which require Due to the high power density in HPPMS [15] and CAD processes [14], high-energy ionized particle fluxes are directed towards the growing film surface. Additionally, a substrate bias potential ranging from tens to hundreds of volts is often applied to control the ion kinetic energy (Ek) during film growth. Therefore, structural defects are ex pected to form and annihilate, of which some are preserved during S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 stability of c-(Ti,Al)N thin films deposited by HPPMS and CAD can be understood based on the ion kinetic energy and, for the first time, based on the orientation-dependent formation of Frenkel defects. growth. In transmission electron microscopy (TEM) studies by Hultman et al. [16,17] of TiN thin films (isostructural to (Ti,Al)N), it has been proposed that dislocation loops observed on the (111) plane are the primary defect types and that they are formed by agglomeration of ion irradiation-induced point defects [17]. However, the energetics of forming these defects preferentially on the (111) planes were not dis cussed. 1. Introduction Since then, follow-up studies showed elastic anisotropy of cubic transition metal nitrides (c-TMN) [18] and cubic transition metal aluminum nitrides (c-(TM,Al)N) [19,20]. For c-TiN, the largest elastic modulus has been predicted along (200) orientation and the lowest along (111) orientation. However, increasing the Al-content strengthens cubic (Ti,Al)N along (200) according to the previous theoretical work [19]. Dependence of other physical properties of c-(Ti,Al)N on preffered orentation remains unfathomed even though it can be learned from other systems that for example thermoelectricity [21], magnetism [22], electrical, and optical properties [23], are affected. 2. Experimental details i Cross-sectional thin film lamellae were prepared by focused ion beam techniques in a FEI Helios Nanolab 660 dual-beam microscope (Hillsboro, OR, USA). The microstructure was characterized using scanning transmission electron microscopy (STEM) at 30 kV and 50 pA within the same microscope and bright field (BF) micrographs were acquired with a STEM III detector. The chemical composition of the thin films was characterized by time-of-flight elastic recoil detection analysis (ToF-ERDA) at the Tan dem Laboratory of Uppsala University [47]. 36 MeV 127I8+ primary ions were directed onto the sample at an angle of 22.5◦with respect to the surface. Scattered and recoiling target particles were detected at 22.5◦ relative to the sample surface, in IBM geometry. The detection telescope was consequently located at 45◦with respect to the primary ion beam and the time-of-flight was measured using thin C foils [48]. Depth profiles were obtained from time-energy coincidence spectra by using the CONTES code [49]. Total maximum measurement uncertainties, obtained by combination of ToF-ERDA and Rutherford backscattering spectrometry (RBS) was 4% relative deviation of the deduced values for N and aliquot fractions for Ti and Al [28]. i Transmission electron microscopy (TEM) analyses were performed using a probe Cs-corrected FEI Titan3 G2 60-300 (Hillsboro, OR, USA) operating at 300 kV accelerating voltage. BF-TEM images and selected area electron diffraction (SAED) patterns for the cross-sections of the samples were obtained with a Gatan CCD camera (Gatan Inc., Pleas anton, CA, USA). The thermal response of (Ti,Al)N powders was investigated in a differential scanning calorimeter (DSC) Jupiter® STA 449 C produced by Netzsch (Selb, Germany) in a continuous heating mode and Ar at mosphere with a purity of 99.9999%. Initially, the powders were out gassed for 30 min at 150 ◦C. The measurements were then started by heating up the powders to 1500 ◦C in 30 sccm flow of Ar. The heating and cooling rates were kept at 20 and 40 K/min, respectively. An inte grated oxygen trap system (OTS®) enabled a low oxygen concentration (<1 ppm) in the measurement atmosphere. For temperature and sensi tivity calibration of the system, high purity standards (≥99.9%) (Zn, Sn, Al, Ag, Au, Pd) were melted. Structural analysis of the thin films were performed with a Siemens D5000 X-ray diffraction (XRD) system (Munich, Germany) using a Cu Kα radiation source, operated at a voltage and current of 40 kV and 40 mA, respectively. 2. Experimental details Time-averaged ion energy distribution functions (IEDFs) of the most dominant positive ions, identified in the mass scans, were subsequently obtained through energy scans at fixed mass-to-charge ratios. The spectrometer orifice 2 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. expansion of the film (9.7 × 10−6 K−1 for Ti0.56Al0.44N and 9.9 × 10−6 K−1 for Ti0.50Al0.50N, calculated in this study) and substrate (5.5 × 10−6 K−1 for α-Al2O3, provided by the substrate supplier). The temperature decrease ΔT after cooling from Ts to room temperature was ~ 430 K for all depositions. Finally, the intrinsic stress (σintrinsic) was calculated by subtracting the σthermal contribution from the σresidual. with a diameter of 100 μm was grounded in all measurements. For the analysis of the HPPMS discharge, the measurements were performed under the same conditions used for the film growth, but without heating. For the CAD discharge, the data were collected at the same pressure- distance product of 128 Pa cm as in the growth condition of the thin films. l i The energy fluxes of the individual ions are calculated using: Elastic modulus E was determined by nanoindentation using a Hysitron (Minneapolis, MN, USA) TI-900 TriboIndenter equipped with a Berkovich geometry-diamond tip with a 100 nm radius. At least 50 quasistatic indents, with a maximum load of 8 mN, corresponding to contact depths between 98 and 108 nm, were performed. The tip area function was determined with a fused silica reference before each measurement series and verified to remain unchanged thereafter. The reduced modulus was acquired from the unloading segment of load- displacement curves using the method of Oliver and Pharr [52]. The elastic moduli of the films were then attained from the reduced moduli data using the Poisson’s ratio of vs = 0.214 [24]. i energy flux = ∫ (E × IEDF(E))dE (1) (1) in which E is the ion energy. The kinetic energy of ionized species arriving at a biased growing film can be expressed as: Ek = E∘ i + qe ( Vplasma −Vs ) (2) (2) where E∘ i denotes the initial kinetic energy of the ions prior to entering the substrate sheath. The kinetic energy gained from acceleration across the substrate sheath is qe(Vplasma − Vs), where q represents the ion charge state, e is the elementary charge, Vplasma is the plasma potential, and Vs is the negative substrate bias potential. 2. Experimental details The X-ray source and the detector were coupled in θ-2θ scans (Bragg-Brentano geometry), scanning a 2θ range from 30◦to 70◦at a step size of 0.05◦, and dwell time of 2 s per step. High energy XRD measurements on the powdered coatings were done in transmission geometry at the beamline P02.1 [50] of the Deutsches Elektronen-Synchrotron (DESY) in Hamburg, Germany, using X-rays with a wavelength of 0.20701 Å. The powders were sealed in fused silica capillaries with a diameter of 1 mm and a wall thickness of 20 μm under Ar (99.9999% purity) atmosphere. The local chemical composition at the nanometer scale was deter mined by atom probe tomography (APT) using a local electrode atom probe 4000X HR (CAMECA, Madison, WI, USA). Field evaporation was assisted by 30 pJ laser pulsing at 200 kHz frequency and the base temperature was 60 K. The detection rate was set to 0.5%. Data recon struction was performed with the IVAS 3.8.8, (CAMECA) using the evolution of the standing voltage and shank angle protocol. The fre quency distribution functions (FDF) were obtained at a bin size of 50, excluding unranged ions. X-ray stress and lattice parameter analyses were performed on the thin films using the sin2ψ method in a ψ geometry (ψ represents the tilt axis in the diffraction plane) over the (200) diffraction peak with a Bruker D8 Discover General Area Diffraction Detection System (Bill erica, MA, USA) by assuming a biaxial stress state [51]. ψ was varied from 0 to 60◦, corresponding to sin2ψ values of 0–0.75. The residual stress state (σresidual) was determined by a least-square fit of the slope of the strain ε as a function of sin2ψ according to: 4.1. Plasma analysis where ETi, EAl, and EN, are total DFT energies of single (isolated) Ti, Al, and N atoms, respectively, m, n, and k, are the number of atoms in the supercells, and ETiAlN, is the total energy of (Ti,Al)N supercells. To determine the temperature-dependent equilibrium volume (lattice parameter), bulk modulus, and linear coefficient of thermal expansion, the Debye-Grüneisen model [61] was used, as described elsewhere [29]. The ion irradiation-induced intrinsic stress state evolution was calcu lated for each bombarded supercell by considering the volume change with respect to the equilibrium volume of the ideal defect-free structure at Ek = 0 eV. Here, the expansion (contraction) of the supercell due to the presence of point defects results in the generation of compressive (tensile) stress. The elastic modulus corresponding to each defect structure was calculated from the bulk modulus within the isotropic approximation, using a Poisson’s ratio of ν = 0.214 [24]. In order to obtain the stress dependence of the elastic moduli, a volume offset from the equilibrium volume was considered for each thermal spike configuration. where ETi, EAl, and EN, are total DFT energies of single (isolated) Ti, Al, and N atoms, respectively, m, n, and k, are the number of atoms in the supercells, and ETiAlN, is the total energy of (Ti,Al)N supercells. To determine the temperature-dependent equilibrium volume (lattice parameter), bulk modulus, and linear coefficient of thermal expansion, the Debye-Grüneisen model [61] was used, as described elsewhere [29]. The ion irradiation-induced intrinsic stress state evolution was calcu lated for each bombarded supercell by considering the volume change with respect to the equilibrium volume of the ideal defect-free structure at Ek = 0 eV. Here, the expansion (contraction) of the supercell due to the presence of point defects results in the generation of compressive (tensile) stress. The elastic modulus corresponding to each defect structure was calculated from the bulk modulus within the isotropic approximation, using a Poisson’s ratio of ν = 0.214 [24]. In order to obtain the stress dependence of the elastic moduli, a volume offset from the equilibrium volume was considered for each thermal spike configuration. The charge states, kinetic energies, and ion fluxes of generated positive ions were characterized and compared for HPPMS and CAD plasmas in this study. The time-averaged IEDFs of gaseous as well as metallic species of Ti and Al in HPPMS and CAD discharges are shown in Fig. 1(a–d). Fig. 3. Theoretical details The defect structures obtained from the low kinetic energy DFT-based MD simulations were used to define the instantaneous energy (Ed), based on the Kinchin-Pease equation [58], N = Ek 2Ed. Here, the number of point defects (N), obtained from the structures after the MD runs, is directly correlated to the initial kinetic energy (Ek) of the irradiating ions. The calculated Ed values for Ti0.5Al0.5N(200), Ti0.56Al0.44N(200), and Ti0.56Al0.44N(111) irradiated surfaces are 4.4 ± 0.4, 4.6 ± 0.5, and 3.8 ± 0.4 eV, respectively. Ed has been shown to be close to the activation energy for diffusion [25,29]. For construction of the surface slab models, the three surfaces (200), (220), and (111) were considered and distinct supercells of bulk c-(Ti, Al)N were generated with 128 atoms, 32 atoms and 96 atoms in the (200), (220) and (111) case, respectively, with the surface to be created always parallel to the ab plane of the simulation cell. We have chosen orthorhombic unit cells for (220) and (111) cases for consistency with structural models used in preceding calculation series. These structures were then fully optimized at 0 K with the lattice parameters of the resulting supercells (a, b, c) of (8.35 Å, 8.35 Å, 16.70 Å), (6.01 Å, 4.14 Å, 11.80 Å), and (5.90 Å, 10.22 Å, 14.46 Å), for (200), (220), and (111) structural models, respectively. From the optimized structures, slab models of the (200), (220), and (111) surfaces were constructed by adding a vacuum region of 10 Å in the c direction of the supercell. The convergence tests were done for different slab thicknesses by success fully adding or removing further atomic layers, with energetic conver gence within numerical limits of the utilized method obtained for all systems using the structural models described above. Thus, the chosen size yields optimum tradeoff between runtime and accuracy. In the case of the (111) surface, the N-terminated variant, corresponding to the orthorhombic structural models used for the thermal spike calculations, was considered. These slab models were in turn optimized with respect to the atomic positions, while keeping cell shape and size constant. The resulting surface energies were then calculated according to the following formula: In order to consider the impact of ion irradiation with Ek covering hundreds of eV, the DFT-based thermal spike model was employed as fully described in our previous works [27,29]. 3. Theoretical details The plasma-surface interactions were investigated by DFT-based [53] molecular dynamics (MD) simulations together with the DFT thermal spike model [29]. The OpenMX code was used for MD simula tions due to its computational speed and simultaneously sufficient ac curacy [54]. The basis functions were in a form of a linear combination of localized pseudoatomic orbitals [55] in conjunction with the gener alized gradient approximation (GGA) [56], and generated using a confinement scheme [55,57] specified as: Ti7.0-s3p2d1, Al7.0-s2p2d1, N6.0-s2p2d1, and Ar5.0-s2p1. Here, Ti, Al, N, and Ar designate the chemical name followed by the cutoff radius (Bohr radius unit) and the corresponding primitive orbitals. A total energy precision of 10−6 Ry atom−1 was reached by choosing an energy cutoff of 150 Ry. Slab models of 512 atoms for (200) surface (equivalent to (100) for the present structures) and 384 atoms for (111) surface (N-terminated) of ε = dψ −d0 d0 = (1 + ν) E σresidual sin2ψ (3) (3) where ν is the Poisson’s ratio and E is the elastic (Young’s) modulus of the thin films. The thermal stress σthermal contribution to the total stress state arising during cooling of the samples from Ts to room temperature, was esti mated by: σthermal = ΔαΔT ( E 1 −ν ) (4) (4) where Δα is the difference between the linear coefficients of thermal where Δα is the difference between the linear coefficients of thermal 3 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. Ti0.50Al0.50N and Ti0.56Al0.44N were used, where the bottom layer of each configuration was frozen to mimic an infinite bulk. The clustered configuration C#3 for c-(Ti,Al)N was considered as proposed by Mayrhofer et al. [2], where the number of Ti–Al second order nearest neighbors was minimized. To describe the Ti0.5Al0.5N(200), Ti0.56Al0.44N(200), and Ti0.56Al0.44N(111) surfaces in the slab configu ration, a 10 Å thick vacuum layer was introduced. Ar atoms with Ek = 30 eV and Ek = 60 eV were primarily placed at atop positions above Ti, Al, and N surface sites at a distance of 3 Å. The MD simulations were performed for a period of 400 fs with a timestep of 1 fs at 723 K (velocity scaling thermostat, canonical ensemble), corresponding to the growth temperature of (Ti,Al)N thin films in this work. 3. Theoretical details The Visualization for Electronic and Structural Analysis (VESTA) code was used for the evaluation of the final atomic configurations [59]. Full structural relaxation of the slabs at 0 K was performed for each defect configura tion. Subsequently, in order to obtain the equilibrium volume of the defect configurations, the irradiation-induced resputtered atoms, which moved above the pristine surface were removed (creating single vacancy sites within the supercells) and structural relaxation at 0 K was per formed. The Birch-Murnaghan equation of state [60] was then used to obtain the equilibrium volume. The cohesive energy (Ecoh) of each irradiated supercell was calculated based on: Esurf = Etot,slab −Etot,bulk 2 ab (6) Esurf = Etot,slab −Etot,bulk 2 ab (6) with Esurf as the surface energy, Etot,bulk and Etot,slab the total DFT energies after optimization of the bulk and corresponding slab models, respec tively, and a and b as the lattice parameters of the respective slab model’s simulation cell. The factor 2 accounts for the cleavage of the bulk crystal and the corresponding generation of two equivalent surfaces. Ecoh = (mETi + nEAl + kEN −ETiAlN) m + n + k (5) 4. Results and discussion (5) 4.1. Plasma analysis 4 Acta Materialia 250 (2023) 118 Fig. 1. Time-averaged IEDFs of (a) gaseous and (b) metallic species of a Ti–Al HPPMS discharge in Ar/N2 atmosphere and (c) gaseous and (d) metallic species Ti–Al cathodic arc discharge in N2 atmosphere. S. Karimi Aghda et al. S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 Acta Materialia 250 (2023) Karimi Aghda et al. 1. Time-averaged IEDFs of (a) gaseous and (b) metallic species of a Ti–Al HPPMS discharge in Ar/N2 atmosphere and (c) gaseous and (d) metallic species in a Al cathodic arc discharge in N atmosphere Fig. 1. Time-averaged IEDFs of (a) gaseous and (b) metallic species of a Ti–Al HPPMS discharge in Ar/N2 atmosphere and (c) gaseous and (d) metallic species in a Ti–Al cathodic arc discharge in N2 atmosphere. seous and (b) metallic species of a Ti–Al HPPMS discharge in Ar/N2 atmosphere and (c) gaseous and (d) metallic species in a mosphere. Fig. 1. Time-averaged IEDFs of (a) gaseous and (b) metallic species of a Ti–Al HPPMS discharge in Ar/N2 atmosphere an Ti–Al cathodic arc discharge in N2 atmosphere. large energy density provided in CAD process is the primary reason for the larger population of the detected doubly charged Ti2+ and molecular N3 + ions [69]. accounts for a higher charge state as well as higher average atomic mass. In addition, as the most probable energy range for both processing routes account for more than 80% of the total ion population, for the discussion of ion kinetic energy we have adopted the concept of average energy for simplicity. The average ion energy increases only slightly from 4.9 to 5.4 for HPPMS and CAD plasmas, respectively. Based on the average charge states and ion energies obtained from the IEDFs, by changing the magnitude of substrate bias potential, the most probable ion kinetic energy (Ek) ranging from 25 to 205 eV for HPPMS and 19 to 236 eV for CAD, are investigated in this work. l The average charge states, atomic masses, and ion energies (Eq. (2)) are calculated from the IEDFs and summarized in Table 1. There is a slight increase in average charge state from 1.01 to 1.15 and in average atomic mass from 39.3 to 42.7 amu comparing the HPPMS and CAD plasmas, respectively. 4.1. Plasma analysis 1 shows that, independent of the plasma source under the here chosen process conditions, the most probable ion energy is in the range of ~ 5 ± 1 eV corresponding to ~80 and ~85% of the total ion population for the HPPMS and CAD processes, respectively. This most probable ion energy is followed by an energy tail extending up to ~70 eV for the metallic species. The extended energy tails originate from the mechanisms of the sputtering or cathodic arc processes and can be associated with the remaining kinetic energy of the species after trans port through the gas phase with no collisions, and hence without change in kinetic ion energy. In addition, in HPPMS, the high energy ions might also originate from the time-variation of the plasma potential during HPPMS pulses and the subsequent acceleration of ions over the modu lated sheath potential [68]. In HPPMS (Fig. 1(a) and (b)), the majority of the ions are singly charged Ar+, N2 +, N+, Ti+, and Al+ ions with the population of detected doubly charged Ar2+, Ti2+ and Al2+ ions being less than 2% of the overall ionic plasma composition. Hence, the HPPMS plasma is mainly dominated by singly charged ions, with Ar+ being the most abundant species with ~52% of the total ionic plasma composi tion. The shape and energy range of the Ti+ and Al+ EDFs are compa rable for both HPPMS and CAE plasmas. However, in CAD (Fig. 1(c) and (d)), there is a significant contribution of doubly charged Ti2+ ions ac counting for ~15% of the energetic high-intensity fluxes. In addition, a significant amount of N3 + ions was detected as the gaseous species. The i The surface energies were obtained using DFT calculations per formed with the Vienna ab initio simulation package (VASP, University of Vienna, version 5.4.4) [62–64], within the framework of the GGA, utilizing projector-augmented waves for basis set representation [65], with the cutoff energy set to 500 eV. Exchange and correlation effects were accounted for using the well-established functional by Perdew, Burke, and Ernzerhof (PBE) [56] and, hence, the same framework as for the OpenMX code. Brillouin zone integration was handled via Bl¨ochl’s tetrahedron method [66] in a k-mesh of sufficient density to ensure energetic convergence, depending on supercell size, generated with the Monkhorst-Pack approach [67]. Spin polarization was not considered as c-(Ti,Al)N is not magnetically active. 4.1. Plasma analysis These values are directly connected to the pres ence of ~15% doubly charged Ti2+ ions in the CAD plasma, which Furthermore, the differences in the ion flux between the processing routes were analyzed by measuring the ion current density at the sub strate and the results are shown in Table 1. The substrate ion current density in the HPPMS process is 0.9 mA/cm2. This is calculated by integrating the measured ion current at the substrate within 100 µs from the target pulse on-time, as the majority of the ions are expected to arrive at the substrate within a short time-span [70]. In contrast, the higher ionization degree in the CAD mode resulted in a continuous ion flux with an ion-current density of 3.0 mA/cm2 which is 3.3 times higher than in the case of HPPMS. Therefore, the ion flux arriving at the growing film surface is significantly larger for CAD than HPPMS, while Table 1 Average charge state, atomic mass, ion energy, and the substrate ion current density in the HPPMS and CAD discharges. Process Average charge state Average atomic mass [amu] Average ion energy [eV] Substrate ion current density (mA/cm2) HPPMS 1.01 39.3 4.9 0.9 CAD 1.15 42.7 5.4 3.0 CAD HPPMS ratio 1.2 1.1 1.1 3.3 Table 1 Average charge state, atomic mass, ion energy, and the substrate ion current density in the HPPMS and CAD discharges. Table 1 Average charge state, atomic mass, ion energy, and the substrate ion current density in the HPPMS and CAD discharges. 5 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. the difference in the average charge state, atomic mass, and ion energy are comparatively small between the processing routes. of film with a (111) preferred orientation is observed. As the Ek is increased, the intensity of the (111) diffraction peak reduces and a random orientation is present at Ek ≥155 eV, which is accompanied by peak broadening. The TEM analysis and SAED pattern of the thin film deposited by HPPMS at Ek = 205 eV are shown in Fig. S2 of the sup plementary materials. This data reveal formation of a single-phase cubic structure of thin film deposited under the maximum ion kinetic energy of 205 eV in HPPMS process. Therefore, the shoulders observed for the peaks at (111) and (220) diffractions might be due to the lattice parameter distortions. In contrast, in CAD, at Ek = 19 eV, a close-to- random orientation is observed, while the intensity of the (200) diffraction peak dominants at Ek = 52 eV. Further increase of Ek to ≥ 144 eV results in a very strong (111) preferred orientation. The observed structural (preferred orientation) evolution of the thin films deposited on Al2O3 substrates are comparable to the thin films which are deposited on Fe foils for the purpose of powered-sample preparation, see Figure S3 of the supplementary materials. Generally, the evolution of preferred orientation during thin film growth is understood to be both thermo dynamically and kinetically controlled [75–77]. in thermodynamic equilibrium, the preferred orientation is defined by the minimization of the sum of surface energy E(hkl) surf and strain energy E(hkl) strain of grains with different orientation. The calculated E(hkl) surf for the metastable cubic Ti0.50Al0.50N in this study shows the lowest surface energy for the (200) plane, E(200) surf = 1.29 J/m2, in comparison to the (220) plane, E(220) surf = 2.64 J/m2, and (111) plane (N-terminated), E(111) surf = 3.20 J/m2. This is consistent with the surface energy data calculated by Forslund et al. [78]. Additionally, the directional elastic anisotropy of c-(Ti,Al)N [20] accounts for the strain energy anisotropy as E(111) strain > E(220) strain > E(200) strain. Therefore, for a defect-free system the (200) plane shows the lowest surface energy as well as the lowest strain energy. 4.2. Thin film composition Average chemical compositions of the thin films were obtained from homogeneous ERDA depth profiles and are presented in Fig. 2. The oxygen content in all thin films was below 0.5 at%. Independent of the processing route and the magnitude of Ek, (Ti,Al)N thin films exhibit overstoichiometric compositions with respect to nitrogen. The measured N content is between 51 and 52 at.% for all the here investigated thin films and this variation is within the maximum total measurement un certainty of 2 at.%. However, the composition of metallic species de pends on the magnitude of Ek. In the following, Al concentrations are presented with the sum of systematic and statistic uncertainties. The Al content in the thin films deposited by HPPMS changes from 25 ± 1 at% at Ek = 25 eV to 23 ± 1 at.% at Ek = 205 eV. A less significant reduction in Al content is observed for the CAD thin films from 22 ± 1 at% to 21 ± 1 at% as the Ek is increased from 19 to 236 eV, respectively. The here observed reduction in the Al content by increasing the magnitude of Ek in case of HPPMS can be rationalized by the ion irradiation-induced preferential resputtering of lighter Al atoms from the surface of the growing film [71,72]. The energy of formation for Al vacancies was calculated to be the lowest amongst all the species in c-(Ti,Al)N [73]. While the target composition for both processing routes was identical (Ti0.5Al0.5), the CAD thin films exhibit, on average, ~13% lower Al content as compared to HPPMS thin films. This can be understood by comparing the pressure-distance product (pd) [74] difference between the HPPMS (pd = 5 Pa cm) and CAD (pd = 128 Pa cm) conditions. The significantly higher pd of CAD may result in stronger scattering of the lighter Al compared to Ti, hence, less Al is incorporated during growth. Based on the data presented in Fig. 2, the dashed lines indicate the average compositions of (Ti0.50Al0.50)0.48N0.52 and (Ti0.56Al0.44)0.48N0.52 as calculated for HPPMS and CAD thin films, respectively, which are used for the remaining discussion. Table 1 However, our cal culations for the high kinetic energy ion irradiation-induced defect structures show that the cohesive energy is defect structure-dependent. We have shown in Fig. 6 (see below) that the ion irradiation-induced generation of point defects would result in a lower cohesive energy along (111) direction in comparison to (200) direction, contrary to the calculated data of defect-free systems by Tasn´adi et al. [19]. Hence, the defect structure-dependent changes in elastic anisotropy, proportional to cohesive energies, results in a lower strain energy along (111) di rection for (Ti,Al)N. Therefore, as thermodynamic equilibrium is approached, both E(hkl) surf and E(hkl) strain are minimized. At low ion irradiation conditions, E(hkl) strain is negligible, thus, a (200) preferred orientation (lowest surface energy) is expected. However, during growth of HPPMS thin films under low mobility condition, Ek = 25 eV, adatoms tend to be preferentially incorporated in the low diffusivity planes, which results in 4.3. Thin film structural analysis The ion energy-dependent phase formation of (Ti,Al)N was charac terized by XRD and is presented in Fig. 3 for the thin films synthesized on Al2O3 substrates by (a) HPPMS and (b) CAD. The diffractograms show the formation of a single-phase metastable cubic (Ti,Al)N solid solution, without presence of the wurtzite AlN, independent of the processing route and magnitude of Ek. However, there are distinct discernible differences in ion-energy-dependent evolution of preferred orientation for HPPMS and CAD thin films. In HPPMS, at Ek = 25 eV, the formation Fig. 2. Chemical composition of (a) HPPMS- and (b) CAD-synthesized (Ti,Al)N thin films as determined by ERDA with respect to the ion kinetic energy. The dashed lines indicate the average films composition for each processing route. Fig. 2. Chemical composition of (a) HPPMS- and (b) CAD-synthesized (Ti,Al)N thin films as determined by ERDA with respect to the ion kinetic energy. The dashed lines indicate the average films composition for each processing route. 6 Acta Materialia 250 (2023) 118 Fig. 3. Structure evolution of (Ti,Al)N thin films deposited on Al2O3 substrates by (a) HPPMS and (b) CAD techniques. S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. Karimi Aghda et al. Fig. 3. Structure evolution of (Ti,Al)N thin films deposited on Al2O3 substrates by (a) HPPMS and (b) CAD techniques. the kinetically limited, upward growth of (111) planes [76,79,80]. Increasing Ek in HPPMS provides enough surface and subsurface mobility to incorporate atoms into the planes with more open crystal channeling, namely (200) and (220) planes in comparison to (111) [75]. These results are consistent with other magnetron sputter-deposited TiN [76], (Ti,Al)N [75,81] and (V,Al)N [27] thin films. In contrast, the 3.3 times higher substrate ion current density accounts for a significantly larger ion flux in CAD. The larger ion flux enhances surface adatom mobility at low ion kinetic energies Ek ≤52 eV. Here, the surface adatom mobility is sufficient to grow low-energy (200)-orientated crystallites during nucleation and coalescence phase [81]. However, at higher irradiation energies Ek ≥144 eV in CAD, where the kinetic limitations should be even smaller than the depositions with low kinetic energies, the (111) evolution is dominant. Here the preferred orientation of (Ti, Al)N film is mainly controlled by the strain energy, hence, the preferred orientation corresponds to the (111) plane with the lowest strain energy. Fig. 4. 4.4. Defect structure To investigate the ion-surface-interactions at the atomistic scale, the thermal spike model [27–29] was used by considering the experimen tally obtained composition (Fig. 2) and preferred orientation evolution (Fig. 3) of the synthesized thin films. Here, the nominal theoretical compositions of Ti0.50Al0.50N and Ti0.56Al0.44N were chosen to represent the average chemical compositions of (Ti0.50Al0.50)0.48N0.52 and (Ti0.56Al0.44)0.48N0.52 corresponding to HPPMS and CAD thin films, respectively. The ion irradiation effects on the final defect structures were systematically investigated in the Ek range from 0 to 203, as well as 0 to 219, and 0 to 205 eV for Ti0.50Al0.50N with a (200) orientation and for Ti0.56Al0.44N with (200) and (111) orientations, respectively. Such comparison is only possible due to minute differences in the average charge state, atomic mass, and ion energy for the HPPMS and CAE processing routes, as argued above. For each of these supercell config urations, the initial and exemplary final atomic configurations after thermal spikes are shown in Fig. 4(a–c). It is evident that ion irradiation with similar kinetic energy results in surface as well as sub-surface diffusion of the atoms, independently of the here investigated compo sitions and surface orientations of the supercells. Formation of self-interstitial/vacancy point-defect pairs (Frenkel pairs) are the dominant type of point defects. We observe not only N but also metallic species as stable Frenkel defects within the c-(Ti,Al)N supercells. Addi tionally, formation of individual lattice vacancies observed as the high-energy ion irradiation also provides enough mobility for the sur face atoms to move above the pristine surface. For the irradiation of the (200) surface (Fig. 4(a) and (b)), resputtering of surface Al atoms is observed. Conversely, the N-terminated (111) polar surface of (Ti,Al)N (Fig. 4(c)) appears to hinder resputtering of Al. This is consistent with the chemical composition analysis in Fig. 2, as the HPPMS thin films showed a significant reduction in Al content with respect to Ek, while the CAD thin films did not show a significant Al reduction and exhibit a dominant (111) surface orientation. The orientation-dependent Frenkel pair concentrations can be rationalized by considering lower instantaneous energy Ed = 3.8 ± 0.4 eV for Ti0.56Al0.44N(111) (34% higher Frenkel pair concentration) in comparison to Ed = 4.6 ± 0.5 eV for Ti0.56Al0.44N(200), as calculated from DFT based MD simulations, see Section 3. The c-TMNs and c-(TM, Al)Ns are well-known to exhibit pronounced elastic anisotropy. Tasn´adi et al. 4.3. Thin film structural analysis Thermal spike model for (a) Ti0.50Al0.50N(200), (b) Ti0.56Al0.44N(200) and (c) Ti0.56Al0.44N(111) N-terminated supercells. The initial kinetic energy of the impinging ion is provided above each structure. Ti0.50Al0.50N(200), (b) Ti0.56Al0.44N(200) and (c) Ti0.56Al0.44N(111) N-terminated supercells. The initial kinetic energy of the structure. Fig. 4. Thermal spike model for (a) Ti0.50Al0.50N(200), (b) Ti0.56Al0.44N(200) and (c) Ti0.56Al0.44N(111) N-terminated supercells. The initial kinetic energy of the impinging ion is provided above each structure. 7 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. ated based on the DFT thermal spike model and the results are sum marized in Fig. 5(a–f). For (200)-terminated Ti0.50Al0.50N, there is a significant increase in the concentration of Frenkel pairs, Fig. 5(a), up to a maximum of 3.7% as Ek is increased to 154 eV, which is followed by a minor reduction to 3.5% at Ek = 203 eV. Here, the dominant Frenkel pairs present in the supercells at Ek ≥154 eV consist of N and Al species. Concurrently, formation of vacancies is evident and their concentration is directly connected to the magnitude of Ek, see Fig. 5 (b). Similarly, for Ti0.56Al0.44N(200) with a slight difference in the chemical composition of the metal sublattice, the same behavior in formation of Frenkel pairs and vacancies with respect to the magnitude of Ek is observed, see Fig. 5 (c) and (d). Here, the highest concentration of Frenkel pairs of 3.5% is observed at Ek = 170 eV, and the continuous increase in vacancy con centration reaches a maximum of 1.8% at Ek = 219 eV. While the ion irradiation-induced formation of point defects on the (111) surface fol lows the same behavior as on the (200) surface for Ti0.56Al0.44N, there is a significant difference in the maximum concentration of Frenkel pairs. As shown in Fig. 5 (e), the maximum concentration of generated Frenkel pairs is 4.7% at Ek = 182 eV, which is ~34% higher than in the case of the irradiated (200) surface at a similar Ek magnitude. While the con centration of Frenkel pairs depends on the orientation of the irradiated surface, the vacancy concentrations are very similar for (111) and (200) surfaces at comparable Ek range, see Fig. 5 (d) and (f). 4.4. Defect structure S. Karimi Aghda et al. 4.4. Defect structure The minute differences between the absolute values of the lattice parameters of the powders in comparison to those of the thin films might be due to the thermal stress contribution of up to +1.0 GPa present in the thin films but not in the powders, as they are considered to be free of macro-stresses. structures along (111) and (200) directions are calculated and the re sults are depicted in Fig. 6. At Ek = 0 eV, corresponding to the defect-free supercells, comparable Ecoh values of 8.23 and 8.22 eV/atom for the (111) and (200)-oriented structures, respectively, are obtained. How ever, by increasing Ek, which consequently results in the formation of point defects, the cohesive energy of (111)-oriented irradiated structure becomes significantly lower than that of the (200)-oriented irradiated one. These results indicate that the elastic anisotropy of Ti0.56Al0.44N is clearly defect structure dependent. The ion energy-dependent defect structures discussed in Figs. 4 and 5 are used to determine the ground state properties of c-(Ti,Al)N. Fig. 7 (a) shows the comparison between experimentally measured lattice pa rameters of the thin films together with calculated lattice parameters at 300 K (near room temperature) as a function of Ek. For HPPMS (Ti0.50Al0.50)0.48N0.52 thin films (blue squares), the lattice parameter increases by 0.8% from 4.202 to 4.231 Å as Ek is increased from 25 to 105 eV. Increasing the ion kinetic energy further to 205 eV, however, results in a 0.4% reduction of the lattice parameter to 4.216 Å. The predicted lattice parameter for Ti0.50Al0.50N(200) (red squares) shows an increase of 0.7% in the Ek range from 0 to 154 eV, followed by a reduction at higher Ek, which is in excellent agreement with the experimentally measured values for lattice parameter evolution. We have recently shown that Frenkel pairs increase the lattice parameter and vacancies contract the lattice [27]. It is evident from the chemical composition data in Fig. 2, that no significant ion irradiation-induced chemical composition change is observed for the here investigated thin films. Therefore, the maximum increase in the lattice parameter by 0.7% in Ti0.50Al0.50N(200) is caused by the Frenkel pair concentration of 3.7% that overcompensates the concurrent vacancy-induced reduction in lattice parameter, see Fig. 5 (a) and (b). 4.5. Thermal stability To probe the effect of ion irradiation-induced defects on the thermal stability of (Ti,Al)N, the heat response of the powders discussed in Fig. 7 (b) and (c) was evaluated by DSC and the results are presented in Fig. 8. The investigated powders show their first enthalpic signals, consistent with recovery processes [7] at ~500 ◦C for HPPMS powders, Fig. 8 (a) and (c), and ~650◦C for CAD powders, Fig. 8 (e) and (g). The DSC signal of the HPPMS powder deposited at Ek = 25 eV, Fig. 8 (a), exhibits a second and third exothermic peak at 900 and 1150 ◦C, respectively, which indicate, according to Mayrhofer et al. [7,82], the onset of spi nodal decomposition and wurtzite phase formation, respectively. These peaks are also observed for the powder deposited at Ek = 155 eV, Fig. 8 (c), at similar temperatures. Comparable to the HPPMS powders, the average onset temperature of spinodal decomposition for the CAD powders occurs at ~900 ◦C, independently of Ek, Fig. 8(e) and (g), while the onset of wurtzite formation for the CAD powder deposited at Ek = 19 eV, Fig. 8 (e), occurs at 1100 ◦C, similar to the case of the HPPMS powders. However, the third exothermic peak, corresponding to the onset of wurtzite formation, is not detected in the DSC signal of the CAD powder deposited at Ek = 144 eV, Fig. 8(g). The XRD patterns of pow ders after annealing are shown in Fig. 8(i) and (j) for HPPMS and CAD powders, respectively. For all four samples, two phases can be detected, corresponding to the c-TiN and the w-AlN reference lines. This indicates that the metastable solid solution has decomposed into the thermody namically stable phases, despite the fact that no exothermic peak cor responding to the wurtzite formation has been observed for the CAD powder deposited at Ek = 144 eV, Fig. 8(g). This anomalous observation may be rationalized by the contemporaneous compensation of the endothermic wurtzite phase formation [7] by the exothermic defect annihilation [25] in films with very high – ion irradiation-induced – defect concentration. Fig. 6. Cohesive energy (Ecoh) of the irradiated Ti0.56Al0.44N for irradiated (200) and (111) surfaces as a function of the ion kinetic energy. 4.4. Defect structure [19] calculated the elastic tensor for Ti1−xAlxN and showed that for Ti0.56Al0.44N the elastic modulus of the (111) direction becomes slightly stronger than the (200) direction. However, these findings pertain to defect-free supercells. To consider the effect of irradiation-induced point defects on the anisotropic behavior of Ti0.56Al0.44N, the cohesive en ergies (Ecoh), as being proportional to elastic modulus, of the irradiated The ion energy-dependent point defect concentrations were evalu Fig. 5. Concentration of Frenkel pairs (top) and vacancies (bottom) from the DFT thermal spike model with respect to the ion kinetic energy for (a) and (b) Ti0.50Al0.50N(200), (c) and (d) Ti0.56Al0.44N(200), (e) and (f) Ti0.56Al0.44N(111). Fig. 5. Concentration of Frenkel pairs (top) and vacancies (bottom) from the DFT thermal spike model with respect to the ion kinetic energy for (a) and (b) Ti0.50Al0.50N(200), (c) and (d) Ti0.56Al0.44N(200), (e) and (f) Ti0.56Al0.44N(111). 8 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. show a 0.8% increase within a similar ion energy range. However, by considering the irradiated (111) surface of Ti0.56Al0.44N (dark purple circles), as the preferred orientation observed for the CAD thin films at high Ek, see Fig. 3 (b), the lattice parameter increase reaches a maximum of 1.1% from 4.196 to 4.241 Å as the Ek is increased from 0 to 182 eV. This larger increase in case of the irradiated (111) surface in comparison to the case of the (200) surface is governed by the 34% higher Frenkel pair concentration at a corresponding Ek. Fig. 7(b) and (c) display the measured lattice parameter of the HPPMS and CAD powders, respec tively, from synchrotron diffraction data together with the theoretically obtained values as a function of Ek. The powders diffraction data (not shown) exhibit polycrystalline cubic structure of (Ti,Al)N without any secondary phase for all the powders investigated here. The lattice parameter increase in the (Ti0.50Al0.50)0.48N0.52/HPPMS powder is 0.5% as Ek is increased from 25 to 155 eV, while for (Ti0.56Al0.44)0.48N0.52/ CAE powders the value is 1.5% upon increasing Ek from 19 to 144 eV. These changes in the lattice parameters of the powders are consistent with the lattice parameter evolution of thin films as discussed in Fig. 7 (a). 4.4. Defect structure Slight deviations between the absolute values of experimentally measured and theoretically obtained lattice parameters with respect to Ek might arise from the deviations between the absolute defect concentrations between the thin films and supercells as the thermal spike model describes one single irradiation event. A similar evolution in lattice parameters of the (Ti0.56Al0.44)0.48N0.52 thin films deposited by CAD (black circles) is observed, Fig. 7(a). However, the maximum increase in the lattice parameter of 1.5% from 4.170 to 4.235 Å upon increasing Ek from 19 to 144 eV is ~2 times larger than the lattice parameter increase measured for the HPPMS deposited thin films. The calculated lattice parameters for Ti0.56Al0.44N based on the irradiation of (200) surface (pink circles) 4.5. Thermal stability Heat flow data of the powders stem from the difference between two heating cycles, to obtain only the powders calorimetric response. (i) and (j) post annealing X-ray diffraction patterns of powders deposited by HPPMS and CAD, respectively. second mass loss occurs at approximately 1260 and 1295 ◦C for the powders deposited at Ek = 25 and 155 eV, respectively, indicating a delay of 35 ◦C in the wurtzite formation temperature. For CAD synthe sized powders, this delay is significantly larger with 206 ◦C, from 1008 to 1214 ◦C, for the films grown at Ek = 19 and 144 eV, respectively. By comparing the total mass loss corresponding to the N2 loss from the thin films, the maximum N2 loss of 5.4 at.% was observed for the highly defective powder deposited by CAD at Ek = 144 eV. Hence, ion irradiation-induced point defects appear to hinder the transformation of the cubic Al-rich (Ti,Al)N structure to the thermodynamically stable wurtzite phase. The here investigated largest defect concentration inferred from the measured lattice parameter of the CAD synthesized powder (Fig. 7 (c)), consistent with predictions obtained with the thermal spike model for Ek = 144 eV (Fig. 4 (e)), results in a thermal stability increase of 206 ◦C. corresponding combined concentration of Frenkel pairs and vacancies causing the lattice parameter increase is calculated based on the thermal spike model, see Figs. 5 and 7, and is shown on the second and third x-axes of Fig. 9, respectively. Within the here employed thermal spike model, formation of vacancies and Frenkel pairs are connected and cannot be separately probed. In Fig. 9, it can be observed that the changes in the lattice parameter due to formation of irradiation-induced point defects scale with the increase in the thermal stability of c-(Ti,Al) N. The irradiation-induced increase in the lattice parameter of 0.5% for the HPPMS synthesized powders in this study is attributed to the pres ence of 1.5% Frenkel pairs and 0.2% vacancies, inferred to be respon sible for the 35 ◦C increase in the onset temperature of the wurtzite phase formation. The lattice parameter increase of 0.9% obtained due to ion irradiation for CAD synthesized powders by Holzapfel et al. [28] is attributed to 2.7% Frenkel pairs and 0.5% vacancies in the lattice, which was shown to enhance the thermal stability of the cubic solid solution by 83 ◦C. 4.5. Thermal stability Fi 6 C h i (E ) f h i di d Ti Al N f i di d The mass as a function of temperature (see thermogravimetric analysis (TGA) signals), Fig. 8(b), (d), (f), and (h), shows a slight reduction at around 450◦C (deposition temperature), which indicates nitrogen loss according to Rovere et al. [83]. This mass loss is signifi cantly larger for the samples deposited at higher ion kinetic energies (Ek = 155 eV for HPPMS powder and Ek = 144 eV for CAD powder), which can be explained by the larger absolute concentration of irradiation-induced point defects present in the growing films, being consistent with [28]. The second mass loss starting at >1000 ◦C is caused by the formation of the wurtzite phase [83]. In HPPMS, the Fig. 6. Cohesive energy (Ecoh) of the irradiated Ti0.56Al0.44N for irradiated (200) and (111) surfaces as a function of the ion kinetic energy. 9 9 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 Fig. 7. Measured lattice parameter of (a) thin films, (b) HPPMS powders, and (c) CAD powders of (Ti,Al)N together with the DFT calculated lattice parameters at 300 K with respect to ion kinetic energy. S. Karimi Aghda et al. Fig. 7. Measured lattice parameter of (a) thin films, (b) HPPMS powders, and (c) CAD powders of (Ti,Al)N together with the DFT calculated lattice parameters at 300 K with respect to ion kinetic energy. Fig. 7. Measured lattice parameter of (a) thin films, (b) HPPMS powders, and (c) CAD powders of (Ti,Al)N together with th K with respect to ion kinetic energy. Fig. 8. DSC and TGA measurements of (Ti0.50Al0.50)0.48N0.52/HPPMS powders deposited at (a) and (b) Ek = 25 eV, (c) and (d) Ek = 155 eV, and (Ti0.56Al0.44)0.48N0.52/ CAD powders deposited at (e) and (f) Ek = 19 eV, (g) and (h) Ek = 144 eV. Heat flow data of the powders stem from the difference between two heating cycles, to obtain only the powders calorimetric response. (i) and (j) post annealing X-ray diffraction patterns of powders deposited by HPPMS and CAD, respectively. Fig. 8. DSC and TGA measurements of (Ti0.50Al0.50)0.48N0.52/HPPMS powders deposited at (a) and (b) Ek = 25 eV, (c) and (d) Ek = 155 eV, and (Ti0.56Al0.44)0.48N0.52/ CAD powders deposited at (e) and (f) Ek = 19 eV, (g) and (h) Ek = 144 eV. 4.5. Thermal stability Furthermore, a much larger lattice parameter increase of 1.5% was obtained in the present work by increasing the kinetic energy of ionized species in the CAD process to 144 eV. This lattice parameter increase is caused – consistent with predictions based on the thermal corresponding combined concentration of Frenkel pairs and vacancies causing the lattice parameter increase is calculated based on the thermal spike model, see Figs. 5 and 7, and is shown on the second and third x-axes of Fig. 9, respectively. Within the here employed thermal spike model, formation of vacancies and Frenkel pairs are connected and cannot be separately probed. In Fig. 9, it can be observed that the changes in the lattice parameter due to formation of irradiation-induced point defects scale with the increase in the thermal stability of c-(Ti,Al) N. The irradiation-induced increase in the lattice parameter of 0.5% for the HPPMS synthesized powders in this study is attributed to the pres ence of 1.5% Frenkel pairs and 0.2% vacancies, inferred to be respon sible for the 35 ◦C increase in the onset temperature of the wurtzite phase formation. The lattice parameter increase of 0.9% obtained due to ion irradiation for CAD synthesized powders by Holzapfel et al. [28] is attributed to 2.7% Frenkel pairs and 0.5% vacancies in the lattice, which was shown to enhance the thermal stability of the cubic solid solution by 83 ◦C. Furthermore, a much larger lattice parameter increase of 1.5% was obtained in the present work by increasing the kinetic energy of ionized species in the CAD process to 144 eV. This lattice parameter increase is caused – consistent with predictions based on the thermal In an effort to correlate the changes in thermal stability of the cubic solid solution with the irradiation-induced structural changes, the thermal stability increase based on the TGA results from Fig. 8 are dis played in Fig. 9 with respect to the lattice parameter increase of the powders from this study and the work by Holzapfel et al. [28]. The 10 Fig. 9. Thermal stability increase as a function of lattice parameter increase obtained experimentally for c-(Ti,Al)N from this study and the work of Hol zapfel et al. [28]. The concentration of Frenkel pairs (ρFrenkel) and vacancies (ρVacancy) corresponding to the increase in lattice parameter are calculated based on the DFT thermal spike model and are shown on the second and third x-axes, respectively. 4.6. Morphological evolution and local chemical composition To understand the effect of Ek on the morphology of the c-(Ti,Al)N deposited in this study, the thin films were characterized at the nano meter scale by cross-sectional BF-STEM, see Fig. 10. All thin films investigated here show no evidence of porosity within the matrix, in dependent of the processing route and ion kinetic energy. However, pores around macroparticles were observed for all CAD synthesized thin films, being consistent with [84,85]. i i For the (Ti0.50Al0.50)0.48N0.52/HPPMS series at low Ek = 25 eV, a fine- grained columnar microstructure is visible, Fig. 10 (a). The increase in kinetic energy of ionized species up to 205 eV, Fig. 10(b) and (c), results in a reduction of column width without significantly altering the columnar growth of the thin films. Reduction in grain size at higher Ek is consistent with the extended structure zone diagram proposed by Anders [86]. Similarly, for (Ti0.56Al0.44)0.48N0.52/CAD thin films, a very fine columnar structure is observed at Ek ≤52 eV, Fig. 10(d) and (e). At Ek = 190 eV, Fig. 10(f), a distinct morphological evolution from the substrate towards the surface of the thin film is observed. Here, at the region close to the substrate, a columnar structure is grown with significantly larger column width than for the thin films deposited at Ek ≤52 eV. Further increase in the film thickness, however, resulted in the termination of the columnar growth with grains having a conical shape. Fig. 9. Thermal stability increase as a function of lattice parameter increase obtained experimentally for c-(Ti,Al)N from this study and the work of Hol zapfel et al. [28]. The concentration of Frenkel pairs (ρFrenkel) and vacancies (ρVacancy) corresponding to the increase in lattice parameter are calculated based on the DFT thermal spike model and are shown on the second and third x-axes, respectively. spike model – by the ion irradiation-induced formation of point defects (predominantly Frenkel pairs), which in turn results in a 206 ◦C enhancement of the thermal stability of the cubic solid solution. Fig. 10. Cross-sectional BF-STEM images of (Ti,Al)N thin films deposited by (top) HPPMS at (a) Ek = 25 eV, (b) Ek = 155 eV, and (c) Ek = 205 eV and (bottom) CAD at (d) Ek = 19 eV, (e) Ek = 52 eV, and (f) Ek = 190 eV. 4.5. Thermal stability S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. Previously, we have shown that the Al mobility is a decisive parameter in defining the thermal stability of c-(Ti,Al)N, while the mobility of N is not required [73]. Holzapfel et al. [28] have computed the activation energies for bulk diffusion of single Ti, Al, and N atoms in Ti0.50Al0.50N structural models with and without a Frenkel pair by means of DFT. The activation energies for bulk diffusion of Ti, Al, and N were calculated to be 3.15, 2.60, and 3.85 eV, respectively. However, the activation energy for bulk diffusion of Al in the vicinity of one Ti, Al, or N interstitial atom increases significantly by 94, 125, and 13%, respectively [28]. Therefore, the here reported Frenkel pair concentration-dependent thermal stability can be rationalized based on the increase in the activation energy for bulk diffusion of Al, thereby influencing the decomposition pathways of metastable cubic (Ti,Al)N. 4.6. Morphological evolution and local chemical composition 5) resulting in a larger mobility for renucleation. Additionally, the near-surface local chemical composition of films was measured by atom probe tomography and the data are in Figure S4 of the supplementary material. Based on the atom tions of Al and the frequency distribution functions, chemical h neity is observed for the HPPMS thin films independent Consequently, a very fine-grained nanocrystalline structure evolves near the surface region of the thin film. Fig. 11 displays higher resolution TEM micrographs together with SAED patterns of the (Ti0.56Al0.44)0.48N0.52/CAD thin films deposited at an Ek of 19 and 190 eV. The cross-sectional TEM micrograph and the SAED pattern of the thin film deposited at 19 eV, Fig. 11(a) and (b), show a columnar polycrystalline cubic structure, consistent with XRD data in Fig. 3(b). From the cross-sectional lamella of the thin film deposited at 190 eV, Fig. 11(c), the near-substrate and near-surface regions are analyzed separately by electron diffraction. The SAED pattern in Fig. 11(d) of the blue square area in Fig. 11(c) shows the single phase cubic structure of (Ti,Al)N at the near substrate regions with a dominant (111) diffraction, consistent with the XRD data in Fig. 3 (b). Fig. 11(e) and (f) display a higher resolution TEM image and SAED pattern of the regions marked by orange and red squares, respectively. From the image in Fig. 11 (e), very fine nano-crystallites embedded in a disordered structure were found. From the SAED pattern in Fig. 11 (f), it is evident that the columnar (111) structure has evolved towards a more polycrystalline nano structure. The near surface nanostructure could not be captured by the resolution of XRD as only (111) diffraction was observed in Fig. 3(b). The evolution of a fine-grained nanocrystalline structure under ion irradiation with Ek = 190 eV may be rationalized by a significant con centration of ion irradiation-induced defects (mostly Frenkel pairs, Fig. 5) resulting in a larger mobility for renucleation. magnitude of Ek. For both HPPMS thin films, the Pearson coefficient µ [87] is comparable and close to a random distribution. For CAD thin films, however, the increase of the Ek to 190 eV results in clustering of Al, with the Pearson coefficient for Al distribution reaching µAl = 0.33. 4.7. Mechanical properties The ion irradiation-induced changes in the structure of c-(Ti,Al)N are mirrored in intrinsic stress (σintrinsic) changes of the thin films. The results are illustrated in Fig. 12 (a). Independent of the processing route, a steep increase in the compressive stress state with respect to Ek is observed. The maximum compressive σintrinsic of -5.9 and -7.2 GPa are obtained for HPPMS and CAD, respectively, within similar Ek of 100–150 eV. This is accompanied by stress relaxation as Ek is increased further. This trend is consistent with the evolution of stress state predicted by the thermal spike model. Ion irradiation-induced stress generation and relaxation are correlated to the generated and annihilated Frenkel pairs and va cancies [27]. The larger maximum compressive σintrinsic observed for the CAD thin films can therefore be rationalized by the larger maximum Frenkel pair concentration on (111)-oriented irradiated surface (Figure 5 (e)) in comparison to the (200)-oriented one (Figure 5 (c)). Additionally, the near-surface local chemical composition of the thin films was measured by atom probe tomography and the data are shown in Figure S4 of the supplementary material. Based on the atomic posi tions of Al and the frequency distribution functions, chemical homoge neity is observed for the HPPMS thin films independent of the To investigate the irradiation-induced changes in elasticity of (Ti,Al) N, the elastic moduli of the thin films was measured and compared to the calculated values from the thermal spike model in Fig. 12 (b). The elastic moduli of HPPMS films vary marginally within the error bars from 514 ± 26 to 501 ± 26 GPa in the Ek range from 25 to 205 eV. Similarly, the Fig. 11. TEM characterization of (Ti,Al)N films deposited by CAD: (a) BF-TEM image of Ek = 19 eV, (b) SAED pattern of (a), (c) BF-TEM image of Ek = 190 eV, (d) SAED pattern of blue region in (c), (e) High resolution (HR) TEM image of orange region in (c), (f) SAED pattern of the red region in (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 11. 4.6. Morphological evolution and local chemical composition The deviation from the random elemental distribution in the CAD thin film at Ek = 190 eV might be correlated to the surface and sub-surface diffusion mediated clustering of atoms in the highly defective structure. 4.6. Morphological evolution and local chemical composition The dashed orange line in (f) shows the morphology alteration for CAD deposited film at Ek = 190 eV. The scale bar in (f) is valid for all the micrographs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 10. Cross-sectional BF-STEM images of (Ti,Al)N thin films deposited by (top) HPPMS at (a) Ek = 25 eV, (b) Ek = 155 eV, and (c) Ek = 205 eV and (bottom) CAD at (d) Ek = 19 eV, (e) Ek = 52 eV, and (f) Ek = 190 eV. The dashed orange line in (f) shows the morphology alteration for CAD deposited film at Ek = 190 eV. The scale bar in (f) is valid for all the micrographs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 11 Acta Materialia 250 (2023) 118864 S. Karimi Aghda et al. Consequently, a very fine-grained nanocrystalline structure evolv the surface region of the thin film. Fig. 11 displays higher res TEM micrographs together with SAED patterns o (Ti0.56Al0.44)0.48N0.52/CAD thin films deposited at an Ek of 19 a eV. The cross-sectional TEM micrograph and the SAED pattern thin film deposited at 19 eV, Fig. 11(a) and (b), show a co polycrystalline cubic structure, consistent with XRD data in Fi From the cross-sectional lamella of the thin film deposited at Fig. 11(c), the near-substrate and near-surface regions are a separately by electron diffraction. The SAED pattern in Fig. 11(d blue square area in Fig. 11(c) shows the single phase cubic stru (Ti,Al)N at the near substrate regions with a dominant (111) diff consistent with the XRD data in Fig. 3 (b). Fig. 11(e) and (f) d higher resolution TEM image and SAED pattern of the regions ma orange and red squares, respectively. From the image in Fig. 11 ( fine nano-crystallites embedded in a disordered structure were From the SAED pattern in Fig. 11 (f), it is evident that the co (111) structure has evolved towards a more polycrystalline structure. The near surface nanostructure could not be captured resolution of XRD as only (111) diffraction was observed in Fi The evolution of a fine-grained nanocrystalline structure un irradiation with Ek = 190 eV may be rationalized by a significa centration of ion irradiation-induced defects (mostly Frenke Fig. 4.7. Mechanical properties Additionally, the ion irradiation-induced changes in elastic modulus can be understood based on the ion energy- and orientation-dependent point defect formation from ab initio calculations. For HPPMS thin films with random orientation, the concurrent effects of ion irradiation- induced formation of Frenkel pairs and generation of compressive stresses resulted in an apparent independence of the elastic modulus on Ek. In contrast, for CAD thin films with (111) preferred orientation, an ~18% reduction in elastic modulus was obtained. It is shown that the reduction of elastic modulus for CAD thin films is due to the significantly larger concentration of Frenkel pairs formed upon irradiation of the experimentally observed (111) orientation in comparison to the (200) orientation. At Ek = 182 eV, irradiation of the (111)-oriented surface leads to ~34% higher Frenkel pair concentration than for the (200)- oriented surface irradiation at similar Ek values. Moreover, we present evidence of ion-energy-dependent thermal stability enhancement of (Ti, Al)N. DSC data reveal that, independent of the processing route, the ion irradiation-induced increase in Frenkel pair concentration leads to an increase in the onset temperature of wurtzite formation by up to 206 ◦C. Therefore, it is evident that ion irradiation-induced changes in elastic modulus and thermal stability of c-(Ti,Al)N thin films can be understood based on the ion kinetic energy and orientation-dependent formation of point defects. calculated elastic moduli of Ti0.50Al0.50N(200) show no apparent alter ation with respect to Ek with values between 463 and 480 GPa. The calculated elastic modulus data combines both the presence of point defects and also the generated intrinsic stress state contribution as depicted in Fig. 12(a). N. Additionally, the ion irradiation-induced changes in elastic modulus can be understood based on the ion energy- and orientation-dependent point defect formation from ab initio calculations. For HPPMS thin films with random orientation, the concurrent effects of ion irradiation- induced formation of Frenkel pairs and generation of compressive stresses resulted in an apparent independence of the elastic modulus on i It has been recently shown for the isostructural c-(V,Al)N [27] that the apparent independence of the elastic modulus of HPPMS thin films from Ek can be explained by the concurrent compressive stress evolution (causing an increase in elastic modulus) and point defect formation (causing a decrease in elastic modulus). Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 4.7. Mechanical properties The elastic modulus of (Ti0.56Al0.44)0.48N0.52/CAD thin films, however, shows an 18% reduction from 527 ± 38 to 421 ± 39 GPa as Ek is increased from 19 to 190 eV. This reduction in elasticity is not observed in the theoretically obtained values from the irradiation of (200)-oriented surface in Ti0.56Al0.44N (200) due to the balancing effects of compressive stress generation and point defect formation. However, the reduction in elastic modulus is captured by considering the irradiation of Ti0.56Al0.44N(111), which is the dominant preferred orientation of CAD thin films at higher Ek > 144 eV (Fig. 3(b)). The 34% higher concentration of generated Frenkel pairs on the irradiated (111) in comparison to the (200) orientation, Fig. 5(c) and (e), causes a reduction in elastic modulus from 461 to 430 GPa in the Ek range from 0 to 182 eV. This reduction can be interpreted as a sig nificant orientation-dependent elastic modulus change of 55 GPa, by considering the elasticity data between Ti0.56Al0.44N(200) of 483 GPa and Ti0.56Al0.44N(111) of 430 GPa, at Ek = 182 eV, see Fig. 12(b). For the polycrystalline HPPMS thin films with no preferred orientation, it is reasonable to expect that the slight influence of (111) orientation on reducing the elastic modulus cannot be resolved experimentally by considering the magnitude of the error bars. Ek. In contrast, for CAD thin films with (111) preferred orientation, an ~18% reduction in elastic modulus was obtained. It is shown that the reduction of elastic modulus for CAD thin films is due to the significantly larger concentration of Frenkel pairs formed upon irradiation of the experimentally observed (111) orientation in comparison to the (200) orientation. At Ek = 182 eV, irradiation of the (111)-oriented surface leads to ~34% higher Frenkel pair concentration than for the (200)- oriented surface irradiation at similar Ek values. Moreover, we present evidence of ion-energy-dependent thermal stability enhancement of (Ti, Al)N. DSC data reveal that, independent of the processing route, the ion irradiation-induced increase in Frenkel pair concentration leads to an increase in the onset temperature of wurtzite formation by up to 206 ◦C. Therefore, it is evident that ion irradiation-induced changes in elastic modulus and thermal stability of c-(Ti,Al)N thin films can be understood based on the ion kinetic energy and orientation-dependent formation of point defects. 4.7. Mechanical properties TEM characterization of (Ti,Al)N films deposited by CAD: (a) BF-TEM image of Ek = 19 eV, (b) SAED pattern of (a), (c) BF-TEM image of Ek = 190 eV, (d) SAED pattern of blue region in (c), (e) High resolution (HR) TEM image of orange region in (c), (f) SAED pattern of the red region in (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 12 S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 Acta Materialia 250 (2023) Fig. 12. Measured and calculated (a) intrinsic stress and (b) elastic modulus of (Ti,Al)N at 300 K with respect to ion kinetic energy. S. Karimi Aghda et al. Fig. 12. Measured and calculated (a) intrinsic stress and (b) ela Measured and calculated (a) intrinsic stress and (b) elastic modulus of (Ti,Al)N at 300 K with respect to ion kinetic energy. N. Additionally, the ion irradiation-induced changes in elastic modulus can be understood based on the ion energy- and orientation-dependent point defect formation from ab initio calculations. For HPPMS thin films with random orientation, the concurrent effects of ion irradiation- induced formation of Frenkel pairs and generation of compressive stresses resulted in an apparent independence of the elastic modulus on Ek. In contrast, for CAD thin films with (111) preferred orientation, an ~18% reduction in elastic modulus was obtained. It is shown that the reduction of elastic modulus for CAD thin films is due to the significantly larger concentration of Frenkel pairs formed upon irradiation of the experimentally observed (111) orientation in comparison to the (200) orientation. At Ek = 182 eV, irradiation of the (111)-oriented surface leads to ~34% higher Frenkel pair concentration than for the (200)- oriented surface irradiation at similar Ek values. Moreover, we present evidence of ion-energy-dependent thermal stability enhancement of (Ti, Al)N. DSC data reveal that, independent of the processing route, the ion irradiation-induced increase in Frenkel pair concentration leads to an increase in the onset temperature of wurtzite formation by up to 206 ◦C. Therefore, it is evident that ion irradiation-induced changes in elastic modulus and thermal stability of c-(Ti,Al)N thin films can be understood based on the ion kinetic energy and orientation-dependent formation of point defects. N. References Moser, P.H. 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Simulations were performed with computing resources granted by the Jülich-Aachen Research Alliance (JARA) HPC section at the IT Center of RWTH Aachen University under the projects JARA0151 and JARA0221 and their support is gratefully acknowledged. Financial support for the operation of the accelerator laboratory in Uppsala by VR-RFI (Contract No. 2017-00646_9) and the Swedish Foundation for Strategic Research (SSF, Contract No. RIF14- 0053) is gratefully acknowledged. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provi sion of experimental facilities. Parts of this research were carried out at PETRA III beamline P02.1. Beamtime was allocated by an In-House By correlatively employing synthesis experiments, integral as well as spatially resolved characterization, and ab initio calculations, the influ ence of ion kinetic energy (Ek) on the structure evolution, mechanical properties, and thermal stability of (Ti,Al)N has been studied. Single- phase cubic metastable (Ti,Al)N thin films were synthesized by HPPMS and CAD within the Ek range from 19 to 236 eV. While for the HPPMS films increasing Ek resulted in irradiation-induced random orientation evolution, the CAD films showed preferred (111) orientation at Ek ≥144 eV. The substrate ion current density of 3.0 mA/cm2 measured for CAD as compared to HPPMS with 0.9 mA/cm2 accounts for a 3.3 times higher energy flux at the growing film surface in the CAD process. Hence, the strong (111) texture evolution in the CAD thin films synthesized at higher Ek can be rationalized based on ion flux- and ion energy-induced minimization of strain energy in defective cubic (Ti,Al) 13 S. Karimi Aghda et al. Acta Materialia 250 (2023) 118864 contingent. contingent. [26] K. Balasubramanian, S.V. Khare, D. Gall, Energetics of point defects in rocksalt structure transition metal nitrides: thermodynamic reasons for deviations from stoichiometry, Acta Mater. 159 (2018) 77–88. 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https://openalex.org/W4292767197	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0273197&type=printable	English	null	Advanced practice providers versus medical residents as leaders of rapid response teams: A 12-month retrospective analysis	PloS one	2,022	cc-by	6,176	Advanced practice providers versus medical residents as leaders of rapid response teams: A 12-month retrospective analysis Herman G. KreeftenbergID1,2☯, Ashley J. R. De Bie1,2,3☯, Eveline H. J. Mestrom2,3☯, Alexander J. G. H. Bindels1,2☯, Peter H. J. van der Voort4,5☯ Herman G. KreeftenbergID1,2☯, Ashley J. R. De Bie1,2,3☯, Eveline H. J. Mestrom2,3☯, Alexander J. G. H. Bindels1,2☯, Peter H. J. van der Voort4,5☯ 1 Dept Internal Medicine, Catharina Hospital Eindhoven, Eindhoven, Netherlands, 2 Dept of Intensive Care, Catharina Hospital Eindhoven, Eindhoven, Netherlands, 3 Dept of Electrical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands, 4 TIAS School for Business and Society, Tilburg University, Tilburg, Netherlands, 5 Dept of Critical Care, University Medical Centre Groningen, Groningen, Netherlands a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * herman.kreeftenberg@catharinaziekenhuis.nl OPEN ACCESS In a time of worldwide physician shortages, the advanced practice providers (APPs) might be a good alternative for physicians as the leaders of a rapid response team. This retrospective analysis aimed to establish whether the performance of APP-led rapid response teams is comparable to the performance of rapid response teams led by a medical resident of the ICU. Citation: Kreeftenberg HG, De Bie AJR, Mestrom EHJ, Bindels AJGH, van der Voort PHJ (2022) Advanced practice providers versus medical residents as leaders of rapid response teams: A 12- month retrospective analysis. PLoS ONE 17(8): e0273197. https://doi.org/10.1371/journal. pone.0273197 Results In total, 179 responses of the APP-led teams were analyzed, versus 275 responses of the teams led by a resident. Per APP, twice as many calls were handled than per resident. Inter- ventions of teams led by APPs, and residents did not differ in number of admissions (p = 0.87), mortality (p = 0.8), early warning scores (p = 0.2) or MAELOR tool triggering (p = 0.19). Both groups scored equally on time to admission (p = 0.67) or time until any performed intervention. Copyright: © 2022 Kreeftenberg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS ONE RESEARCH ARTICLE Editor: James Mockridge, PLOS: Public Library of Science, UNITED KINGDOM Editor: James Mockridge, PLOS: Public Library of Science, UNITED KINGDOM Received: November 5, 2021 Accepted: August 4, 2022 Published: August 23, 2022 Received: November 5, 2021 Accepted: August 4, 2022 Published: August 23, 2022 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0273197 Material and methods In a retrospective single-center cohort study, the electronic medical record of a tertiary hos- pital was queried during a 12-months period to identify patients who had been visited by our rapid response team. Patient- and process-related outcomes of interventions of rapid response teams led by an APP were compared with those of teams led by a medical resi- dent using various parameters, including the MAELOR tool, which measures the perfor- mance of a rapid response team. Editor: James Mockridge, PLOS: Public Library of Science, UNITED KINGDOM PLOS ONE PLOS ONE Study design and setting We performed a single-center retrospective cohort study over a period of 12 months. This time period was chosen to reduce the influence of confounders, such as changes within the organiza- tion of the hospital and the ward, for instance the implementation of a completely new opera- tional Electronic Medical Record (EMR) in the hospital or the use of continuous monitoring devices within certain departments. The study was performed in the Catharina Hospital Eind- hoven, a Dutch tertiary teaching hospital which houses all specialties except for neurosurgery and transplantation surgery. The hospital has a 33-bedded ICU, which facilitates a mix of post- operative cardiac and oncologic surgery and, on the other hand, specialties such as, neurology, pulmonology and gynecology and internal medicine, including dialysis. During the period of the study, clinical protocols regarding the rapid response system remained unchanged. PLOS ONE PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams Funding: The author(s) received no specific funding for this work. Funding: The author(s) received no specific funding for this work. Introduction Hospital medicine is dealing with patients with increasingly complex disorders that require a highly efficient and high-quality healthcare organization [1, 2]. Rapid response systems with teams led by physicians have been shown to reduce in-hospital cardiopulmonary arrests and mortality [3, 4]. However, the organization of these rapid response systems is subject to the worldwide emerging shortages of physicians, especially in rural areas [5, 6]. Competing interests: The authors have declared that no competing interests exist These shortages force numerous hospitals to reorganize their rapid response systems and other teams in order to be able to continue to provide a 24/7 coverage. One option that has been considered is that a rapid response team might be led by different health care professionals, ranging from attending physicians to nurses. Limited scientific evi- dence suggests that teams led by a physician perform better than teams led by non-physicians [7, 8]. In practice however, an increasing proportion of in-hospital acute and emergency care is delivered by junior clinicians in the first years of their training, including the responsibility of leading a rapid response team, which might reduce the efficacy and quality of these teams. One of the potential solutions is to reallocate this responsibility to physician assistants or nurse practitioners, also called advanced practice providers (APP). This profession is gaining recog- nition in critical care which is supported by clinicians who recognize their quality and continu- ity of their work [9, 10]. Very few experiences with APPs as leaders of a rapid response team have been reported [11, 12]. Two previous studies provided some guidance about the outcomes of patients visited by a team led by an advanced practice provider, but inter-comparability is hampered by differences between the considered health care systems and by the lack of validated outcome parameters. A third, retrospective single center study comparing outcome data of rapid response teams led by a nurse practitioner and by a registrar showed an improved hospital mortality in the nurse practitioner -led group after propensity matching. This study mainly reported patient out- comes [13]. The main objective of the present study was, to establish whether the performance of APP- led rapid response teams is comparable to the performance of teams led by a medical resident of the ICU, focusing on process- as well as patient-related outcomes. Conclusion This retrospective analysis showed that the quality of APP-led rapid response teams was similar to the quality of teams led by a resident. These findings need to be confirmed by pro- spective studies with balanced outcome parameters. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 Rapid response team organization The organization of the local rapid response system has been described in the COMET study, a multicenter study that evaluated the implementation of structured rapid response systems in the Netherlands [3]. Since implementation of the rapid response team in the Catharina Hospi- tal Eindhoven in 2013, These teams have consisted of an ICU nurse and a team leader, the leader being either a medical resident working on the ICU or an advanced practice provider. One of the medical residents or advanced practice providers on the ICU manages the pager for the rapid response team during duty. A call to the rapid response team can be made if a patient on the ward scores a modified early warning score of 3 points or if a nurse experienced a substantiated sense of worry about a patient. This call can either be made by medical residents or by registered nurses on the general ward. Usually, when the modified early warning score indicates a critically ill patient, the nurse on the ward informs the resident, who in turn decides if a rapid response team assessment is necessary. The rapid response team call is postponed if the cause of the high early warning score is known and additional treatment, such as an opera- tion, has been planned or if a sepsis can be treated on the normal ward. The team carries a basic set of materials, which consists of resuscitation fluids, masks for supplemental oxygen and lifesaving medication, such as glucose or phenylephrine. Following the first assessment, the leader of the rapid response team discusses the case with the intensivist on call for the ICU. This consultation results either in ICU admission or in rec- ommendations for treatment on the general ward, which can include changing treatment limi- tations, such as the “do not resuscitate” (DNR) code or the “do not intubate” (DNI) code and other treatment limitations. If it was decided that the patient is to be admitted to the ICU, the rapid response team transports the patient and admits him or her to the ICU. After the admis- sion to the ICU the leader of the rapid response team remains responsible for the care of the patient until the end of his duty, together with a dedicated ICU nurse. Shortly after ICU admis- sion, the intensivist visits the patient in the ICU. Patient selection The EMR was queried to identify patients who had been assessed by the rapid response team. Patients and assessments were eligible if a bed-side assessment had been performed by the 2 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams rapid response team in patients aged 18 years of older. Medical consultations by telephone were excluded. Rapid response team triggering, modified early warning score and the MAELOR tool On the wards, the modified early warning score is used as described by v Galen et al. [14] to identify deteriorating patients or patients in need of advanced care. In short, this tool assigns points to abnormal physiological parameters and in turn triggers a rapid response team call. It also provides the opportunity to call in support if there is a sense of unease about the condition of the patient. The MAELOR tool is a validated tool to measure and quantify the performance of a rapid response team [15]. This tool consists of a flow chart which is triggered if the patient has a high modified early warning score and stops triggering if a patient is admitted within 4 hours after the initial call or has a resolution of critical clinical symptoms within 48 hours. The tool also stops triggering if there are treatment limitations and if ICU admittance is not warranted. Th MAELOR l fl di i d i d i Fi 1 Cli i l i bl f h The MAELOR tool flow diagram is depicted in Fig 1. Clinical variables necessary for the MAELOR tool were only recorded until 48 hours after the initial call. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 Rapid response team organization The hospital has a separate team for non- ICU-related in-hospital cardiac arrests. In case of a cardiac arrest the rapid response team is involved if the patient experiences a return of spontaneous circulation and will be admitted to the ICU or the cardiac care unit (CCU). 3 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams Fig 1. Flow chart MAELOR tool. Advanced practice providers and medical residents The advanced practice providers who work in the ICU are qualified as physician assistants. They received a 2.5-year training in the medical domain, which grants them a master’s degree, and after graduation, they are qualified to perform all ICU tasks autonomously. The APPs all worked as ICU-nurse before their training to become an APP. They work in collaboration with intensivists. The medical residents originate from the following disciplines: internal medi- cine, cardiology, pulmonology, and surgery. The medical residents attend an internship during a period ranging from 3 months to 1 year. Shifts The ICU ward uses a system of rotating shifts with a minimum of four clinicians in each shift (advanced practice providers and medical residents). Usually, six of these clinicians are present during the day shift. During the evening shift, two or three of these clinicians are present, and Fig 1. Flow chart MAELOR tool. https://doi.org/10.1371/journal.pone.0273197.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 4 / 11 Fig 1. Flow chart MAELOR tool. Fig 1. Flow chart MAELOR tool. https://doi.org/10.1371/journal.pone.0273197.g001 Advanced practice providers and medical residents The advanced practice providers who work in the ICU are qualified as physician assistants. They received a 2.5-year training in the medical domain, which grants them a master’s degree, and after graduation, they are qualified to perform all ICU tasks autonomously. The APPs all worked as ICU-nurse before their training to become an APP. They work in collaboration with intensivists. The medical residents originate from the following disciplines: internal medi- cine, cardiology, pulmonology, and surgery. The medical residents attend an internship during a period ranging from 3 months to 1 year. Data Data of all the consecutive rapid response team visits over a period of 12 consecutive months (2017–2018) were retrospectively extracted from the hospital data base. The patient variables collected were age sex, diagnosis, hospital admission, discharge data, death, Apache IV score on admission and after 24 hours, all blood samples before and after the rapid response team consultations and treatment limitations. Additionally, the composition of the team was noted. Since there are no mandatory rules for the composition and organization of a rapid response team, the performance and efficiency of different teams are difficult to measure and compare. We gathered several parameters to measure the efficiency of the process. The param- eters were categorized in three groups. First, the patients’ outcome data: length of stay, mortal- ity, and if applicable, treatment limitations. Second, parameters of team performance: to measure team performance, we used the time until change in early warning system score together with the time until various interventions: the time between the consultation of the rapid response team and the arrival on the ICU, the time between the consultation and inter- ventions such as central or arterial catheterization or intubation. Arterial and central venous lines can be inserted both by residents and by APPs. Intubations by residents are performed under supervision. Third, the MAELOR tool, a validated instrument to assess the performance of rapid response teams was scored. Statistics The data were analyzed with SPSS statistical package version 25 (IBM corporation, Armonk, NY, USA). Means are reported with standard deviations for normal distributions and medi- ans with interquartile range are reported for other distributions. Parameters which were recorded once every hour were considered continuous. Categorical independent variables were compared using the Chi-square test with Yates continuity correction. Categorical and continuous independent non-parametric variables were compared with the Mann Whitney U test, and for the independent parametric variables were analyzed with the independent samples t-test. Categorical variables with two continuous variables at different points were compared using the mixed between-within subjects analysis of variance was used. A p-value of p< 0.05 was considered statistically significant. Ethics Approval for the study was obtained from the national and regional Ethics committee in accor- dance with Dutch and European legislation (Medical Research Ethics Committees United (MEC-U); W17.095). A local applicability permission was obtained separately. This article was prepared using the Standards for Quality Improvement Reporting Excellence (SQUIRE) guidelines [16]. Shifts The ICU ward uses a system of rotating shifts with a minimum of four clinicians in each shift (advanced practice providers and medical residents). Usually, six of these clinicians are present during the day shift. During the evening shift, two or three of these clinicians are present, and 4 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams during the night shift, two. The number of FTE in the entire group of advanced practice pro- viders is 4.99, and in the MR group it is 10.69. Results All 454 consecutive rapid response team calls during the assessed period were included in the analysis. Because not all patients received every treatment that was assessed in this study, data on antibiotic change, central venous access, arterial catheters and intubation were not available for all patients. 5 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams The team was led by an advanced practice provider in 179 cases and by a medical resident in 275 cases. Of the 454 rapid response team calls, 296 resulted in the patient being admitted to the ICU. This represents approximately 10% of the total yearly ICU admittances. The percent- ages of rapid response calls resulting in an admission to the ICU were comparable between teams led by an APP and those led by a medical resident (118 (65%) vs 178 (66%), p = 0.78). The level of experience of the APPs was a median of 6.25 years (3.33y-8.25y). In general, an APP handled twice as many calls as a MR. Table 1 presents the baseline characteristics of patients assessed by APP-led teams and by teams led by a medical resident. Most patients were assessed in the emergency department and on the internal medicine ward. The APACHE IV score of the patients indicates a high severity of illness. No significant differences were found between these two groups except for diastolic blood pressure, which was significantly higher in patients assessed by MR-led teams. Both the patient- and the process-related outcomes are described in Table 2. The baseline characteristics of the patients assessed by teams led by advanced practice pro- viders and medical residents demonstrate that no statistically significant differences were encountered between the groups except for diastolic blood pressure. The differences in blood pressure were very small and are therefore considered clinically unimportant. Concerning the validated MAELOR tool, we were able to retrieve the MAELOR tool out- come in 451 of the 454 cases. Three cases were excluded due to insufficient data. In the analysis of the MAELOR tool outcomes, no significant differences were found between patients treated by teams led by an advanced practice provider or by a medical resident. APP: advanced practice provider, MR: medical resident, IQR: interquartile range Results In the 296 patients admitted to the ICU, the time until change of antibiotics after ICU admission was not significantly associated with the rapid response team leader being an APP or a resident (Table 2). In addition, no association was found between the leader of the rapid response team being an APP or a medical resident and the time until antibiotics administration after ICU admission. The ICU LOS was determined for 283 of the 296 ICU admitted patients. No significant dif- ference in ICU LOS was found between the patients attended to by APP-led teams and those attended to by rapid response teams led by a medical residents. The early warning score was assessed at two different points in time (on admission and after 24h). There was no significant difference between the rapid response team leader and the early warning score, or the reduction in early warning score after 24 hours. Of the 158 patients who remained on the general ward after the rapid response team visit, we were able to extract the early warning score after 24 hours for 123 patients. The warning scores after 24 hours and the reduction in warning scores did not differ between the APP-led teams and the resident-led teams. In 126 out of 452 patients new treatment limitations were applied after the rapid response team visit. There was no significant association between the instigation of treatment limita- tions and the rapid response team leader (Table 2). In addition, no effect of rapid response Table 2. Outcome variables. Results Since the rapid response team leader in our ICU model remains responsible for the care provided to the admitted ICU patients, the efficiency of the team could be reflected in the time until arterial line insertion, the time until central venous catheter insertion and the time until Table 1. Patient characteristics on arrival of the rapid response team. Data are given as numbers with percentages or as medians with IQR. Leader of rapid response team p-value APP Median (IQR) n = 179 MR Median (IQR) n = 275 Age (years) 68 (56–76) 70 (58–78) 0.19 Sex (male) 99 (55%) 168 (61%) 0.26 Sex (female) 80 (45%) 107 (39%) 0.26 Apache IV predicted mortality 58 (42–86) 62 (37–76) 0.89 Temperature (degrees Celsius) 37.3 (36.9–38.5) 37.4 (37.0–38.4) 0.80 Systolic Blood Pressure (mmHg) 120 (99–140) 128 (109–151) 0.06 Diastolic Blood Pressure (mmHg) 69 (50–75) 70 (60–80) 0.03 Pulse (rate/min) 108 (86–124) 100 (85–119) 0.28 Respiratory Rate (rate/min) 25 (18–30) 20 (16–30) 0.37 Location of outreach for ICU-admitted patients APP Number (%) MR Number (%) Surgery 18 (6.1%) 26 (8.8%) 1.00 Internal Medicine 21 (7.1%) 24 (8.1%) 0.40 Cardiology 4 (1.4%) 6 (2.0%) NA Pulmonology 14 (4.7%) 24 (8.1%) 0.82 Cardiothoracic surgery 7 (5.9%) 2 (1.1%) NA Neurology 2 (0.7%) 8 (2.7%) NA Gastroenterology 7 (2.4%) 7 (2.4%) 0.61 Emergency department 38 (12.8%) 73 (65.8%) 0.16 Other 7 (5.9%) 8 (4.5%) NA Table 1. Patient characteristics on arrival of the rapid response team. Data are given as numbers with percentages or as medians with IQR. Leader of rapid response team p-value the rapid response team. Data are given as numbers with percentages or as medians with IQR. t characteristics on arrival of the rapid response team. Data are given as numbers with percentages or as medians with IQR Table 1. Patient characteristics on arrival of the rapid response team. Data are given as numbers with percentages or as m PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 6 / 11 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams intubation. These variables did not differ significantly between patients attended to by a team led by an APP or by a resident (Table 2). To determine the efficiency of non-technical procedures, we also compared the times until change of administered antibiotics in the first 12 hours after admission to the ICU. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 Discussion The present study provides insight into the performance of APPs as leaders of rapid response teams in direct comparison with medical residents. In this retrospective study, we found no differences in either process-related or patient-related outcomes between rapid response teams led by APPs and teams led by medical residents. This comparability included the trend of the early warning score after the call and the triggering of the MAELOR tool 48 hours after the call, a tool validated for assessing the quality of rapid response team assessments [17]. To measure patient- and process-related outcomes, we used a wide variety of parameters, ranging from the standardized measurement tools that were validated for these rapid response team assessments to the times until interventions and general outcome data. Moreover, the environment and organization of the rapid response teams we assessed are in line with those in a multi-center trial that established a standard deployment of the rapid response teams which reduced in-hospital morality rates [3]. In our study, this organizational structure was considered efficient, based on the high number of calls that resulted in ICU admissions (60– 70%) suggesting an effective afferent limb (detection), and on the relative fast reaction time as a parameter for the efferent limb (response) [18]. The absence of significant differences in outcomes between teams led by APPs and teams led by medical residents suggests that APPs are suitable alternatives for medical residents in leading rapid response teams. This finding is also supported by the higher number of calls han- dled per FTE by the APPs compared to the number of calls handled per FTE of medical resi- dents of the ICU: one FTE APP handled approximately 2.5 times more calls than one FTE medical residents. An explanation for this substantial difference might be the continuity of care that APPs provide. This continuity is established by the APPs’ continuous coverage in most shifts together with their presence alongside the residents. This continuity of care proba- bly also explains the observation that APPs more easily decide to respond to the calls than the rotating medical residents, who regard these calls as stressful events (personal communication). The outcomes of this study are in line with the outcomes of the few previously published studies, although the settings of those studies were different, which makes a sound comparison is difficult. Results Leader of rapid response team p-value Response by APP: N(%), Med (IQR) Response by MR: N(%), Med (IQR) Number of calls 179 275 Admission ICU 118 (66%) 178 (65%) 0.87 Time to ICU (hours) 1.19 (0.56–1.75) 1.16 (0.59–1.75) 0.67 Within 24h: Hospital mortality 13 (7%) 17 (6%) 0.80 ICU mortality 6 (5%) 10 (6%) 1.00 Time to insertion arterial line (hours) 1.68 (0.87–2.94) 1.54 (0.78–2.72) 0.50 Time from visit to insertion Central venous catheter(hours) 2.17 (1.24–5.78) 1.71 (0.92–3.30) 0.30 Time from visit to intubation (hours) 3 (1.5–16) 2 (1.07–10.50) 0.24 MAELOR not triggering anymore (good outcome) 165 (92%) 261 (96%) 0.19 MEWS admission 4.04 (2.03–6.29) 3.92 (1.98–6.56) 0.90 MEWS at 24 hours 2.13 (1.04–3.46) 1.63 (0.51–3.06) 0.12 Δ MEWS between leaders 0.20 Change in antibiotics 27 (30%) 40 (29%) 1.00 Time to change of antibiotics (hours) 1.33 (0.62–2.25) 1.40 (0.65–2.30) 0.77 ICU LOS (days) 1.00 (0.20–2.79) 1.10 (0.17–3.43) 0.80 Treatment limitation initiated (%) 54 (30%) 72 (26%) 0.44 MEWS: Modified Early Warning Score, APP: advanced practice provider, MR: medical resident, IQR: interquartile range https://doi org/10 1371/journal pone 0273197 t002 Table 2. Outcome variables. 7 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams team leadership on mortality was not found in the patients who were deceased on the ward or in the ICU within 24 hours after the RRT visit. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 Discussion One study showed no differences in quality after the introduction of a nurse practi- tioner as leader of a rapid response team [12]. The study compared a situation with and with- out a rapid response team, but in the group of patients treated by a rapid response team, the nurse practitioner was not always available, and deployment restrictions for the rapid response team were in place. Additionally, another publication reported an shorter time until admission to the ICU in a APP-led rapid response team [11]. Despite this improvement, however, the time span was much longer than the 4 hours suggested to be adequate by the MAELOR tool. Also our results are in line with the findings of a recent retrospective single-center analysis. This study compared an APP-led rapid response team with a resident-led rapid response team. This study found no differences between the groups except for a shorter in-hospital stay of the patients visited by the APP-led group but after propensity matching [13]. The study mainly focused on patient outcomes and less on process outcomes. In accordance with this study, there is growing evidence that APPs are a valuable substitute for a physicians as leaders of a rapid response team. Although the implementation of an RRT in this study is the general method used in the Netherlands, comparability between countries and healthcare systems remains cumbersome PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 8 / 11 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams because different RRT models that are used [3]. Apparently, so far, an optimal composition and implementation of a rapid response team has not yet been established [8, 19]. This fact and the lack of validated measurement tools except for the MAELOR tool probably explains why the literature reports different success rates and struggles with aligning outcome data when reporting on the performance of rapid response teams [3, 8, 13, 20, 21]. The main limitations of the present study are the retrospective design and the single-center setting. Before extrapolating our results to other hospitals, the local situation should be consid- ered. Another limitation is that patients might have been missed if they did not fit our query, or were not registered in the database. However, we know that the database is used consistently and that registration is a central part of the workflow for all APPs and medical residents. Discussion It is therefore unlikely that selection is a major bias of this study. Another important limitation is that the patient- and process-related outcome measures were chosen arbitrarily, although they are clinically relevant. When focusing on time before intubation for example, there is a differ- ence between the groups in time from admission to intubation without reaching significance. This is probably caused by the fact that there are a lot of oxygen therapies available which can be applied as initial treatment and if the included sample size is too small, significant differ- ences in patient-related outcomes are difficult to detect. In addition, the outcomes are affected by many other variables related to the patient, the pathology, and the organization of the ward and the ICU. Especially, an ICU nurse may have a substantial influence on the rapid response team’s performance. Necessary critical steps such as oxygen administration or positioning of the patient to enable adequate breathing are steps often overlooked by junior clinicians. Even simple treatment recommendations provided by the nurse, can be very valuable for junior cli- nicians. These steps are often independently covered by the accompanying ICU nurse. Regarding the use of the MAELOR tool, this tool was validated to evaluate the performance of a rapid response team. However, the acquaintance of researchers with this tool is limited and therefore its use to compare between studies is limited as well. In addition, the added value of APPs might have been underestimated because certain benefits of APPs might have been missed, such as patient satisfaction, communication skills, team guidance, situational awareness, and other non-technical skills. This same issue has been addressed in the literature before [22]. Prospective cohort studies are therefore needed to confirm the outcomes of the present study and to assess the potential additional benefits of APP-led rapid response teams. Conclusion In this observational retrospective single-center study on process- and patient-related outcome parameters of RRTs led by APPs and MRs, we have shown that APPs perform at least as well as MRs in leading a rapid response team. As the performance of rapid response teams is influ- enced by the organization of healthcare systems prospective studies in other institutions are needed to confirm our findings. S1 Data. Author Contributions Conceptualization: Ashley J. R. De Bie, Alexander J. G. H. Bindels, Peter H. J. van der Voort. Data curation: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mestrom. References 1. Clark AV, LoPresti CM, Smith TI. Trends in Inpatient Admission Comorbidity and Electronic Health Data: Implications for Resident Workload Intensity. J Hosp Med 2018; 13(8):570–2. https://doi.org/10. 12788/jhm.2954 PMID: 29578553 2. Steventon A, Deeny S, Friebel R, Gardner T, Thorlby R. Emergency Hopsital admissions in England: which may be avoidable and how? The Health Foundation—Publications 2018. 3. Ludikhuize J, Brunsveld-Reinders AH, Dijkgraaf MG, Smorenburg SM, de Rooij SE, Adams R, et al. Outcomes Associated With the Nationwide Introduction of Rapid Response Systems in The Nether- lands. Crit Care Med 2015; 43(12):2544–51. https://doi.org/10.1097/CCM.0000000000001272 PMID: 26317569 4. Maharaj R, Raffaele I, Wendon J. Rapid response systems: a systematic review and meta-analysis. 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Kreeftenberg HG, Pouwels S, Bindels A, de Bie A, van der Voort PHJ. Impact of the Advanced Practice Provider in Adult Critical Care: A Systematic Review and Meta-Analysis. Crit Care Med 2019; 47 (5):722–30. https://doi.org/10.1097/CCM.0000000000003667 PMID: 30720539 10. Kleinpell RM, Grabenkort WR, Kapu AN, Constantine R, Sicoutris C. Nurse Practitioners and Physician Assistants in Acute and Critical Care: A Concise Review of the Literature and Data 2008–2018. Crit Care Med 2019; 47(10):1442–9. https://doi.org/10.1097/CCM.0000000000003925 PMID: 31414993 11. Gershengorn HB, Xu Y, Chan CW, Armony M, Gong MN. The Impact of Adding a Physician Assistant to a Critical Care Outreach Team. PLoS One 2016; 11(12):e0167959. https://doi.org/10.1371/journal. pone.0167959 PMID: 27942002 12. Scherr K, Wilson DM, Wagner J, Haughian M. Evaluating a new rapid response team: NP-led versus intensivist-led comparisons. AACN Adv Crit Care 2012; 23(1):32–42. https://doi.org/10.1097/NCI. 0b013e318240e2f9 PMID: 22290088 13. Gupta S, Balachandran M, Bolton G, Pratt N, Molloy J, Paul E, et al. Comparison of clinical outcomes between nurse practitioner and registrar-led medical emergency teams: a propensity-matched analysis. Crit Care 2021; 25(1):117. Author Contributions 9 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams Formal analysis: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mestrom. Investigation: Herman G. Kreeftenberg, Ashley J. R. De Bie, Peter H. J. van der Voort. Methodology: Alexander J. G. H. Bindels, Peter H. J. van der Voort. Project administration: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mestrom. Resources: Herman G. Kreeftenberg. Supervision: Herman G. Kreeftenberg, Alexander J. G. H. Bindels, Peter H. J. van der Voort. Validation: Ashley J. R. De Bie, Eveline H. J. Mestrom. Writing – original draft: Herman G. Kreeftenberg. Writing – review & editing: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mes- trom, Alexander J. G. H. Bindels, Peter H. J. van der Voort. Investigation: Herman G. Kreeftenberg, Ashley J. R. De Bie, Peter H. J. van der Voort. Methodology: Alexander J. G. H. Bindels, Peter H. J. van der Voort. Methodology: Alexander J. G. H. Bindels, Peter H. J. van der Voort. Project administration: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mestrom. Resources: Herman G. Kreeftenberg. Project administration: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mestrom. Resources: Herman G. Kreeftenberg. Supervision: Herman G. Kreeftenberg, Alexander J. G. H. Bindels, Peter H. J. van der Voort. Validation: Ashley J. R. De Bie, Eveline H. J. Mestrom. Supervision: Herman G. Kreeftenberg, Alexander J. G. H. Bindels, Peter H. J. van der Voort. Validation: Ashley J. R. De Bie, Eveline H. J. Mestrom. Writing – original draft: Herman G. Kreeftenberg. Writing – review & editing: Herman G. Kreeftenberg, Ashley J. R. De Bie, Eveline H. J. Mes- trom, Alexander J. G. H. Bindels, Peter H. J. van der Voort. PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 References https://doi.org/10.1186/s13054-021-03534-4 PMID: 33752731 14. van Galen LS, Dijkstra CC, Ludikhuize J, Kramer MH, Nanayakkara PW. A Protocolised Once a Day Modified Early Warning Score (MEWS) Measurement Is an Appropriate Screening Tool for Major 10 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0273197 August 23, 2022 PLOS ONE Advanced practice providers and physicians as leaders of rapid response teams Adverse Events in a General Hospital Population. PLoS One 2016; 11(8):e0160811. https://doi.org/10. 1371/journal.pone.0160811 PMID: 27494719 Adverse Events in a General Hospital Population. 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Introduction of the medical emer- gency team (MET) system: a cluster-randomised controlled trial. Lancet 2005; 365(9477):2091–7. https://doi.org/10.1016/S0140-6736(05)66733-5 PMID: 15964445 21. Chan PS, Jain R, Nallmothu BK, Berg RA, Sasson C. Rapid Response Teams: A Systematic Review and Meta-analysis. Arch Intern Med 2010; 170(1):18–26. https://doi.org/10.1001/archinternmed.2009. 424 PMID: 20065195 22. Sarani B, Sonnad S, Bergey MR, Phillips J, Fitzpatrick MK, Chalian AA, et al. Resident and RN percep- tions of the impact of a medical emergency team on education and patient safety in an academic medi- cal center. Crit Care Med 2009; 37(12):3091–6. https://doi.org/10.1097/ccm.0b013e3181b09027 PMID: 19938331 11 / 11
https://openalex.org/W2934571706	https://www.biorxiv.org/content/biorxiv/early/2019/04/09/600593.full.pdf	English	null	The <i>Prevotella copri</i> complex comprises four distinct clades that are underrepresented in Westernised populations	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	19,478	. CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint The Prevotella copri complex comprises four distinct clades that are underrepresented in Westernised populations , Task Force on Microbiome Studies, University of Naples Fe p gy 10, Departments of Biology and Computer Science, New York University, NY 10003, USA. 10, Departments of Biology and Computer Science, New York University, NY 10003, USA. 11, National Institute for Medical Research, Tanga Centre, Tanzania. 11, National Institute for Medical Research, Tanga Centre, Tanzania. 12, Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Techn 13 Hardin Hall School of Natural Resources University of Nebraska-Lincoln NE USA 12, Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Ghana. 13, Hardin Hall, School of Natural Resources, University of Nebraska-Lincoln, NE, USA. umasi Centre for Collaborative Research in Tropical Medicin 12, Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Ghana. 13, Hardin Hall, School of Natural Resources, University of Nebraska-Lincoln, NE, USA. y - Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, University of Vienna sse 14, 1090 Vienna, Austria. 14, CUBE - Division of Computational Systems Biology, Department of Microbiology and Ecosystem Science, Univers Althanstrasse 14, 1090 Vienna, Austria. 15, Centre for Translational Microbiome Research, Department of Microbiology Tumor and Cell Biology, Karolinska Institutet, St kh l S d 15, Centre for Translational Microbiome Research, Department of Microbiology Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 16, Institute for Mummy Studies, EURAC Research, Viale Druso 1, 39100 Bolzano, Italy Institute for Mummy Studies, EURAC Research, Viale Druso 17, Institute of Agrochemistry and Food Technology-National Research Council (IATA-CSIC), 46980, Paterna, Valencia, Spain. 17, Institute of Agrochemistry and Food Technology-National Research Council (IATA-CSIC), 46980, Paterna, Valencia, Spain. 18, Department of Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine, 20359, Hamburg, Germany. 19, German Center for Infection Research, Hamburg-Borstel-Lübeck-Riems, 20359, Hamburg, Germany. 17, Institute of Agrochemistry and Food Technology-National Research Council (IATA-CSIC), 46980, Paterna, Valenci 18, Department of Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine, 20359, Hamburg, g y gy ( ) 18, Department of Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Med 18, Department of Infectious Disease Epidemiology, Bernhard Nocht Institute for Tropical Medicine, 20359, Hamburg, 19, German Center for Infection Research, Hamburg-Borstel-Lübeck-Riems, 20359, Hamburg, Germany. 19, German Center for Infection Research, Hamburg-Borstel-Lübeck-Riems, 20359, Hamburg Correspondence: Adrian Tett (adrianjames.tett@unitn.it); Nicola Segata (nicola.segat The Prevotella copri complex comprises four distinct clades that are underrepresented in Westernised populations Adrian Tett1, Kun D. Huang1,2, Francesco Asnicar1, Hannah Fehlner-Peach3, Edoardo Pasolli1,4, Nicolai Karcher1, Federica Armanini1, Paolo Manghi1, Kevin Bonham5,6, Moreno Zolfo1, Francesca De Filippis7,8, Cara Magnabosco9, Richard Bonneau9,10, John Lusingu11, John Amuasi12, Karl Reinhard13, Thomas Rattei14, Fredrik Boulund15, Lars Engstrand15, Albert Zink16, Maria Carmen Collado17, Dan R. Littman3, Daniel Eibach18,19, Danilo Ercolini7,8, Omar Rota-Stabelli2, Curtis Huttenhower5,6, Frank Maixner16, Nicola Segata1* 1, CIBIO Department, University of Trento, Italy. p y y ment of Sustainable Agro-Ecosystems and Bioresources, Fondazione Edmund Mach, San Michele all'Adige, Italy. l C t f Bi l d M di i f th Ski b ll I tit t N Y k U i it S h l f M di i N Y k N 2, Department of Sustainable Agro-Ecosystems and Bioresources, Fondazione Edmund Mach, San Michele all'Adige, Italy. 3, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, NY 10016, USA 2, Department of Sustainable Agro-Ecosystems and Bioresources, Fondazione Edmund Mach, San Michele all'Adige, 3, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New 2, Department of Sustainable Agro-Ecosystems and Bioresources, Fondazione Edmund Mach, San Michele all'Adige, Italy. 3, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, NY 10016, 2, Department of Sustainable Agro Ecosystems and Bioresources, Fondazione Edmund Mach, San Michele all Ad 3, The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, N USA. 4, Current address: Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy. 4, Current address: Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy 5, The Broad Institute, Cambridge, USA. 5, The Broad Institute, Cambridge, USA. 6, Harvard T.H. Chan School of Public Health, Boston, USA , , , 7, Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy. 7, Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy. 7, Department of Agricultural Sciences, University of Naples p g y p y 8, Task Force on Microbiome Studies, University of Naples Federico II, Naples, Italy. g y y 8, Task Force on Microbiome Studies, University of Naples Federico II, Naples, Italy. 1, CIBIO Department, University of Trento, Italy. Introduction Prevotella copri is a frequently observed inhabitant of the human intestinal microbiome and it displays a large inter-individual variation (Human Microbiome Project Consortium, 2012; Qin et al., 2010; Truong et al., 2017). Being 39.1% prevalent in healthy individuals from current metagenomic profiles (Pasolli et al., 2017), P. copri is not ubiquitous. But when present, it is often the most abundant species identified (34% of instances) and it represents the clearest reproducible gut microbiome structure (Arumugam et al., 2011; Koren et al., 2013). P. copri also came to prominence due to its reported association with inflammatory diseases, including new-onset rheumatoid arthritis (Scher et al., 2013), ankylosing spondylitis (Wen et al., 2017) and intestinal inflammation in chronic HIV-1 infection (Dillon et al., 2014). It has also been shown to induce insulin resistance and glucose intolerance (Pedersen et al., 2016). Conversely, others have linked P. copri with improved glucose and insulin tolerance in diets rich in fibre (De Vadder et al., 2016; Kovatcheva-Datchary et al., 2015) which suggests the beneficial effects of P. copri could be diet dependent (Pedersen et al., 2016). As previously expressed (Cani, 2018; Ley, 2016), such conflicting reports regarding the benefits of P. copri suggest that it is an important but enigmatic member of the microbiome. Higher prevalence of Prevotella has been consistently reported in non-Westernised populations (De Filippo et al., 2010; Hansen et al., 2019; Obregon-Tito et al., 2015; Schnorr et al., 2014; Smits et al., 2017; Yatsunenko et al., 2012) and metagenomic studies, capable of species-level resolution, have shown P. copri to be particularly prevalent (Pasolli et al., 2019; Vangay et al., 2018). Non-Westernised populations follow a traditional lifestyle and typically consume diets rich in fresh unprocessed food (vegetables and fruits) and high in fibre compared to Western populations (De Filippo et al., 2010; Segata, 2015; Statovci et al., 2017). This association may further support the hypothesis of diet being an important factor in selecting and shaping Prevotella populations as it has been shown for the overall diversity of microbial ecosystems (Smits et al., 2017; Sonnenburg and Bäckhed, 2016; Sonnenburg et al., 2016). The high prevalence in societies with more traditional rather than high-fat diets may also support the health benefit of P. copri. Despite the importance of P. Abstract Prevotella copri is a common inhabitant of the human gut. Interest in P. copri has gathered pace due to conflicting reports on whether it is beneficial or detrimental to health. In a cross-continent meta-analysis exploiting >6,500 available metagenomes supported by new isolate sequencing and recovery of high-quality genomes from metagenomes, we obtained >1,000 P. copri genomes. This 100-fold increase over existing isolate genomes allowed the genetic and global population structure of P. copri to be explored at an unprecedented depth. We demonstrate P. copri is not a monotypic species, but encompasses four distinct clades (>10% inter-clade vs. <4% intra-clade average single nucleotide variants) for which we propose the name P. copri complex, comprising clades A, B, C and D. We show the complex is near ubiquitous in non-Westernised populations (95.4% versus 29.6% in Westernised populations), where all four clades are typically co-present within an individual (61.6% of the cases), in contrast to Westernised populations (4.6%). Genomic analysis of the complex reveals substantial and complementary functional diversity, including the potential for utilisation of complex carbohydrates, suggestive that multi-generational dietary modifications may be a driver for the reduced P. copri prevalence in Westernised populations. Analysis of ancient stool microbiomes highlights a similar pattern of P. copri presence consistent with modern non-Westernised populations, allowing us to estimate the time of clade delineation to pre-date human migratory waves out of Africa. Our analysis reveals P. copri to be far more diverse than previously appreciated and this diversity appears to be underrepresented in Western-lifestyle populations. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Results and discussion Analysis of >1,000 P. copri genomes reveals four clades comprising the P. copri complex Analysis of >1,000 P. copri genomes reveals four clades comprising the P. copri complex Figure 1. (A). Whole-genome phylogenetic tree of a representative subset of the four distinct P. copri clades comprising the P. copri complex in relation to other sequenced members of the genera Prevotella, Alloprevotella and Paraprevotella. Magenta circles indicate P. copri sequenced isolates. The phylogeny containing all P. copri genomes is available as Supplementary Data File 1 (see Data Availability). (B) Genetic distance within clade (intra-clade), between clades (inter-clade) and between clades and other species (denoted OS) of Prevotella, Alloprevotella and Paraprevotella (inter-species), shown as pairwise average nucleotide identity distances (ANI distance). The dotted line denotes 5% ANI distance. (C-D) Pairwise SNV distances based on core gene alignment, and Jaccard distance based on pairwise gene content (see Methods) between and within the P. copri clades. Analysis of >1,000 P. copri genomes reveals four clades comprising the P. copri complex Figure 1. (A). Whole-genome phylogenetic tree of a representative subset of the four distinct P. copri clades comprising the P. copri complex in relation to other sequenced members of the genera Prevotella, Alloprevotella and Paraprevotella. Magenta circles indicate P. copri sequenced isolates. The phylogeny containing all P. copri genomes is available as Supplementary Data File 1 (see Data Availability). (B) Genetic distance within clade (intra-clade), between clades (inter-clade) and between clades and other species (denoted OS) of Prevotella, Alloprevotella and Paraprevotella (inter-species), shown as pairwise average nucleotide identity distances (ANI distance). The dotted line denotes 5% ANI distance. (C-D) Pairwise SNV distances based on core gene alignment, and Jaccard distance based on pairwise gene content (see Methods) between and within the P. copri clades. To investigate the global distribution and population structures of P. copri, we performed an analysis of 6,874 publicly available metagenomes from 36 individual datasets (Supplementary Table S1), representing six continents and over 20 different countries. By means of a novel assembly and mapping-based computational approach, we expanded the total number of available P. copri genomes to 1,023 and all metagenomically assembled genomes can be defined as high-quality according to current guidelines (Bowers et al., 2017) (estimated completeness >95%, contamination <5%, see Methods). This approach, (see Methods) involved collating a highly representative set of genomes comprising our newly sequenced P. Introduction copri and the open-ended question regarding its role in health and disease there is a lack of available reference genomes and much of what is currently known relies on studies of the single type strain P. copri DSM18205 (Hayashi et al., 2007). Recent reports have begun to highlight a degree of strain level heterogeneity within P. copri (De Filippis et al., 2019; Truong et al., 2017; Vangay et al., 2018). Indeed, sub-species strain variation may account for at least some of the differences in the reported benefits or detriments of P. copri. Yet to date there has been no large scale concerted effort to explore the distribution and genetic variation within P. copri. Here we use a combination of isolate sequencing and large scale metagenomic assembly and strict quality control to recover over 1,000 P. copri genomes from publicly available metagenomes spanning multiple host-geographies, disease and lifestyles. We also expand the catalogue of non-Westernised sampled populations with additional metagenomic sequencing of individuals from Ghana, Ethiopia and Tanzania and further profile P. copri in ancient intestinal samples from a European natural ice-mummy and stools of pre-Columbian amerinds. These datasets and analyses . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint provide an unprecedented comprehensive insight into the genetic diversity, global population structure and evolutionary history of P. copri. Results and discussion copri isolates (n=15), publicly available references (n=2), as well as a set of carefully curated and manually-guided metagenomically assembled genomes from diverse populations (n=55). This set of 72 genomes was used as a pangenomic reference to bin via mapping metagenomically-assembled contigs from single samples into whole P. copri genomes (n=949) (see Methods). The obtained genomes were . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint subjected to strict quality control including estimation of within sample strain heterogeneity (see Methods) and resulted in genomes with assembly characteristics comparable with those of isolate sequencing and manually curated metagenomic assembled genomes (Supplementary Figure S1; Supplementary Table S2). This genome catalogue spans multiple host geographies, populations and lifestyles and can thus be mined to ask fundamental questions regarding the population genomic structure of P. copri. P. copri is strikingly revealed by this analysis to be not a monotypic species, but comprised of four distinct clades when placed in phylogenetic context with the closest publicly available representatives of the wider Prevotella, Alloprevotella, and Paraprevotella genera (Figure 1A). These four clades are clearly distinct to other Prevotella and to the other considered species, each clade is supported by at least one of our newly sequenced genomes from isolates (Figure 1A) and all clades are represented in our three newly sequenced non-Westernised datasets (98 new genomes, see below). The average nucleotide identity (ANI) distances between the P. copri genomes revealed a limited intra-clade distance (mean 2.55% s.d. 0.35% for Clade B to 4.16% s.d. 0.78% for Clade C). Conversely, the inter-clade distances were very high, with values ranging from 13.0% to 21.4%. In comparison, each of the four P. copri clades were >23.0% distant to any other Prevotella, Alloprevotella and Paraprevotella species, indicating that the four distinct P. copri clades are genetically closer to each other than genomes outside the P. copri complex (Figure 1B). Results and discussion The high inter-clade genetic distance observed suggests the genome catalogue could represent four distinct species. Studies have sought to place a threshold at which ANI values between genomes equate to the delineation of strains into species, with a broad consensus being values above 5-6% distance (Goris et al., 2007; Jain et al., 2018; Konstantinidis and Tiedje, 2005; Pasolli et al., 2019). All members of all four clades fall well below this threshold when compared to other P. copri clades (>10% ANI distance) (Figure 1B). The distinction of the four clades is further supported based on core genome single nucleotide distance (>10% distance) (Figure 1C), by the separation of the clades based purely on gene content (Figure 1D) as well as based on phylogeny (Figure 1A). Respecting the clear distinction of the P. copri clades and being conscious of the difficulties in advising separation into species we propose the naming of P. copri to encompass these four distinct clades. Therefore we recommend the term “Prevotella copri complex” for which there are four genetically distinct Clades A, B, C and D, named sequentially based on the decreasing number of genomes recovered (Figure 1A). he four clades are globally distributed with instances of country-specific subtype In this study, P. copri genomes were recovered from 22 different countries offering a unique opportunity to investigate the biogeographical population structure. These clades were not strictly separated based on geographical location, i.e. all of the four P. copri clades were identified in multiple countries and spanning multiple continents (Figure 2). However, within several clades we did observe geographical stratification. In Clade A, for which the most genomes were recovered, we identified three sub-types which were either exclusively or nearly so to samples of Chinese origin, and in addition there was also a cluster exclusive to Israel. In Clade B a specific cluster was identified which can be attributed to Fiji. For Clade C and D it is difficult to ascertain if there is stratification due to the lower number of genomes recovered for these two P. copri clades. While geographical stratification was evident for some intra-clade sub-types, most sub-types appeared to be multi-country and even multi-continental, indicating that not only at the clade level but also at intra-clade level P. copri is widely geographically distributed. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Figure 2 Phylogenetic representation of all 1,023 P. copri genomes separated for each clade of the P. copri complex. Outer ring is coloured by continent of origin and inner ring is further divided by country. Radial grey bars indicate newly sequenced isolate genomes and publicly available reference genomes are denoted by black stars. Figure 2 Phylogenetic representation of all 1,023 P. copri genomes separated for each clade of the P. copri complex. Outer ring is coloured by continent of origin and inner ring is further divided by country. Radial grey bars indicate newly sequenced isolate genomes and publicly available reference genomes are denoted by black stars. Reconstruction of 98 additional genomes from non-Westernised samples expands the diversity of the P. copri clades with fewer representatives Most of our understanding of the microbiome has been accumulated from Westernised populations. While small in comparison to Westernised populations, a number of publicly available datasets have been generated from non-Westernised populations inhabiting Peru (Obregon-Tito et al., 2015), Fiji (Brito et al., 2016), Mongolia (Liu et al., 2016) and two from Tanzania (Rampelli et al., 2015; Smits et al., 2017) totaling 340 metagenomes. The term “Westernisation” encompasses many factors including lifestyle, environment and diet (for full description see Methods). A common feature of non-Westernised datasets has been the frequent observation of high Prevotella prevalence (De Filippo et al., 2010; Hansen et al., 2019; Obregon-Tito et al., 2015; Schnorr et al., 2014; Smits et al., 2017; Yatsunenko et al., 2012) and particularly P. copri in these populations (Pasolli et al., 2019; Vangay et al., 2018). To further investigate the prevalence and abundance of P. copri in non-Westernised populations, we also considered our recently sequenced dataset of non-Westernised adults from Madagascar (110 metagenomes) (Pasolli et al., 2019) and three new non-Westernised cohorts. These included paired infant and mother samples from Ethiopia (50 metagenomes) and extended families from Ghana and Tanzania (44 and 68 metagenomes respectively) (see Methods). From these additional 272 metagenomes (Supplementary Table S3), we recovered 98 high quality P. copri complex genomes expanding the clades represented by fewer reconstructed members (Clade A, B, C and D were expanded by 3.4%, 34.7%, 17.6% and 40% respectively) (Supplementary Figure S5; Supplementary Table S4). An additional feature of the three newly sequenced datasets was that they included sampling within families. This offered the potential to establish if transmission occurs and if so to what extent within families. When P. copri genomes were recovered from more than one family member, we compared the genetic distances to estimate the level of intra-family strain sharing. Using normalised phylogenetic tree distances and cutoffs proposed previously (Truong et al., 2017) (see Methods) in 5 of 26 cases (19.2%) we identified the same strain, suggesting both horizontal and vertical transmission of the P. copri complex within family units, and therefore familial prevalence of P. copri as a potential important source in acquisition. Associating P. copri clades with metagenomically investigated human diseases ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint considered if the distribution of the P. copri complex could be associated with other factors such as body mass index (BMI) and age (Supplementary Figures S3 and S4). Similar to disease, we note potential batch and cohort effects but no significant differences with all four clades being identified across all age groups and BMI categories. considered if the distribution of the P. copri complex could be associated with other factors such as body mass index (BMI) and age (Supplementary Figures S3 and S4). Similar to disease, we note potential batch and cohort effects but no significant differences with all four clades being identified across all age groups and BMI categories. Associating P. copri clades with metagenomically investigated human diseases ociating P. copri clades with metagenomically investigated human diseases A question that remains to be resolved is whether P. copri is of benefit or detriment to human health as there are studies reporting both scenarios (De Vadder et al., 2016; Dillon et al., 2014; Kovatcheva-Datchary et al., 2015; Pedersen et al., 2016; Scher et al., 2013; Wen et al., 2017). Here, in a meta-analysis of available disease phenotypes we found no strong indicator that they were associated with the four clades of the complex. Specifically, to investigate the association of the P. copri complex with different diseases, we analysed the prevalence and abundance of the four clades for each cohort where the study design included both case and controls. In total there were 10 datasets including colorectal cancer (CRC) (Feng et al., 2015; Vogtmann et al., 2016; Yu et al., 2017; Zeller et al., 2014), type-2-diabetes (T2D) (Karlsson et al., 2013; Qin et al., 2012), hypertension (Li et al., 2017), liver cirrhosis (Qin et al., 2014) and inflammatory bowel disease (IBD) (He et al., 2017; Nielsen et al., 2014). To identify and estimate the abundance of each of P. copri clades within a sample the metagenomic reads were mapped to a panel of unique clade-specific markers designed for each of the four clades (see Methods). The most significant changes in abundance and prevalence of P. copri and specifically the four clades, were identified in the CRC and adenoma cohort of FengQ et al. with both Clade A and C being associated with disease (Supplementary Figure S2). However, three other CRC cohorts considered (Vogtmann et al., 2016; Yu et al., 2017; Zeller et al., 2014) and an overall CRC meta-analysis comprising 7 cohorts failed to support this observation (Thomas et al., 2019). Generally, while there were some weak associations of the P. copri clades in disease, across the control samples of the different datasets we observe heterogeneity in both abundance and prevalence suggesting significant batch effects. As such, at the clade level there is no clear evidence to suggest P. copri is associated with the etiology of these diseases. We further . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations We detected the P. copri complex in all 40 datasets considered, but the prevalence in non-Westernised populations was nearly ubiquitous (95.4% prevalence) in contrast to Westernised populations (29.6% prevalence) (Figure 3A, Supplementary Figure S6). Extending this to consider each of the four P. copri clades separately, all four were found to be significantly higher (p-value < 1.1e-12, Welch’s t-test) in the non-Westernised datasets with respect to Westernised, with the P. copri complex Clade A being the most prevalent (91.5% in non-Westernized vs 26.9% in Westernised populations) followed by C (88.2% vs 8.35%), B (73.5% vs 6.2%) and D (68.8% vs 2.7%, Figure 3A). The agreement that all four P. copri clades are always higher in non-Westernised populations spanning multiple countries and continents compared to Westernised populations is remarkable, with the only exception being from Mongolia (Liu et al., 2016). The Mongolian cohort sampled both urban dwellers and rural non-Westernised populations. While the urban dwellers have a prevalence closer to that of the non-Westernised populations (Clade A . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint prevalence; 100%, B; 73.3%, C; 80%, D; 42.2%), this is still generally lower than the rural non-Westernised Mongolian population (Clade A prevalence; 98.5%, B; 76.9%, C; 84.6%, D; 56.9%). Although P. copri was observed at a much lower abundance in Westernised populations, all four clades were detected, and among those Clade A was the most prevalent type (Figure 3A). Figure 3. Prevalence of the P. copri complex and its association with non-Westernised populations. A) P. copri prevalence in individual non-Westernised and Westernised datasets, “All” refers to the prevalence of any of the four clades being present. B) Prevalence and abundance of the P. copri complex in the four newly sequenced non-Westernised datasets. C) Percentage of individuals harbouring multiple P. copri clades. D) P. Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations copri pangenome sizes for non-Westernised individuals by dataset compared to Westernised individuals. Figure 3. Prevalence of the P. copri complex and its association with non-Westernised populations. A) P. copri prevalence in individual non-Westernised and Westernised datasets, “All” refers to the prevalence of any of the four clades being present. B) Prevalence and abundance of the P. copri complex in the four newly sequenced non-Westernised datasets. C) Percentage of individuals harbouring multiple P. copri clades. D) P. copri pangenome sizes for non-Westernised individuals by dataset compared to Westernised individuals. Considering the high prevalence of the four P. copri clades in non-Westernised populations we next sought to identify if these clades are mutually exclusive or able to co-inhabit in the intestine. Analysis of our newly sequenced datasets clearly displayed multiple clades within individuals (Figure 3B), which confirms the results observed when analysing all the other available datasets (Figure 3C). Strikingly, for the 95.4% of non-Westernised individuals with at least one P. copri complex clade, in 61.6% of these all four clades were detectable, in 82.0% at least three, and at least two in 93.8% of individuals. The high-percentage of individuals carrying multiple clades was a consistent feature observed across all non-Westernised datasets, spanning four continents (Figure 3C). In comparison, for the smaller fraction of 29.6% of Westernised individuals with at least one P. copri complex clade, only 4.6% had all four clades, 12.5% at least three and 32.1% more than one. Therefore, we demonstrate that not only is P. copri prevalence higher in non-Westernised populations but the pattern of multi-clade co-presence in these populations is also a defining characteristic. Because of the existence of multiple clades within an individual (Figure 3B and 3C), and the observation of sizeable inter-clade diversity based on gene content (Figure 1D), we decided to estimate the sum of unique P. copri complex genes within each individual, or rather “the within . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint functional profiles (Figure 4A), which is consistent with the inter-clade genetic diversity discussed above (Figure 1B-D). Some of the distinguishing functionalities included sulphur metabolism and assimilation, which were enriched in all clades relative to B (Supplementary Table S5). Similarly, in carbohydrate metabolism, beta-galactosidase was found to be absent in Clade B while being prevalent in all other clades (at least >60% prevalence). In the metabolism of cofactors and vitamins, genes responsible for folate metabolism were depleted in Clade D. Interestingly, Clade D also had the least diversity of antimicrobial resistance genes lacking 5 out of 7 identified as differentially prevalent in the other three clades. Differences were also noticeable in membrane transporters; for instance, the polyamine spermidine/putrescine ABC transporter (potABCD) was present in almost all members of Clade A, C and D but never observed in Clade B. Conversely, an energy coupling factor (ECF) type ABC transporter that could be responsible for micronutrient uptake was solely found in a subset of genomes of Clade B (27% of genomes). One reported feature of P. copri is its effect on glucose homeostasis (De Vadder et al., 2016; Kovatcheva-Datchary et al., 2015; Pedersen et al., 2016), with one recent study suggesting a positive benefit via succinate production (De Vadder et al., 2016). While potential succinate production was observed in all clades the genes responsible were less prevalent in Clade B (p-value < 5.2e-37, Bonferroni-corrected Fisher-exact test). On the contrary, high-levels of circulating branched chain amino acids (BCAA), linked to the development of insulin resistance, have been associated to higher levels of P. copri in the gut microbiome (Pedersen et al. 2016). In addition, the prevalence of genes for BCAA biosynthesis in the P. copri pangenome has been recently shown to be diet-dependent and associated to actual urinary BCAA levels (De Filippis et al., 2019). Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint individual P. copri pangenome” (see Methods). As expected, individuals with multiple clades tended towards a larger number of unique P. copri genes (Figure 3D), and as multiple clades is a feature of being non-Westernised, a considerably larger P. copri functional potential was revealed in these populations (Figure 3D, Supplementary Figure 7). individual P. copri pangenome” (see Methods). As expected, individuals with multiple clades tended towards a larger number of unique P. copri genes (Figure 3D), and as multiple clades is a feature of being non-Westernised, a considerably larger P. copri functional potential was revealed in these populations (Figure 3D, Supplementary Figure 7). Evidence of distinct carbohydrate metabolism repertoires in the four P. copri complex clades Figure 4. Functional diversity of the P. copri complex. A) Presence and absence of eggNOG functions significantly different between the four P. copri clades (yellow present, black absent) (see Methods). B) Multidimensional scaling (MDS) ordination based on CAZy families present in each genome showing distinct inter and intra clustering in the P. copri complex. C) The ten most enriched CAZy families for each clade compared to all other clades (grey bars). For full list of CAZy prevalence see Supplementary Table S6. Evidence of distinct carbohydrate metabolism repertoires in the four P. copri complex clades Evidence of distinct carbohydrate metabolism repertoires in the four P. co Evidence of distinct carbohydrate metabolism repertoires in the four P. copri complex clades Figure 4. Functional diversity of the P. copri complex. A) Presence and absence of eggNOG functions significantly different between the four P. copri clades (yellow present, black absent) (see Methods). B) Multidimensional scaling (MDS) ordination based on CAZy families present in each genome showing distinct inter and intra clustering in the P. copri complex. C) The ten most enriched CAZy families for each clade compared to all other clades (grey bars). For full list of CAZy prevalence see Supplementary Table S6. To investigate the potential functional diversity of the P. copri complex, we first annotated the open reading frames (ORFs) for each genome using the eggNOG database (Huerta-Cepas et al., 2017) (see Methods). Between and even within the four clades of the complex we observed considerable diversity, with Clade B being the most dissimilar based on the overall distance of the eggNOG . Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations Here we found that BCAA biosynthesis genes were widespread across all four clades of the P. copri complex, with >85% prevalence in all cases. The P. copri complex is strongly associated with non-Westernised populations which have diets that tend to be higher in fibre and complex carbohydrates and lower in fats and animal protein compared to typical Western diets (De Filippo et al., 2010; Segata, 2015; Statovci et al., 2017). To specifically look at the P. copri complex for potential carbohydrate utilisation, the genomes were also screened for carbohydrate active enzymes (CAZymes) (Lombard et al., 2014) (see Methods). While many of these CAZy families were found to be prevalent across all four P. copri clades (Supplementary Table S6), considerable variability in the presence of these families was seen between and even within the different clades (Figure 4B, 4C and Supplementary Figure S8). To focus on families potentially associated with plant-derived carbohydrate degradation (e.g. cellulose, hemicellulose and pectin) each family was ascribed a broad substrate specificity via manual curation (Supplement Figure S9). While all clades were found to have the potential to degrade plant-derived carbohydrates, not all CAZy families were represented or equally distributed throughout the four clades (Supplementary Figure S9); for example, the polysaccharide pectin-degrading families PL9 and PL10 were highly prevalent and nearly exclusively present in Clade B. In Clade D, the GH9 CAZy family of cellulases was particularly enriched compared to the other clades (Figure 4C). The distinct clustering based on CAZy gene content (Figure 4B) displaying inter- and intra-clade functional differences suggests overlapping but potential heterogeneity in carbohydrate metabolism. The frequent co-presence of all four clades in non-Westernised populations would suggest they are non-competing and therefore niche separated. While it cannot be discounted that these four clades are spatially separated in the intestine, the ability to utilise a differing array of carbohydrates or substrate specificities could potentially be the driver of this separation. Within an individual the presence of multiple clades collectively offers a larger and perhaps complementary functionality to efficiently metabolise a wide range of dietary fibre. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . P. copri diversity in ancient human gut contents resembles that of non-Westernised populations and gives insights into its evolutionary history To test whether the high P. copri prevalence and co-presence of the four clades of the complex in non-Westernised populations reflects the composition in ancient human gut microbiomes, we analysed four archaeological gut contents for the presence of P. copri. We studied material from the lower intestinal tract and lung tissue of the Iceman, a 5,300-year-old natural ice mummy (Spindler, 1994). The Iceman genetically belongs to the Early European Farmers and originated and lived in Southern Europe, in the Eastern Italian Alps (Haak et al., 2015; Keller et al., 2012; Lazaridis et al., 2014; Müller et al., 2003). (Figure 5A). We also analysed three coprolite samples (fossilized faeces) (Figure 5A) recovered from the pre-Columbian (1300 ± 100 B.P.) site “La Cueva de los Muertos Chiquitos” from Durango, a Northwestern state of Mexico (Brooks et al., 1962) (see Methods). We found the P. copri complex to be prevalent both in the Mexican and the European ancient gut metagenomes (Figure 5B). All samples had at least two P. copri clades (Clades A and C) and in two coprolites all four clades could be detected. The higher prevalence of clade A and C in our ancient samples mirrors the tendency of modern day populations (both Westernised and non-Westernised) where these two clades are more prevalent (Figure 3A). To discount the possibility of a non-ancient gut origin, we verified that the P. copri reads displayed damage patterns indicative of ancient DNA (Supplementary Figure 10) (Orlando et al., 2015) and in a control sample (Iceman lung tissue) no P. copri clades were detected (positive for only a single marker of the 2,448 P. copri complex specific markers) (Figure 5B). Two characteristics point toward a similarity between the ancient samples and modern non-Westernised populations. First, P. copri is highly prevalent in the ancient samples as in non-Westernised samples. Second, the ancient samples are characterised by a high clade co-presence (presence of 2 to 4 distinct clades) as observed in non-Westernised individuals. Although we have analysed only a small number of ancient metagenomes, these observations suggest that the P. copri carriage in non-Western populations is more akin to our ancestors. To calibrate a P. copri phylogeny we screened all ancient samples and found that one coprolite (sample 2180, radiocarbon dated A.D. 673 to 768, see Methods) had sufficiently high coverage of Clade A for clocking (see Methods). Co-presence of multiple P. copri complex clades is typical in individuals from non-Westernized populations CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Figure 5 Ancient microbiomes and the evolutionary history of the P. copri complex A) Ancient Mexican coprolite samples and intestinal and lung tissue sampled from the Iceman, a natural ice mummy. B) Percentage of positive P. copri clade specific markers identified in each ancient metagenomic sample. C) Time measured phylogenetic tree of the P. copri complex, Figure 5 Ancient microbiomes and the evolutionary history of the P. copri complex Ancient Mexican coprolite samples and intestinal and lung tissue sampled from the Iceman natural ice mummy. B) Percentage of positive P. copri clade specific markers identified in ea ancient metagenomic sample. C) Time measured phylogenetic tree of the P. copri comple magenta star indicates ancient coprolite sample, 2180 (see Methods). Figure 5 Ancient microbiomes and the evolutionary history of the P. copri complex A) Ancient Mexican coprolite samples and intestinal and lung tissue sampled from the Iceman, a natural ice mummy. B) Percentage of positive P. copri clade specific markers identified in each ancient metagenomic sample. C) Time measured phylogenetic tree of the P. copri complex, magenta star indicates ancient coprolite sample, 2180 (see Methods). Figure 5 Ancient microbiomes and the evolutionary history of the P. copri complex A) Ancient Mexican coprolite samples and intestinal and lung tissue sampled from the Iceman, a natural ice mummy. B) Percentage of positive P. copri clade specific markers identified in each ancient metagenomic sample. C) Time measured phylogenetic tree of the P. copri complex, magenta star indicates ancient coprolite sample, 2180 (see Methods). . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Discussion We demonstrate that P. copri is not a monotypic species but four clearly defined clades, each spanning a diversity that is typical of distinct species (Figure 1), and all four clades have the potential to reside either solely or in combination within an individual (Figure 3C). We propose to name this group the P. copri complex, comprising clades A, B, C and D. The insights that we have gained into the P. copri complex genetics and population genomics relied on isolate sequencing, on new sequencing from underrepresented non-Westernised populations, and largely on the tremendous resource of publicly available metagenomic datasets, covering multiple geographies, diseases and lifestyles. This led to the observation that the P. copri complex is globally distributed (Figure 2), but with a highly structured distribution, both in terms of prevalence and the presence of multiple clades within an individual, with non-Westernised populations (Figure 3). While we concede the term Westernised versus non-Westernised serves to demarcate what may be better seen as a continuum along multiple lifestyle parameters, the distinction nonetheless has its merits, as interest grows in comparing our Westernised microbiomes to those presumed more akin to our ancestral microbiomes. Recent studies have expanded our understanding of the microbial diversity of non-Westernised populations (Hansen et al., 2019; Pasolli et al., 2019), and the rapid loss of diversity with Westernisation (Vangay et al., 2018). What is still to be determined is the consequences of this microbial impoverishment with respect to the wider gut microbial ecosystem and its impact on human health. Evidence from the analysis of ancient stool samples (Figure 5) points towards P. copri diverging into four clades prior to the first human migration events out of Africa. The fact that we consistently observe high prevalence in globally disparate non-Westernised populations and in ancient microbiomes suggest the loss of P. copri might be a result of Westernisation. A major element of Westernisation has been a shift in diet over the course of the last two centuries with the advent of industrialisation and food processing, from one high in fibre and complex carbohydrates to one high in sodium, fat, and simple sugars and low in fibre. It was previously shown that P. copri provides a host benefit in response to a high-fibre diet (De Vadder et al., 2016; Kovatcheva-Datchary et al., 2015), but not one high in fat (Pedersen et al., 2016). The P. P. copri diversity in ancient human gut contents resembles that of non-Westernised populations and gives insights into its evolutionary history copri in Westernised populations is the consequence of its loss in response to Westernisation, which has been rapidly occurring in an almost infinitesimal time frame relative to host-microbe coevolution. rather than horizontal acquisition of P. copri between the populations. This, together with the estimation of clade divergence, implies the P. copri complex has been a long-standing feature of the human microbiome. This analysis suggests the underrepresentation of P. copri in Westernised populations is the consequence of its loss in response to Westernisation, which has been rapidly occurring in an almost infinitesimal time frame relative to host-microbe coevolution. P. copri diversity in ancient human gut contents resembles that of non-Westernised populations and gives insights into its evolutionary history Model selection indicated that this dataset is best modelled by a strict clock suggesting a constant rate of evolution through time and in different P. copri clades (see Methods). All our divergence estimates converged satisfactorily on clear posterior means (Figure 5C) and the age estimates indicate that P. copri began to diversify (split of clade B) ~6.5 million years ago (Figure 5C). This split is concurrent with the proposed divergence of human and chimpanzees (Patterson et al., 2006a). The diversification of clades A, D and C is estimated to have occured between ~3.5 and ~2.5 million years ago when the first archaic species of the genus Homo developed (Ungar and Sponheimer, 2011; Wood and Collard, 1999). The differentiation within each of the clades is instead relatively recent with the median estimates following that of the emergence of Homo sapiens circa 315 ka (Hublin et al., 2017). Despite the range in estimated clade divergence, even at the lowest estimation (420 ka) this occurred well before the first human migration waves out of Africa circa 90 to 194 ka years ago (Grün et al., 2005; Hershkovitz et al., 2018). This would indicate that the four clades of the P. copri complex were a feature of our pre-migratory human ancestors. Further support of P. copri clade diversification prior to migration is that the high prevalence of all four clades and multi-clades within an individual is a consistent feature of disparate non-Westernised populations in Africa, Oceania, South America and Asia. This observation in non-Westernised populations that are geographically and socially restricted would suggest vertical . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint rather than horizontal acquisition of P. copri between the populations. This, together with the estimation of clade divergence, implies the P. copri complex has been a long-standing feature of the human microbiome. This analysis suggests the underrepresentation of P. Discussion copri complex shows a diversity in plant-derived carbohydrate utilisation (Figure 4) which may also suggest diet is a key driver responsible for its ultimate demise in Westernised populations. Diet in particular seems to play a pivotal role in the case of P. copri, yet it is extremely difficult to study this influence in the context of long-term human dietary modifications spanning multiple generations. Given previous work associating multi-generational microbial impoverishment with dietary changes in mice (Sonnenburg et al., 2016), clearly more work is required both in silico and using in vitro studies to functionally associate and characterise the P. copri complex with respect to long-term dietary exposures, transmission, and retention. In part, P. copri has come to attention based on its association with disease, but in this study we found no clear evidence that particular clades are associated with the subset of health conditions available for meta-analysis. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Nevertheless, it cannot be disregarded that such associations may exist, possibly only at the sub-clade level, but such an investigation would require the power of a far larger number of disease-specific cohorts than are currently available. Finally, it is particularly notable that the analysis approach taken here is generalisable to other microbial species in instances where there are minimal reference isolate sequences available. This is in principle the case for species that are understudied due to being recalcitrant to cultivation or because they have not been the focus of sequencing efforts. The ever-increasing number of publicly available metagenomes will serve this end, as well as likely add clarity to whether P. copri is considered either a positive or negative influence on health in the context of other microbiome members, diet, lifestyle and host genetic factors. This study reveals P. Discussion copri is far more complex than previously imagined and it will be important in future studies to appreciate this in order not to oversimplify and underestimate the potential P. copri diversity within the human gut microbiome. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Metagenomic assembly Metagenomic samples were assembled using metaSpades (Version 3.10.1) (Nurk et al., 2017) using default parameters, chosen due to its reported performance compared to other assemblers (Forouzan et al., 2018; Pasolli et al., 2019; van der Walt et al., 2017). Samples that exceeded permitted memory requirements (>1Tb of RAM) or those for which only unpaired reads were available were assembled using Megahit (Version 1.1.1) (Li et al., 2015) using default parameters. Only assembled contigs ≥ 1kb were considered further. Material and Methods The overall aim of the study was to identify and recover P. copri genomes in publicly available intestinal metagenomic datasets and new non-Westernised datasets presented in this work. These datasets represent multiple countries, host-conditions and lifestyles. The process involved collating a panel of 72 high-quality P. copri genomes using manually guided metagenomic genome binning and sequencing new P. copri isolates and publicly available reference sequences. The resulting panel of genomes was used to automatically recover additional P. copri genomes from single-sample assembled metagenomes. In addition, clade-specific markers were designed to accurately identify the prevalence and abundance of the P. copri clades across datasets. Constructing a P. copri genome panel with newly sequenced isolates and manually curated genomes from metagenomes A panel of 72 P. copri genomes were collated consisting of two publicly available reference genomes (RefSeq assembly accessions: GCF_000157935.1, GCF_002224675.1), 15 newly described isolate genomes (see below) and 55 manually curated metagenomically assembled genomes. P. copri strains were isolated from stool from healthy subjects and new onset rheumatoid arthritis patients. Stool was collected into anaerobic transport media (Anaerobe Systems), then streaked on BRU and LKV plates (Anaerobe Systems). After 24-48h, individual colonies were picked and screened with Prevotella-specific PCR primers, and the 16S rRNA V3-V4 sequence was confirmed by Sanger sequencing (Fehlner-Peach et al., manuscript in preparation). Prevotella-positive isolates were grown on BRU plates, and mature colonies were collected for genomic DNA isolation with the DNeasy PowerSoil Kit (Qiagen). Libraries were prepared for sequencing on the HiSeq2500 platform with the TruSeq DNA PCR-free Library Prep Kit (Illumina). In total 83 P. copri isolates were sequenced. As this included multi-sampling from the same individual, 15 isolates were selected for this study that represented the total genetic diversity of the isolate dataset. The 55 genome bins were recovered using anvi’o (Version 2.3.2) (Eren et al., 2015) applied on a set of assembled metagenomes. Anvi’o provides a platform for metagenomic genome binning and offers the ability to manually asses and curate those bins, potentially increasing accuracy compared to automated binning methods, but at the expense of being low throughput. Briefly, the 100 metagenomic samples determined to have a high abundance of P. copri based on MetaPhlan2 (Truong et al., 2015) were selected. The metagenomic samples were assembled (see above) and reads mapped back to the contigs using Bowtie2 (version 2.2.9, using “very-sensitive-local” parameter) (Langmead and Salzberg, 2012). Contigs (>2.5kb) were clustered by anvi’o based on . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint coverage and tetranucleotide frequency, and manually curated. Constructing a P. copri genome panel with newly sequenced isolates and manually curated genomes from metagenomes All recovered bins were subjected to strict quality control (see below), resulting in 55 high-quality genome bins. Automated recovery of P. copri genomes from >6500 metagenomes To automatically recover P.copri genomes from metagenomes, we first assembled each metagenome (see above) and for each assembly its contigs were mapped against the panel of 72 high-quality reference genomes representing the four clades of the P. copri complex (described above) using Blast (version 2.6.0+) (Altschul et al., 1990). Only contigs with a nucleotide identity ≥95% and an alignment ≥50% were considered further and placed into one of the four P. copri bins (Clade A, B, C or D) based on the membership of the reference genome. On the rare occasion a contig was ≥95% identical and aligned over ≥50% to multiple reference genomes representing different clades, the contig was placed into a single clade bin based on best BitScore, if this score was ≥10% than any other competing clade(s). If the BitScore threshold was not satisfied, the contig could not confidently be placed and was not considered further. All recovered P. copri metagenomic genome were assessed for quality (see below). P. copri genome quality control All P. copri genomes were strictly quality controlled. QC involved four steps 1) genome size 2) estimated completeness, 3) estimated contamination and 4) level of strain heterogeneity. Only genome bins >2.5 Mb <5.0 Mb and composed of <500 contigs were considered. CheckM (Parks et al., 2015) was used to estimate the completeness and level of contamination. High quality genomes were those >95% completeness, <5% contamination, except for P. copri clade D where a completeness of >90% was used to be more inclusive. For the newly sequenced non-Westernised datasets (see section: westernisation and additional Non-Westernised datasets, below) and the manually curated metagenomically assembled genomes a threshold of >90% completeness was also selected. We also investigated strain-level diversity for each of the P. copri clades within a sample as this could indicate contig chimeric assembly. Strain-level heterogeneity was estimated using an in-house developed tool, CMseq, available here: https://bitbucket.org/CibioCM/cmseq/commits/41082ef. Firstly, protein coding genes of the contigs were predicted with prodigal (Hyatt et al., 2010) implemented in the prokka pipeline (Version 1.11) (Seemann, 2014). To avoid overestimating strain-heterogeneity due to genes in common across the four P. copri clades, only the clade specific genes (see below) were considered as a proxy to estimate strain heterogeneity. Secondly, metagenomic reads were mapped to the assembled contigs using Bowtie2 (Langmead and Salzberg, 2012) (version 2.2.9, using “very-sensitive-local” parameter) and for each coding nucleotide base calls were only considered if there was >10X coverage and a PHRED quality score of ≥30. Each position was considered non-polymorphic if the frequency of the dominant allele was >80%. When calculating the overall contig polymorphic rate only the non-synonymous positions were considered. Phylogenetic analysis The phylogenetic analyses were performed with PhyloPhlAn (Segata et al., 2013) using the new version available in the "dev" branch of the repository (commit 7c38e19, https://bitbucket.org/nsegata/phylophlan). The phylogeny in Figure 1A was built using the 400 universal marker genes as identified by PhyloPhlAn using the following parameters: “--diversity low --fast”. The set of external tools with their respective options is reported below: their respective options is reported below: ● Diamond version v0.9.9.110, (Buchfink et al., 2015), with “blastx” for the nucleotide-based mapping, “blastp” for the amino-acid based mapping, and “--more-sensitive --id 50 --max-hsps 35 -k 0” in both cases ● MAFFT version v7.310, (Katoh and Standley, 2013), with “--localpair --maxiterate 1000 --anysymbol --auto” options ● trimAl version 1.2rev59, (Capella-Gutiérrez et al., 2009), with “-gappyout” option ● FastTree version 2.1.10, (Price et al., 2010), with “-mlacc 2 -slownni -spr 4 -fastest -mlnni 4 -no2nd -gtr -nt” options ● RAxML version 8.1.15 (Stamatakis, 2014), with “-p 1989 -m GTRCAT -t <FastTree phylogeny>” options The tree was built on a total of 90 genomes comprising a subset of 25 representative genomes for the three clades A, B and C whereas for clade D all 15 genomes were considered. The tree was built on a total of 90 genomes comprising a subset of 25 representative genomes for the three clades A, B and C whereas for clade D all 15 genomes were considered. The phylogeny in Supplementary Data File S1 is based on the 210 set of core genes screened to be monophyletic as described in the section below on molecular dating. The phylogeny has been reconstructed using PhyloPhlAn with the following parameters: “--diversity low --trim greedy --remove_fragmentary_entries”. Additionally, the set of external tools with their options is reported below: ● Blastn version 2.6.0+, (Altschul et al., 1990), with “-outfmt 6 -max_target_seqs 1000000” options ● Blastn version 2.6.0+, (Altschul et al., 1990), with “-outfmt 6 -max_target_seqs 1000000” options ● MAFFT, trimAl, FastTree, and RAxML were run with the same options as reported a ● MAFFT, trimAl, FastTree, and RAxML were run with the same options as reported above. ● MAFFT, trimAl, FastTree, and RAxML were run with the same options as repo The phylogenies in Figure 2 and in Supplementary Figure S6 were built with PhyloPhlAn using the set of core genes for each P. Genetic distance between and within the P. copri complex and related species Average nucleotide distance (ANI) pairwise were calculated using pyani (version 0.2.6; option ‘-m ANIb’) (Pritchard et al., 2015) considering representative subset of P. copri genomes of the four clades (25 for clade A, B and C, and all the 15 genomes of clade D) and publicly available reference genomes of the Prevotella, Alloprevotella and Paraprevotella genera available from NCBI RefSeq. Distances scores were filtered to include only pairwise comparison where alignment . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint lengths exceeded 500,000 bp. Pairwise core genome distances based on comparisons of single nucleotide polymorphisms were calculated on the core genome alignment of all 1,023 P. copri genomes. The core genome alignments were produced utilising PRANK (Löytynoja, 2014) as part of the Roary pipeline (version 3.11) (Page et al., 2015) with the parameters of 90% similarity identity for gene clustering and present in 90% of genomes for defining core genes. The pangenome-based matrix also produced from Roary was used to compare the pairwise gene content similarity calculated using the Jaccard similarity coefficient as part of the SciPy package (Bressert, 2012). To infer instances of strain sharing between individuals, normalised phylogenetic distances on the P. copri phylogeny were compared and called as the same strain based on a 0.2% identity threshold as previously described (Truong et al., 2017). The P. copri pangenome and evaluating prevalence and abundance The protein coding regions for the 72 P. copri genome panel (see above) were predicted using Prodigal (Hyatt et al., 2010) as part of the prokka pipeline (Version 1.11) (Seemann, 2014) and the total P. copri pangenome determined using Roary with 90% similarity identity parameter (Version 3.11) (Page et al., 2015). Markers specific to each clade of the P. copri complex where defined as present in >95% of the P. copri genomes of a given clade but absent in all others. This gave for Clade A n=430 markers, for Clade B n=954, for Clade C n=479 and for Clade D n=585. To determine if a P. copri clade is present in a metagenomic sample reads were mapped to the clade specific markers using Bowtie2 (Langmead and Salzberg, 2012) and mappings processed using PanPhlAn (Scholz et al., 2016). A marker was scored present if had a coverage ≥0.5X, and a clade present if ≥50% of the clade specific markers were hit. Estimation of P. copri clade relative abundance was calculated thus: (Mean clade marker coverage x approximated genome size(bp)) / total metagenome size (bp). Phylogenetic analysis copri clade (>95% prevalent across all genomes within a clade) determined using Roary (version 3.11) (Page et al., 2015) with a minimum gene identity of 90%. PhyloPhlAn was run using the following parameters: "--mutation_rates --min_num_entries <97% of the number of input genomes> --diversity low". The set of external tools used is: Blastn, MAFFT, . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint trimAl, FastTree, and RAxML, and they were executed with the same options as reported above. The phylogenetic trees in Figure 2, and Supplementary Figure S6 were visualized using GraPhlAn (version 1.1.3, (Asnicar et al., 2015)). Iceman samples and Mexican coprolite material In this study we metageomically analyzed archaeological gut contents for the presence of P. copri. The analyzed material includes gut content and lung tissue (negative control) of the Iceman, a European Copper Age ice mummy (Figure 5). The Iceman, commonly referred to as “Ötzi”, is one of the oldest human mummies discovered. His body was preserved for more than 5,300 years in an Italian Alpine glacier before he was discovered by two German mountaineers at an altitude of 3,210 m above sea level in September 1991. The mummy is now conserved at the Archaeological Museum in Bolzano, Italy, together with an array of accompanying artifacts (www.iceman.it). The Iceman was naturally mummified by freeze-drying (Lynnerup, 2007). Therefore, his body tissues and intestines still contain well preserved ancient biomolecules (DNA, proteins, lipids) that allowed e.g. the reconstruction of the Iceman's genome (Keller et al., 2012), the genomic analysis of the stomach pathogen Helicobacter pylori (Maixner et al., 2016), and the molecular reconstruction of the Iceman's last meal (Maixner et al., 2018). In addition, we subjected three ancient coprolite samples from a Mexican cave to metagenomics analysis (Figure 5). The archaeological site “La Cueva de los Muertos Chiquitos” in the northern Durango region of el Zape, Mexico, was excavated by Brooks and colleagues in the early 1960s (Brooks et al., 1962). The sub-humid climate in this natural cave at an altitude of approx. 1,800 m above sea level provided favourable conditions for the preservation of various ancient remains including human skeletons, botanical artefacts, quids and coprolites. The site was dated by previous radiocarbon dating of a single wood sample from one of the oldest levels (square B4, level 24-28) to A.D. 600 (1300 ± 100 B.P.) (Brooks et al., 1962). The ancient remains have been previously subjected to botanical (Brooks et al., 1962), dietary (Hammerl et al., 2015; Meade, 1994), parasitological (Camacho et al., 2018; Jiménez et al., 2012; Morrow and Reinhard, 2016), and molecular analysis (Tito et al., 2008, 2012). All three coprolite samples used in this study were discovered in square B4 in two different levels (Supplementary Table S7). The samples were stored in the Pathoecology Laboratory in the School of Natural Resources at the University of Nebraska-Lincoln in Lincoln, Nebraska. We obtained radiocarbon dates at the Curt-Engelholm-Centre for Archaeometry, Mannheim, Germany for coprolite sample 2180 from level 16-20 (A.D. Westernisation and additional Non-Westernised datasets CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint sequenced on the Illumina HiSeq2500 100nt paired end platform with a target depth of 5Gb/sample. Genome functional potential analysis We performed the functional annotation using the EggNOG mapper (version 1.0.3) (Huerta-Cepas et al., 2017) that is based on the EggNOG orthology system (Huerta-Cepas et al., 2016) and the sequence searches performed using HMMER (Eddy, 2011). We used the KEGG Brite Hierarchy to screen the EggNOG annotations that are shown in Figure 4A. In this figure, for each clade, we report only the eggNOGs that are significantly different in each of its three pairwise comparisons to the other clades (p-value < 0.01, Bonferroni corrected Fisher-exact test). CAZy enzymes (Lombard et al., 2014) (http://www.cazy.org/) were predicted with HMMSEARCH (Version 3.1b2) (Eddy, 2011) against dbCAN HMMs v6 using default parameters and applying post-processing stringency cut-offs as suggested (Yin et al., 2012). Westernisation and additional Non-Westernised datasets Westernisation as the adoption of a Westernised lifestyle and culture can trace its origins to industrialisation and its promotion of urbanisation over the past two centuries. Westernisation has had a profound effect on human populations, due to access to healthcare and pharmaceutical products, hygiene and sanitation, changes in diets (processed, high-fat, low in complex carbohydrates but rich in refined sugars and salt), population density increase and reduced exposure to livestock. Westernisation is nonetheless difficult to ascribe as it demarcates populations which are clearly on a continuum. In this study definition of “Westernised” and “non-Westernised” is considered based on how populations differ based on the above criteria and how the samples were reported in the original publication. In this study five previous studies were considered where non-Westernised populations have been sampled from Fiji (Brito et al., 2016), Peru (Obregon-Tito et al., 2015), Tanzania (Rampelli et al., 2015; Smits et al., 2017) and Mongolia (Liu et al., 2016). In addition, we recently sequenced a population of adults from a rainforest region in North-eastern Madagascar (110 metagenomes) (Pasolli et al., 2019). We expanded upon a dataset of 5 samples sequenced from an established cohort in Ethiopia from Gimbichu in the Oromia region (Pasolli et al., 2019) with 45 additional samples. This cohort included 24 mothers and their infant(s) for a total of 50 metagenomes. We also sequenced two new non-Westernised populations from Ghana and Tanzania. In Ghana 12 extended families from the Asante Akim North district region were sampled where the local occupation is subsistence farming and the wider economy based on farming cash crops such as cocoa and plantain and there is also a commercial poultry industry. (44 metagenomes). From Tanzania samples were collected from 18 families from Korogwe District region where local employment and economy is based on agriculture particularly based on sisal fibres, cashew nuts and cotton (68 metagenomes). For all samples DNA was extracted using the PowerSoil DNA isolation kit (MoBio) as previously described (Human Microbiome Project Consortium, 2012). Libraries were constructed using the NexteraXT DNA Library Preparation Kit (Illumina) and . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . Iceman samples and Mexican coprolite material 673 to 768, 1,284 ± 16 B.P.) that confirms the previous direct dating of the pre-Columbian archaeological site (Supplementary Table S8). . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint The molecular analysis of the Iceman samples and of the ancient human coprolites was conducted at the ancient DNA laboratory of the EURAC Institute for Mummy Studies in Bolzano, Italy. Sample preparation and DNA extraction was performed in a dedicated pre-PCR area following the strict procedures required for studies of ancient DNA: use of protective clothing, UV-light exposure of the equipment and bleach sterilization of surfaces, use of PCR workstations and filtered pipette tips. DNA was extracted from the archaeological specimen using a chloroform-based DNA extraction method according to the protocol of (Tang et al., 2008). Libraries for the sequencing runs were generated with a modified protocol for Illumina multiplex sequencing (Kircher et al., 2012; Meyer and Kircher, 2010). Libraries of the Mexican coprolite samples were sequenced on Illumina HiSeq2500 platforms using 101–base pair paired-end sequencing kits. The Iceman samples were sequenced on an Illumina HiSeqX platform using the 150–base pair paired-end sequencing kit. Paired Illumina reads were quality-checked and processed (adapter removal and read merging) as previously described in (Maixner et al., 2018). Reads were mapped using Bowtie2 (Langmead and Salzberg, 2012) to the human genome (build Hg19, default mapping parameters) (Rosenbloom et al., 2015), the human mtDNA reference genome (rCRS, mapping parameter --very-sensitive-local) (Andrews et al., 1999), and selected P. copri genomes from the four clades. For details to the mapping results please refer to Supplementary Table S9. To deduplicate the mapped reads we used the DeDup tool (https://github.com/apeltzer/DeDup). The minimum mapping and base quality were both 30. The resulting bam files were used to check for characteristic aDNA nucleotide misincorporation frequency patterns using mapDamage2 (Jónsson et al., 2013). P. copri genome reconstruction from ancient gut metagenomes and molecular dating To reconstruct ancient P. copri genomes, we utilized our in-house scripts (https://bitbucket.org/CibioCM/cmseq) to build 4 ancient P. copri genome “scaffolds”, extracting consensus sites of aligned reads of sample 2179 and 2180 (the two samples with all four copri clades detected, Figure 5B) to representative genomes, one for each of the four clades for P. corpi complex. Sites covered by ancient reads were filled with gaps if one of following quality criteria was violated: (1) mapping quality is less than 30, (2) coverage is less than 5-fold, (3) the length of aligned read is less than 50nt, (4) minimum identity for the read is less than 97%, (5) minimum dominant allele frequency is less than 80%. Phylogeny of each core gene of a total of 540 was analyzed separately using BEAST v2.5.1 (Bouckaert et al., 2014). Core genes (n=210) supporting monophyly of the 4 P. copri clades (thus are unlikely to have been subject of horizontal gene transfer) were kept for searching for orthologs in ancient P. copri and their modern counterparts. We searched for these orthologs by aligning selected core genes against 4 “scaffolds” representative of the clades using BLASTn (Altschul et al., 1990) with parameter -word_size of 9. Mapping hits with either length less than 30 bp or e-value over 1e-10 were excluded. We kept orthologs shared by all 72 modern P. copri genomes and at least 1 ancient P. copri genome “scaffold”, and subsequently applied multiple sequence alignment using mafft (Katoh et al., 2002), with parameter --maxiterate of 1000 and --globalpair, to each of orthologs. Single-ortholog alignments were manually curated excluding mis-aligned sites (we consider continuous variant nucleotides observed in the alignment as artificially mis-aligned sites) and were then merged into one concatenation alignment. Out of eight ancient strains, we chose only the one with the best overall coverage which was the Clade A strain from sample 2180 (Supplementary Figure 12). This sample was accurately radiocarbon dated (A.D. 673 to 768, 1284 ± 16 B.P). The alignment composed of the selected ancient P. copri starin and 72 modern strains, which was further processed to automatically remove gappy columns using trimAl(Capella-Gutiérrez et al., 2009). The final alignment included 214,399 nucleotide positions. BEAST v2.5.1(Bouckaert et al., 2014) was used to infer divergence times of P. copri clades, using a GTR model of nucleotide substitution (with 4 gamma categories). Iceman samples and Mexican coprolite material CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint SAMtools mpileup (Li et al., 2009) and PileupCaller (https://github.com/stschiff/sequenceTools/tree/master/src-pileupCaller) for the Mexican specimen with the highest endogenous human content (2179, 2180) at loci that over-lapped with the Affymetrix Human Origins SNP array data (Patterson et al., 2012) and merged them to a modern European, Asian and Native American subset (n=2068) (Lazaridis et al., 2016). Principal Component Analysis (PCA) (Patterson et al., 2006b; Price et al., 2006) on the resulting SNP dataset show that the human DNA form the two coprolite samples has the greatest genetic affinity with modern Native Americans (Supplementary figure S11). This result highly supports the haplogroup assignment of the uniparental marker and genetically allocates the specimens to the American continent. SAMtools mpileup (Li et al., 2009) and PileupCaller (https://github.com/stschiff/sequenceTools/tree/master/src-pileupCaller) for the Mexican specimen with the highest endogenous human content (2179, 2180) at loci that over-lapped with the Affymetrix Human Origins SNP array data (Patterson et al., 2012) and merged them to a modern European, Asian and Native American subset (n=2068) (Lazaridis et al., 2016). Principal Component Analysis (PCA) (Patterson et al., 2006b; Price et al., 2006) on the resulting SNP dataset show that the human DNA form the two coprolite samples has the greatest genetic affinity with modern Native Americans (Supplementary figure S11). This result highly supports the haplogroup assignment of the uniparental marker and genetically allocates the specimens to the American continent. SAMtools mpileup (Li et al., 2009) and PileupCaller (https://github.com/stschiff/sequenceTools/tree/master/src-pileupCaller) for the Mexican specimen with the highest endogenous human content (2179, 2180) at loci that over-lapped with the Affymetrix Human Origins SNP array data (Patterson et al., 2012) and merged them to a modern European, Asian and Native American subset (n=2068) (Lazaridis et al., 2016). Iceman samples and Mexican coprolite material Both human and bacterial reads display low but already increased frequencies of C to T substitutions close to the fragment ends characteristic of ancient DNA (Orlando et al., 2015) (Supplementary Figure S10). Reads of the Iceman lung tissue metagenome that mapped to one single marker of the 2,448 P. copri complex specific markers display no DNA damage. The sex of the mapped human reads was assigned using a Maximum likelihood method, based on the karyotype frequency of X and Y chromosomal reads (Skoglund et al., 2013) (Supplementary Table S9). Estimation of human contamination rates using Schmutzi (Renaud et al., 2015) and ANGSD (Korneliussen et al., 2014) was in most samples not possible due to low damage pattern rates in the mitochondrial reads and due to the low coverage of the X chromosome (sample 2180), respectively. The Iceman samples with sufficient X-chromosome coverage show low contamination in the autosomal DNA when using ANGSD (Supplementary Table S9). Analysis of the human mitochondrial and autosomal variants provided further evidence for the sample origin and authenticity of the data. Variants in the mitochondrial genome were called using SAMtools mpileup and bcftoools (Li et al., 2009) with stringent filtering options (quality>30). Visual inspection of the called variants identified only less than 1% low-frequency variants that could be indicative for contamination.The haplogroup was identified by submitting the variant calling file to the HaploGrep website (Weissensteiner et al., 2016) (Supplementary Table S9). The human mitochondrial genomes in both Iceman samples carry the same variants as reported in previous Iceman genomic studies and belong to the K1f haplotype (Ermini et al., 2008; Keller et al., 2012). Importantly, in the Mexican coprolite samples 2179 and 2180 both detected mitochondrial haplogroups (C1b and B2) belong to the four main pan-American mtDNA lineages (Achilli et al., 2008; Bodner et al., 2012; Perego et al., 2010; Tamm et al., 2007). Furthermore, both haplogroups have been detected in previous studies in ancient human remains from Meso- and South America (Fehren-Schmitz et al., 2015; Gómez-Carballa et al., 2015; Llamas et al., 2016; Posth et al., 2018; Tackney et al., 2015) and haplogroup C1b has still nowadays its highest frequency in Peru and Mexico (Gómez-Carballa et al., 2015). We extended our analysis to the human autosomal data and called pseudodiploid genotypes using . Iceman samples and Mexican coprolite material Principal Component Analysis (PCA) (Patterson et al., 2006b; Price et al., 2006) on the resulting SNP dataset show that the human DNA form the two coprolite samples has the greatest genetic affinity with modern Native Americans (Supplementary figure S11). This result highly supports the haplogroup assignment of the uniparental marker and genetically allocates the specimens to the American continent. Data availability The 15 isolate P. copri genomes (and the extended set of 83 isolate, see above) and all metegenomically assembled metagenomes (MAGS) are available here: http://segatalab.cibio.unitn.it/data/Pcopri_Tett_et_al.html and metadata given in Supplemnetary Tables S2, S4. The full P. copri phylogeny of 1023 genomes is available as Supplementary Data File 1 (Newick format) at http://segatalab.cibio.unitn.it/data/Pcopri_Tett_et_al.html. Metadata for the three sequenced non-Westernised dataset is given in Supplementary Tables S11, S12, S13 and is also available as part of the curatedMetagenomicData package (Pasolli et al., 2017). The metagenomic reads for these datasets are available under NCBI-SRA BioProject ids; PRJNA529124 (Ghana), PRJNA529400 (Tanzania), PRJNA504891 (Ethiopia). The Data for the ancient metagenomic samples are available from the European Nucleotide Archive under accession no. PRJEB31971. P. copri genome reconstruction from ancient gut metagenomes and molecular dating To choose the best clock and demographic models we performed a model selection comparing coalescent constant, coalescent exponential, coalescent bayesian skyline, and coalescent extended bayesian skyline models (for the demographic priors) and strict and relaxed lognormal (for the clock prior). Model selection (Supplementary Table S10) was performed by comparing AICM from BEAST analyses with 100,000,000 Markov Chain Monte Carlo (MCMC) states for each model and sampling every 10,000 states. Convergence of posteriors was assessed by visualising . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint log files with Tracer v1.7(Rambaut et al., 2018). The most fitting combination of models was a coalescent constant population, with strict clock: this analysis was run longer for 204,000,000 iterations and effective sample size (ESS) of all parameters was over 200. Acknowledgements We thank Levi Waldron and the members of his laboratory for the effort in developing and supporting curatedMetagenomicData (Pasolli et al., 2017), and all the members of the Segata laboratory for fruitful discussions and support. We thank the GeNaPi Project, a collaborative study between Emalaikat Foundation, University of Valencia and IATA-CSIC, for data and sample collection from the Ethiopia cohort, and particularly Mari Olcina and Lourdes Larruy. We thank all field workers and laboratory technicians involved in the sample collection in Ghana and Tanzania. This work was supported by NIH NHGRI grant R01HG005220, NIDDK grant R24DK110499, NIDDK grant U54DE023798, CMIT grant 6935956 to C.H., the NYU-HHC CTSI grant (1TL1TR001447) to H.F.P., the U.S. National Institute of Health grant R01-DK103358-01 to R.B. and D.R.L., the Howard Hughes Medical Institute to D.R.L. Further support was provided by the Programma Ricerca Budget prestazioni Eurac 2017 of the Province of Bolzano, Italy to F.M., by the EU-H2020 (DiMeTrack-707345) to E.P. and by the European Research Council (ERC-STG project MetaPG-716575), MIUR ‘‘Futuro in Ricerca’’ RBFR13EWWI_001, and the European Union (H2020-SFS-2018-1 project MASTER-818368) to N.S. . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Supplementary Material Supplementary Tables/figures as a single pdf file Supplementary Tables/figures as a single pdf file CC-BY 4.0 International license under a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint . 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It is made available The copyright holder for this preprint (which was not this version posted April 9, 2019. ; https://doi.org/10.1101/600593 doi: bioRxiv preprint Supplementary Tables/figures as a single pdf file Table S1, Metagenomic datasets included in this study Table S1, Metagenomic datasets included in this study Table S3, Non-Westernised datasets recently sequenced and sequenced as part of this study Table S3, Non-Westernised datasets recently sequenced and sequenced as part of th Figure S1, Genome statistics for automatically metagenimically assembled genomes (MAGs), manually curated mategenomic assembled genomes (MC-MAGs) and isolate sequences Figure S2, Prevalence and abundance of the P. copri complex in publicly available datasets for which there are case and control samples Figure S3, Prevalence of P. copri complex in healthy publicly available metagenomes for which Body-Mass-Index (BMI) information was available Figure S3, Prevalence of P. copri complex in healthy publicly available metagenomes for which Body-Mass-Index (BMI) information was available Figure S4, Prevalence of P. copri complex in healthy publicly available metagenomes for which age of participants was available Figure S4, Prevalence of P. copri complex in healthy publicly available metagenomes for which age of participants was available Figure S5, Phylogeny of all 1121 P. copri genomes recovered in this study (including 98 new non-Westernised genomes) Figure S5, Phylogeny of all 1121 P. copri genomes recovered in this study (including 98 new non-Westernised genomes) Figure S6, Prevalence of P. copri clades in healthy individuals for datasets considered in this study Figure S7, The within sample P. copri complex pangenome for datasets considered in this study Figure S8, Presence absence of CAZy families in each of the 1121 P. copri genomes Figure S11, PCA plot of two Mexican coprolite samples and selected modern European, Asian and Native American . 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https://openalex.org/W4225654210	https://sciendo.com/pdf/10.2478/amns.2021.1.00102	English	null	Analysis of the relationship between industrial agglomeration and regional economic growth based on the multi-objective optimisation model	Applied mathematics and nonlinear sciences	2,021	cc-by	4,841	Abstract Against the background of the new era, with improvement at the socio-economic development level, scientific research scholars have begun to analyse the relations between industry agglomeration and regional economic growth, and thus of innovation for different parts of the industry upgrading and economic development to provide effective countermeasure. In order to promote the development of the regional economy in our country, there can be coordinated development. Therefore, on the basis of understanding the research status of the relationship between industrial agglomeration and regional economic growth at home and abroad, according to the multi-objective optimisation model and structural equation model, this paper conducts an in-depth discussion of the two, and finally concludes that industrial agglomeration will further promote regional economic growth. : multi-objective, optimisation model, industrial agglomeration, regional economic growth, structural equation model Submission Info Communicated by Juan Luis García Guirao Received June 6th 2021 Accepted September 24th 2021 Available online November 22nd 2021 Applied Mathematics and Nonlinear Sciences 7(2) (2022) 423–432 Applied Mathematics and Nonlinear Sciences 7(2) (2022) 423–432 Applied Mathematics and Nonlinear Sciences 7(2) (2022) 423–432 Applied Mathematics and Nonlinear Sciences https://www.sciendo.com Applied Mathematics and Nonlinear Sciences 7(2) (2022) 423–432 Applied Mathematics and Nonlinear Sciences https://www.sciendo.com Analysis of the relationship between industrial agglomeration and regional economic growth based on the multi-objective optimisation model Analysis of the relationship between industrial agglomeration and regional economic growth based on the multi-objective optimisation model Zetian Chen† School of Economics and Finance, South China University of Technology Guangdong, Guangzhou, 510641, China 1 Introduction Since the era of neoclassical economics, economists around the world have paid more and more attention to the phenomenon of industrial agglomeration. The term “accumulation” was originally coined in the early 1920s by Marshall, and is mainly used in this paper to mean the geographical position of close enterprise and industry concentration. A few words can produce positive external effects, and can be technology spillovers from labour, intermediate products, three aspects of analysis the cause of the industry gathered. With the constant improve- ment in the industry agglomeration theory, in the mid-1990s Porter and others put forward the typical “diamond” model during the study of national competitive advantage. There is clear information about the industry agglom- eration influence on international competitiveness in the regional industry; industrial concentration can further Open Access. © 2021 Chen, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 4.0 License. †Corresponding author. Email address: 1395845531@qq.com ISSN 2444-8656 doi:10.2478/AMNS.2021.1.00102 Open Access. © 2021 Chen, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 4.0 License. ISSN 2444-8656 doi:10.2478/AM Open Access. © 2021 Chen, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 4.0 License. ISSN 2444-8656 doi:10.2478/AM Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 424 enhance the level of regional competition and increase economic speed. At present, there is no systematic theo- retical system for research on the relationship between industrial agglomeration and regional economic growth, and most of them mainly exist in different disciplines such as neoclassical economics, regional economics and development economics. However, since the middle and late 1990s, especially in recent years, China has begun to conduct in-depth research on the phenomenon of industrial agglomeration, and has made some achievements. Generally speaking, the core research of Chinese researchers on industrial clusters at the present stage mainly focuses on the phenomenon of industrial clusters and the in-depth exploration of relevant theories of Western scholars. For example, on the basis of studying and introducing the evolution of the theory of enterprise com- munity abroad, Feng Delian and Wang Lei et al. sort out and analyse the relevant enlightenment according to the development trend of regional economy and industry in China. 1 Introduction Yang Baoliang integration in the domes- tic market, under the guidance of geographic gathered from industry and under the open economy of the two aspects of industry agglomeration and international trade, the increasingly innovation system to study the geog- raphy environment industry gathered phenomenon and the international economic and trade problems, and find the main industrial geographic concentration of the overall upward trend. From the perspective of the develop- ment of multinational enterprises, Ren Shenggang et al. made an in-depth discussion on the dynamic evolution process of their participation in clusters. In the opinion of Wang Jici and others, industrial agglomeration can only be considered from the perspectives of economics, sociology and innovation, and has unique advantages to promote the rapid development of regional economy and enhance their own competitiveness. Nowadays, industrial agglomeration, as the focus of research scholars at home and abroad, has attracted the attention of scholars in different fields and started to conduct in-depth discussion from multiple perspectives. However, after understanding the relevant literature, it is found that there are few related topics that consider the relationship between industrial agglomeration and regional economic growth from the perspective of economic openness, which makes it difficult to correctly consider the real causes of industrial agglomeration in various places from a macro perspective. Against the background of the new era, with the increase of content and form of finan- cial data, the traditional technology concept has been unable to meet the demands of practice development, so research scholars in their experiments to explore based on multi-objective optimisation theory put forward the quantitative trading strategy generated, as shown in Figure 1, the technical methods, and in the insurance, fund, stock and other financial products market brought about a positive role [1, 2]. Therefore, this paper also uses the multi-objective optimisation model to conduct in-depth discussion on the relationship between industrial agglomeration and regional economic growth, in order to provide an effective basis for subsequent scientific research and exploration [3]. 2.1 Multi-objective optimisation model Second, the objective optimisation mathematical model should be constructed The objective function is min f = f (f1, f2,..., fs) The constraint condition is 2.1 Multi-objective optimisation model For the multi-objective optimisation model, the existence of a solution is very complex, because there is a certain complexity between the objective function and the multi-personality. Because the objective function cannot reach the maximum or minimum values at the same time under many conditions, it is difficult to obtain the optimal solution for multi-objective problems. However, the actual problem needs to make a choice and get an optimal solution. In essence, the optimal solution refers to the point that conforms to the constraint conditions and allows all objective functions to reach the maximum or minimum value, which belongs to the optimal solution of the multi-objective optimisation problem. Generally speaking, even if there is an optimal solution for a multi-objective optimisation problem, the computational process is also very difficult, so it needs to be studied by using a search method. In order to satisfy the calculation requirements of the optimal solution, several new conditions need to be proposed to obtain the conditional optimal solution. The usual ways are mainly divided into the following [4]:i the single objective solution. The original multi-objective optimisation problem contains nition 1. First, the single objective solution. The original multi-objective optimisation problem 425 Analysis of the relationship between industrial agglomeration and regional economic growth Analysis of the relationship between industrial agglomeration and regional economic growth Fig. 1 Quantitative trading strategy generation technology method based on multi-objective optimisation theory Fig. 1 Quantitative trading strategy generation technology method based on multi-objective optimisation theory 1 Quantitative trading strategy generation technology method based on multi-objective optimisation theo multiple objective functions into the objective optimisation problem with only one objective function. The specific ideas are as follows: multiple objective functions into the objective optimisation problem with only one objective function. Th specific ideas are as follows: First, build a new target function: f = f (f1, f2,..., fs) Proof. Comply with the property requirements: within the scope of constraint conditions. The above objective function f = f (f1, f2,..., fs) belongs to the monotonic increment function of . It should be noted that, in the construction of the new objective function, it should also be analysed in combination with practical problems, and the f = f (f1, f2,..., fs)is defined as the f1, f2,..., fs undecreasing function. ( )i g Theorem 1. The constraint condition is gi (x1,x2,...,xn) = 0i = 1,2,...,m Proof. Finally, the mathematical model of multi-objective optimisation is calculated, and the optimal solution or conditional optimal solution of the original multi-objective optimisation problem is obtained. Proof. Finally, the mathematical model of multi-objective optimisation is calculated, and the optimal solution or conditional optimal solution of the original multi-objective optimisation problem is obtained. In order to better calculate the optimal solution of the single objective function, the following theorems should be combined to study the relationship between the two: Assuming that f = f (f1, f2,..., fs) is the monotone increasing function of f1, f2,..., fs, then the original multi-objective optimisation problem has an optimal solution, and the corresponding single-objective function Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 426 optimisation problem is as follows: optimisation problem is as follows: min f = f (f1, f2,..., fs) gi (x1,x2,...,xn) = 0i = 1,2,...,m gi (x1,x2,...,xn) = 0i = 1,2,...,m Proposition 2 There is a certain optimal solution which is also the optimal solution of the original multi- objective optimisation problem. Proposition 2 There is a certain optimal solution which is also the optimal solution of the original multi- objective optimisation problem. Assuming that the optimal solution of the original multi-objective optimisation problem is X∗, then the objective function in this region f1, f2,..., fs can obtain the minimum value. Lemma 3. Assuming that the optimal solution of the single-objective optimisation problem is Y∗, then the objective function in this region can obtain the minimum value f (Y∗). Proof. It can be concluded that: fi (X∗) ≤fi (Y ∗)i = 1,2,...,s f (X∗) ≥f (Y ∗) fi (X∗) ≤fi (Y ∗)i = 1,2,...,s f (X∗) ≥f (Y ∗) And because f(f1, f2,..., fs) is the monotone increasing function of f1, f2,..., fs, according to the analysis of the above results, we can get: f (X∗) ≤f (Y ∗) and f (X∗) = f (Y ∗) Therefore we can get: And because f(f1, f2,..., fs) is the monotone increasing function of f1, f2,..., fs, according to the analysis of the above results, we can get: f (X∗) ≤f (Y ∗) and f (X∗) = f (Y ∗) Therefore, we can get: Therefore, we can get: fi (X∗) = fi (Y ∗)i = 1,2,...,s Corollary 4. Thus, Y∗represents the optimal solution of the original multi-objective optimisation problem. The constraint condition is The second optimisation is as follows: min fi2 (x1,x2,...,xn)where.fi2is.some.function.in.f1, f2,..., fs (x1,x2,...,xn) ∈D1 min fi2 (x1,x2,...,xn)where.fi2is.some.function.in.f1, f2,..., fs (x1,x2,...,xn) ∈D1 Proof. The solution set D2 is then computed. Proof. The solution set D2 is then computed. Similarly, S-repeat optimisation is carried out, then the S-repeat optimisation is: min fi (x1,x2,...,xn) (x1,x2,...,xn) ∈Ds−1 min fi (x1,x2,...,xn) ( ) ∈D (x1,x2,...,xn) ∈Ds−1 The solution set, dS, is the set of optimal solutions for multiple optimisation problems. In other words, the optimal solution set or conditional optimal solution set of the original multi-objective optimisation problem is DS. It should be noted that it is not necessary to carry out multiple optimisation for all objective functions, but it can also carry out multiple optimisation according to certain objective functions, or even only need to select a certain objective function for optimisation. Note 7. Third is the target correlation function method. In the calculation and analysis based on the mathematical model of multi-objective optimisation problem, it is assumed that only a certain objective function is selected as the target, and the other objective functions are called constants within the expected value range, then the following mathematical model can be obtained: Proof. Where f represents one of the target functions. fik (x1,x2,...,xn) = aikk = 2,3,...,s Among them, I1,I2,I3,...,IS stands for 1,2,.... Some permutation of s gi(x1,x2,...,xn) = 0 i = 1,2,...,m Assuming that there is an optimal solution to the above problem, then min f1 (x1,x2,...,xn) is directly related to the value range of the objective function. In other words, the calculation method of the objective correlation function is to fix the value range of the objective function of one part, so as to obtain the optimal solution of the objective function of another part. Thus, the optimisation mathematical model of the binocular object is as follows: min f1 (x1,x2,...,xn) min f2 (x1,x2,...,xn) gi (x1,x2,...,xn) = 0i = 1,2,...,m gi (x1,x2,...,xn) = 0i = 1,2,...,m Assuming that the optimal value min f1 (x1,x2,...,xn) is F, then F is a function of t, and F = F (t) can also be obtained. Thus, this function can be called the objective correlation function of the first objective function with respect to the second objective function, and also the objective correlation function of the primary objective with respect to the sub-objective. The constraint condition is From the point of view of the common objective function of single objective, it can be seen that when the multi- objective optimisation problem does not have the optimal solution, the design of objective function is the most important to obtain the conditional optimal solution by using the solution method of bullet screen package. The new objective function can directly show the relationship between the original multiple objectives, so it must be combined with real problems to design, in order to intuitively and accurately show the properties of the actual problems. Here are some common forms of the objective function: Equilibrium optimisation function is: f(f1, f2,..., fs) = f1 + f2 +...+ fs Proof. The weight optimisation function is: f(f1, f2,..., fs) = ω1 f1 +ω2 f2 +...+ωs fs And ω1,ω2,...,ωs are all weight coefficients greater than 0 The optimisation function of the sum of squares is: And ω1,ω2,...,ωs are all weight coefficients greater than 0 The optimisation function of the sum of squares is: f(f1, f2,..., fs) = s ∑ i=1 f 2 i And are all weight coefficients greater than 0 Conjecture 5. Second, multiple optimisation metho And are all weight coefficients greater than 0 Conjecture 5. Second, multiple optimisation method. And are all weight coefficients greater than 0 C j 5 S d l i l i i i h d And are all weight coefficients greater than 0 Conjecture 5. Second, multiple optimisation method. Conjecture 5. Second, multiple optimisation method. Combined with the property analysis of the actual problem, the original multi-objective optimisation prob- lem is transformed into multiple single-objective optimisation problem. This calculation method needs to be combined with the analysis of the objective function properties of the packet optimisation problem, and thus clear the objective and order to be optimised. At the same time, multiple optimisation mathematical models should be built, as shown below: 427 Analysis of the relationship between industrial agglomeration and regional economic growth Analysis of the relationship between industrial agglomeration and regional economic growth Proof. The first optimisation is as follows: Proof. The first optimisation is as follows: min f = f (f1, f2,..., fs)where.fi1is.some.function.in.f1, f2,..., fs min f = f (f1, f2,..., fs)where.fi1is.some.function.in.f1, f2,..., fs gi (x1,x2,...,xn) = 0i = 1,2,...,m The solution set D1 is then computed p Example 6. 2.2 Analysis of industrial agglomeration in regional economic growth Combined with the analysis of the multi-objective optimisation model, the following hypotheses are ob- tained: First, opening to the outside world has a positive and direct impact on the composition of industrial agglom- eration; second, the regional economic growth of opening to the outside world has a positive impact on the role; third, industrial agglomeration has a positive and positive effect on the factor level of the agglomeration area. Fourth, industrial agglomeration has a positive and positive effect on regional growth; Fifth, factor input has a positive effect on the regional economic growth. In this paper, 4 latent variables and 11 observed variables are proposed, and their specific contents and meanings are shown in Table 1 [5,6]: i At the same time, combined with the assumptions and variables proposed in this study, the equation model as shown in Figure 2 can be obtained: Due to the high requirement of model research on the number of samples, the practical analysis takes prefecture-level cities in China as the research target and carries out cross-section research on the basis of collecting and accumulating relevant data. Finally, the sample size reaches 283, which is mainly derived from the statistical yearbooks of provinces and cities. During the data analysis and processing, in order to avoid the phenomenon of false regression caused by unstable variables, this paper implemented unit root detection for all variables using the ADF method. Assuming that the variables are stationary, the following analysis can be performed directly. Otherwise, relationship testing shall be conducted in accordance with the specified requirements, and the final test results are as shown in Table 2 [7]: Combined with the analysis in Table 2, it can be seen that most variables are controlled at the significance level of 1%, which meets the requirements of unit root test, and the single integer order is 0. It is proved that all the variables in the unit root test results belong to a stationary sequence and meet the basic requirements of equation model analysis, so subsequent analysis can be carried out [8]. In the normal distribution test, the maximum likelihood estimation method should be used for research, and all variables should conform to the normal distribution. The final results are shown in Table 3. The constraint condition is It should be noted that the value range of t can be analysed as follows: min f2 (x1,x2,...,xn) ≤t ≤max f2 (x1,x2,...,xn) min f2 (x1,x2,...,xn) ≤t ≤max f2 (x1,x2,...,xn) 428 Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 Assuming that the correlation function of the first objective with respect to the second objective is F = F (t), then there is an optimal solution for the original dual-objective optimisation problem, which can be obtained at the corresponding point in .t = min f2 (x1,x2,...,xn) If the objective correlation function F = F (t) cannot ob- tain the minimum value in the t = min f2 (x1,x2,...,xn)region, then there is no optimal solution for the original dual-objective optimisation problem. When a fixed value t is studied, the value point range of the objective cor- relation function F = F (t) is the conditional optimal solution obtained by the original dual-objective optimisation problem under the condition of a fixed second objective. pi j When considering the superior and inferior solutions, it is assumed that X∗and Y∗represent feasible solu- tions, and if the following conditions are met, X∗is a better solution than Y∗. f1 (X∗) ≤f1 (Y ∗), f2 (X∗) < f2 (Y ∗) f1 (X∗) < f1 (Y ∗), f2 (X∗) ≤f2 (Y ∗) f1 (X∗) < f1 (Y ∗), f2 (X∗) ≤f2 (Y ∗) 3 Result analysis The analysis path shown in Figure 2 is studied using the AMOS7.0 software and estimated data to analyse the model data. Combined with the above picture analysis, it can be seen that the Chi-square test probability value of the model outlined in this paper is 0.215, which is more than 0.05, which proves that the model fitting data conforms to the original hypothesis. 3 Result analysis At the same time, combined with the analysis of parameter estimation results as shown in Table 4, it can be seen that model evaluation should first judge whether the parameters 429 Analysis of the relationship between industrial agglomeration and regional economic growth Table 1 Connotation analysis of latent variables and observed variables Latent variables Observed variables Variable meaning Economic growth Per capita gross regional product (RJGDP) (yuan) Ratio of GDP to total population at year-end Average salary (PJGZ) (Yuan) Weighted average wages of state-owned units, urban collective units and other units Per capita fiscal revenue (RJCZSR) (yuan) Ratio of annual general budget revenue to year-end population Industrial agglomeration Proportion of industrial added value (GYBZ) (%) The proportion of industrial added value in regional GDP Proportion of Manufacturing Enterprises (CYRS) (%) Proportion of the number of manufacturing enterprises in the total number of regional enterprises Agglomeration Index (JJZS) Calculate according to relevant formulas Opening to the outside world Proportion of Actual Utilisation of Foreign Investment (SJLYWZ) (%) The proportion of FDI actually utilised in GDP Proportion of Processing Trade (JGMYBZ) (%) Processing trade as a share of a region’s total foreign trade, which also measures the extent to which a region is integrated into the global production network Trade Dependence (MyYCD) (%) Ratio of total regional imports and exports to GDP Factor input Growth of Fixed Asset Investment (GDZCTZ) (%) (Total Fixed Assets Investment of Current Year – Total Fixed Assets Investment of Last Year)/Total Fixed Assets Investment of Last Year – 1 Human capital (RLZB) (%) Growth rate of employment at year-end Table 2 Analysis of test results based on ADF unit root Variable Test statistics P values Lag The whole order Rjgdp −4.473965 0.0003 0 I(0) Pjgz −5.019404 0.0002 0 I(0) Rjczsr −4.965987 0.0003 0 I(0) Gybz −3.573293 0.0068 11 I(0) Cyrs −3.979609 0.0017 11 I(0) Jjzs −5.419012 0 0 I(0) Sjlywz −4.460539 0.0003 0 I(0) Jgmybz −4.455309 0.0003 0 I(0) Myycd −5.131048 0 0 I(0) Gdzctz −4.145615 0.00015 0 I(0) Rlzd −5.241372 0.00024 0 I(0) Table 2 Analysis of test results based on ADF unit root 430 Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 Fig. 2 Structure diagram of equation model Fig. 3 Result analysis 2 Structure diagram of equation model Table 3 Test and analysis results of normalised data Pjgz Rigdp Jgmybz Silywz Mzzcd Gybz Cyrs Jjzs Rjczsr Gdzctz Rlzb N 283 283 283 283 283 283 283 283 283 283 283 Mean 1.84 1.41 48.6 2.87 64.13 47.3 23.86 1.28 24.19 25.7 5.89 SD 1.0064 0.0539 0.1544 0.1915 0.0744 0.264 0.0038 0.0362 0.1464 0.1028 0.0546 Skewness 0.0001 0 0 0 0 0 0.0002 0 0 0 0 Kurtosis 2.9704 2.9714 2.9705 2.9725 2.9732 2.9687 2.9745 2.9709 2.9689 2.9704 2.9717 Jarque Bera 0.0120 0.0116 0.0118 0.0117 0.0124 0.0117 0.0106 0.0113 0.0116 0.0115 0.0135 Probability 0.9941 0.9942 0.9938 0.9924 0.9854 0.9868 0.9947 0.9944 0.9958 0.9524 0.9673 Table 3 Test and analysis results of normalised data contained in it have statistical significance, and then conduct significance test and analysis on each coefficient. In this paper, a simple test method in AMOS 7.0 was selected as CR, and it was found that the significance test of the final parameters and variance results was completed [9].i contained in it have statistical significance, and then conduct significance test and analysis on each coefficient. In this paper, a simple test method in AMOS 7.0 was selected as CR, and it was found that the significance test of the final parameters and variance results was completed [9].i It can be seen that the first hypothesis proposed in this paper is valid, and the opening to the outside world has a direct impact on the phenomenon of industrial agglomeration, and the corresponding influence coefficient reaches 0.12. The significance level of the second hypothesis put forward in this paper reaches 0.034, which proves that the hypothesis is valid and has direct and indirect effects on economic growth. The significance level of the third hypothesis proposed in this paper reaches 0.004, which proves that the hypothesis is also valid, and the direct influence coefficient of industrial agglomeration on the accumulation of factors reaches 0.3. 3 Result analysis Based on the analysis from the perspective of practical development, the influencing factors of industrial agglomeration on regional economic growth can be divided into two situations: one is direct influence, the other is indirect influence, and the direct influence of industrial agglomeration on economic growth is greater; in Hypothesis 5, factor input also has a direct impact on the regional economic growth, and the significance level reaches 0.006. But from a practical point of view, the impact is small. At the same time, combined with the analysis of observation variables proposed in this paper, it can be seen that industrial agglomeration is closely related to regional economic growth, and will form a direct or indirect impact from multiple perspectives. 3 Result analysis Therefore, it can be seen that industrial agglomeration can lead to the agglomeration of population, technology, Analysis of the relationship between industrial agglomeration and regional economic growth 431 Table 4 Parameter estimation analysis results The normalised path coefficient S.E C.R P Industrial agglomeration < – Opening to the outside world 123 139 924 003 Factor input < – Industrial agglomeration 301 165 966 034 Economic growth < – Industrial agglomeration 467 302 2.877 004 Economic growth < – factor input 073 704 363 002 Economic growth < – Opening to the outside world 005 252 041 006 Jgmybz< – Opening to the outside world 301 215 2.066 039 Myycd< – Opening to the outside world 974 219 1.135 026 Sjlywz< – Opening to the outside world 378 Gybz< – Industrial agglomeration 830 161 8.294 * Jjzs< – Industrial agglomeration 847 268 8.476 * Pjgz< – Economic growth 648 120 4.246 * Rjgdp< – Economic growth 732 Gdzctz< – factor input 390 Rlzd< – factor input 644 2.888 944 045 Rjczsr< – Economic growth 706 193 4.385 * Cyrs< – Industrial agglomeration 871 Note: ※※※ P < 0.001 Table 4 Parameter estimation analysis results capital and other factors. Hypothesis 4 proposed in this paper is also valid, and the actual significance level reaches 0.002. Based on the analysis from the perspective of practical development, the influencing factors of industrial agglomeration on regional economic growth can be divided into two situations: one is direct influence, the other is indirect influence, and the direct influence of industrial agglomeration on economic growth is greater; in Hypothesis 5, factor input also has a direct impact on the regional economic growth, and the significance level reaches 0.006. But from a practical point of view, the impact is small. At the same time, combined with the analysis of observation variables proposed in this paper, it can be seen that industrial agglomeration is closely related to regional economic growth, and will form a direct or indirect impact from multiple perspectives. capital and other factors. Hypothesis 4 proposed in this paper is also valid, and the actual significance level reaches 0.002. 4 Conclusion To sum up, in the new era, in order to grasp better the relationship between regional economic growth and industrial optimisation and upgrading, researchers should start from the multi-objective optimisation model to strengthen the research on the relationship between the two, and pay attention to learning from the conclusions of research topics at home and abroad, so as to put forward effective suggestions for the current economic development. Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 432 Chen Applied Mathematics and Nonlinear Sciences 7(2022) 423–432 ] Di Ye. An Empirical Study on the Relationship between Industrial Agglomeration Effect and Regio Growth in China – Taking Industry as an Example [J]. Economic Perspective, 2013(11):135-137.i References [1] Xinzhu Zhang. New Business Week, 2019, 000(012):P.12-12. (in Chinese) [2] Di Ye. An Empirical Study on the Relationship between Industrial Agglomeration Effect and Regional Economic Growth in China – Taking Industry as an Example [J]. Economic Perspective, 2013(11):135-137.i [2] Di Ye. An Empirical Study on the Relationship between Industrial Agglomeration Effect and Regional Economic Growth in China – Taking Industry as an Example [J]. Economic Perspective, 2013(11):135-137.i [3] Jialiang He, Jiandong Zhao. Research on the relationship between financial agglomeration and regional economic growth – A case study of Anhui Province [J]. Journal of Heilongjiang Institute of Technology (comprehensive edition), 2019(8). [3] Jialiang He, Jiandong Zhao. Research on the relationship between financial agglomeration and regional economic growth – A case study of Anhui Province [J]. Journal of Heilongjiang Institute of Technology (comprehensive edition), 2019(8). [4] Xin’an Qi. Journal of Anhui Agricultural University (Social Science Edition), 2001(3):8-10. (in Chin [ ] Q g y ( ), ( ) ( ) [5] Ying Sun, Li-xia Zhu,Qiu-xian Ding, et al. Research of Agricultural Modernization, 2016, 37(02):247-254.] [5] Ying Sun, Li-xia Zhu,Qiu-xian Ding, et al. Research of Agricultural Modernization, 2016, 37(02):247-254.] g g The Impact of Financial Agglomeration Spatial Distribution on Regional Economic Growth – An Empirica d on Spatial Dupin Model [J]. Regional Finance Research, 2019, No.567(11):13-19. [6] Fanfan Li. The Impact of Financial Agglomeration Spatial Distribution on Regional Economic Growth – An Empirical Study Based on Spatial Dupin Model [J]. Regional Finance Research, 2019, No.567(11):13-19. [7] Yingjun Feng. Fuzzy solutions for multi-objective optimization problems [J]. Chinese Science Bulletin, 1981(17):1028- 1030. (in Chinese) Study Based on Spatial Dupin Model [J]. Regional Finance Research, 2019, No.567(11):13-19. [7] Yingjun Feng. Fuzzy solutions for multi-objective optimization problems [J]. Chinese Science Bulletin, 1981(17):1028- 1030. (in Chinese) p p g ( ) ng. Fuzzy solutions for multi-objective optimization problems [J]. Chinese Science Bulletin, 1981(17):1028- hinese) ( ) [8] In the gold. Innovative Education Research, 2019, 7(5):7. (in Chin ( ) [8] In the gold. Innovative Education Research, 2019, 7(5):7. (in Chinese) g ( ) ( ) [9] Gongyue Wu. Research on optimal problem of multi-objective programming based on obstacle function. Think Tank Times, 2019, 202(34):292+299.
https://openalex.org/W4321842987	https://ajas.uoanbar.edu.iq/article_120061_572f343c1fec3cf75886e0e775829e6e.pdf	Arabic	null	Effect of potassium fertilizer and foliar application with Copper in growth and yield of broad bean Vicia Faba L.	Maǧallaẗ al-Anbār li-l-ʿulūm al-zirāʿiyyaẗ/Maǧallaẗ al-anbār li-l-ʻulūm al-zirāʻiyyaẗ	2,015	cc-by	7,779	ISSN: 1992-7479 ISSN: 1992-7479 ةص خال ال Basheer Hamad Abdullah AL Duleimi Anmar Ismael Ali Fayadh AL Fahdawi College of Agriculture – University of AL-Anbar Basheer Hamad Abdullah AL Duleimi Anmar Ismael Ali Fayadh AL Fahdawi College of Agriculture – University of AL-Anbar ةص خال ال نفذت تجربة حقلية في تربة ذات نسجه مزيجة طينية غرينية في الم وسم الشتوي لعام4102 في محافظة األنبار- منطقة حصيبه الشرقية التابعة لقضاء الخالدية الواقعة على خط طول24 o ودائرة عرض44 o لمعرفة تأثير الرش بعنصر النحاس بأربعة تراكيز1 و01 و41 و41 ملغمCu لتر- 0 وأربعة مستويات من البوتاسيوم1 و01 و011 و001 غم ك K ه- 0 أضيفت إلى التربة في نمو وحاصل ونوعية الباقالء صنف إسبانيLuz De Otono . استخدم في تنفيذ التجربة ترتيب األلواح المنشقةSplit-Plot وفق تصميم القطاعات العشوائية الكاملة ( R.C.B.D) وبأربعة مكررات. وتلخص ت :نتائج التجربة باآلتي- أثرت تراكيز النحاس معنوياً في جميع الصفات ال مدروسة وقد أعطى التركيز41 ملغمCu لتر- 0 أعلى متوسط الرتفاع النبات ونسبة البوتاسيوم في األوراق وطول الجذر وعدد القرنات بالنبات ووزن011 ( بذرة021.40 ( غم) وحاصل بذور النبات90.16 غم) ونسبة البروتين ( في البذور40.64 % ) بينما أعطى التركيز41 ملغمCu لتر- 0 أعلى ن سبة للنحاس في األوراق، وأعطى التركيز 01 ملغمCu لتر- 0 .أعلى متوسط للمساحة الورقية في النبات أثرت مستويات البوتاسيوم معنوياً في جميع الصفات المدروسة وقد أعطى المستوى011 كغمK ه- 0 أعلى متوسط للمساحة الورقية في النبات وطول الجذر ووزن 011 ( بذرة040.90 غم) وحاصل ( بذور النبات40.49 غم. نبات- 0 ) بينما تفوق المستوى001 كغمk ه- 0 في ارتفاع النبات وتركيز النحاس والبوتاسيوم في األوراق وعدد القرنات بالنبات ونسبه البروتين في البذور ( 46.96 % ). أثر التداخل بين مستويات البوتاسيوم وتراكيز النحاس معنوياً في جمع الصفات المدروسة باستثناء ( نسبة البروتين في البذور. وقد أعطى التداخل بين المستوى العالي لكال العنصرين001 كغمk ه- 0 + 41 ملغم Cu لتر- 0 ( ) أعلى متوسط لعدد القرنات بالنبات06.16 ( قرنه) وحاصل بذور النبات04.20 غم. نبات- 0 .) Effect of potassium fertilizer and foliar application with Copper in growth and yield of broad bean Vicia Faba L. Effect of potassium fertilizer and foliar application with Copper in growth and yield of broad bean Vicia Faba L. Abstract A field experiment was carried out in loamy-clay silty in winter season of 2014 in Anbar Governorate-Hisaiba region/Al-Khalidiyah district that located on 43° البحث مستل من رسالة ماجستير للباحث الثاني * 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 longitude and 33° latitude, in order to know the effect of foliar application with four concentrations of copper 0, 10, 20, 30 mg Cu L-1 and four concentration with potassium 0, 50, 100 and 150 Kg ha-1 were added to the soil in growth, yield and its quality of broad bean- Spanish variety (cv. Luz De Otono). Split-plot arrangement. Was used in Randomized Complete Block Design (R.C.B.D.) with four replications . The experiment results were summarized as follows: Copper concentrations significantly affected in all studied traits. The concentration of 30 mg Cu L-1 gave highest average of plant height, potassium ratio in leaves, root length, pod number, 100 seeds weight (140.75g) grain yield of plaint (68.09g) percentage in seeds (28.97%), while 20 mg Cu L-1 gave highest average of cu percentage in leaves and 10 mg Cu L-1 gave highest average of leaf area.Potassium levels significantly affected in all studied traits. 100 kg K ha-1 gave the highest average of, leaf area for plant, root length 100- seed weight(138.59g), seed yield in plaint(71.76g), while 150 kg K.ha-1 was superior in plant height, concentration of Cu and K in leaves, number of pod in plant and protein percentage in seeds (29.69%). The interaction between K levels and Cu significantly affected significantly in all studied traits, except the, protein percentage in seeds. The combination of 150 Kg K ha-1 + 30 mg Cu L-1 gave highest average of number of pod in plant (19.09 pod), seed yield in plaint ( 83.41g). المقدمة تعد الباقالءVicia faba L. Abstract أحد نباتات العائلة البقوليةFabaceae المهمة في العديد من بلدان العالم كونها مصدر مهم من مصادر المواد الغذائية ل ل ماليين من سكانها ولدى المجتمعات الفقيرة منها وذوي الدخل المحدود خاصة الحتواء بذورها على نسبة عالية من البروتي ن عالي الجودة التي لها أثر مهم في تقليل نقص البروتين الحيواني في تلك المجتمعات فضالً عن احتوائها على الكاربوهيدرات وبعض العناصر المعدنية .والفيتامينات يعاني محصول الباقالء من مشكلة كبيرة وهي ارتفاع نسبة األزهار المتساقطة والتي تصل نسبتها 91 - 01 % من األزهار( الكلية04 ،) وهذه النسبة العالية تؤدي إلى انخفاض إنتاجيته .وأحد معالجات هذه المشكلة هي استخدام التغذية المتوازنة والسيما عنصري البوتاسيوم والنحاس أن التسميد بعنصر البوتاسيوم له دور مهم في نمو النبات وا نتاجيته من خالل عملة على تنشيط أكثر من01 ،أنزيم وفي ف تح وغلق الثغور وتنظيم الجهد االزموزي للخاليا النباتية وزيادة نفاذيتها والمساهمة في عملية التمثيل الضوئي وانتقال نواتجه وانقسام الخاليا ومقاومه النبات لالضطجاع واألمراض ،النباتية كما يساهم في تكوين األحماض النووية والبروتينات واألنزيمات. Abstract أن حاجة النبات للبو تاسيوم تفوق حاجته ألي من العناصر الغذائية األخرى عدا،النتروجين وقد يفوق ه ( في بعض مراحل النمو06 .) وأن نقصه يؤدي إلى إحداث خلل في العمليات .الفسيولوجية والتي تنعكس سلباً في نمو وحاصل النبات ونوعيته كما ان للتغذية الورقية بالنحاس دور مهم في تحسين أداء النب ات وان نقصه يسبب خلل في نمو النبات من خالل اشتراكه في عملية الفسفره الضوئية في عملية التمثيل الضوئي و عمليات األكسدة واالختزال في سلسلة 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 النقل اإللكتروني ( Electron transport chain ( ) في التنفس الهوائي1 و42 و42 )، فضالً عن ذلك اشتراك النحاس في األنزيمات الت ي تساعد في التفاعالت التي تختزل جزيئة األوكسجين وتشمل هذه األنزيمات أنزيم Cytochrome oxidase Lactase وTyrosinase وDiamine oxidase وAscorbic acid oxidase ِوهذه األنزيمات تساهم في عمليات األكسدة واالختزال التي تحدث في خاليا أنسجة النبات وتؤدي إلى تنشيط عمل يات حيوية كالتنفس وتكوين الكلوروبالست والبروتين واختزال النترات وتكوين فيتامينC ( 9 و11 و24 )، النقل اإللكتروني ( Electron transport chain ( ) في التنفس الهوائي1 و42 و42 )، فضالً عن ذلك اشتراك النحاس في األنزيمات الت ي تساعد في التفاعالت التي تختزل جزيئة األوكسجين وتشمل هذه األنزيمات أنزيم Cytochrome oxidase Lactase وTyrosinase وDiamine oxidase وAscorbic acid oxidase ِوهذه األنزيمات تساهم في عمليات األكسدة واالختزال التي تحدث في خاليا أنسجة النبات وتؤدي إلى تنشيط عمل يات حيوية كالتنفس وتكوين الكلوروبالست والبروتين واختزال النترات وتكوين فيتامينC ( 9 و11 و24 )، و للنحاس أهمية في عملية تكوين البروتين من خالل دوره في زيادة تثبيت النتروجين الجوي وكذلك من خالل رفع قدرة النبات على زيادة تكوين البروتين واألحماض النوويةDNA وRNA المهمة في عملية تكوين البروتين فقد لوحظ تجميع لألمونيوم وكذلك انخفاض مستوياتDNA في األجزاء النباتية التي تعاني من نقص ( النحاس04 ً). وبناء على أهمية ما تقدم طبقت تجربة حقلية في الموسم الشتوي لعام4102 تضمنت دراسة أربعة مستويات من البوتاسيوم بتأثير أربع ة تراكيز للتغذية الورقية بالنحاس بهدف تحديد أفضل مستوى من السماد البوتاسي والتغذية الورقية بالنحاس ًوصوال إلى .زيادة النمو الخضري والحاصل ومكوناته وتحسين نوعيته المواد وطرائق العمل جدول1 بعض الصفات الكيميائية والفيزيائية لتربة أرض التجربة قبل الزراعة وعلى عمق0 - 00 :سم الصفة الرمل (غم كغم- 3 ) الغرين (غم كغم- 3 ) الطين (غم كغم- 3 ) النسجة المادة العضوية (غم كغم- 3 ) التوصيل الكهربائي (PH) درجة التفاعل N الجاهز(ملغم كغم- 3 ) P الجاهز(ملغم كغم- 3 ) K (الجاهز ملغم كغم- 3 ) Cu الجاهز(ملغم كغم- 3 ) القيمة 120 490 260 مزيجة طينية غرينية 9.7 2.7 7.9 70 9.8 65 0.4 أجري التحليل في مختبرات وزارة العلوم والتكنولوجيا-بغداد أجري التحليل في مختبرات وزارة العلوم والتكنولوجيا-بغداد ( أضيف السماد النتروجيني على شكل يوريا%46 N ) وبواقع91 كغم Nه- 0 ( 00 ) على دفعتين األولى بعد أجراء عمل ية الخف مباشرة والثانية عند مرحلة التزهيز . تم تعشيب حقل التجربة2 مرات يدويا خالل موسم ،النمو و بعد مرور أربعة عشر يوما من الرشة األخيرة للنحاس (والتي كانت في بداية تكوين األزهار) أخذت ستة نباتات بصورة عشوائية من كل وحدة تجريبية لدراسة صفة المساحة الورقية(دسم4. نبات- 0 ) والتي تم قياسها بطريقة األقراص، أذ اخذ91 قرص بقطر0.0 سم من91 ورقة من أوراق النباتات الستة ثم جففت األوراق :واألقراص كالً على حده واخذ وزنها الجاف ثم قدرت المساحة الورقية باستخدام المعادلة التالية الوزن الجاف ألورا ق النبات المساحة الورقية للنبات = ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــ ــــــ ـــــ × مساحة91 قرص ( 31 ) الوزن الجاف لـ91 قرص ( أضيف السماد النتروجيني على شكل يوريا%46 N ) وبواقع91 كغم Nه- 0 ( 00 ) على دفعتين األولى بعد أجراء عمل ية الخف مباشرة والثانية عند مرحلة التزهيز . تم تعشيب حقل التجربة2 مرات يدويا خالل موسم ،النمو و بعد مرور أربعة عشر يوما من الرشة األخيرة للنحاس (والتي كانت في بداية تكوين األزهار) أخذت ستة نباتات بصورة عشوائية من كل وحدة تجريبية لدراسة صفة المساحة الورقية(دسم4. المواد وطرائق العمل نفذت تجربة حقلية في الموسم الشتوي لعام 4102 في محافظة األنبار منطقة حصيبه الشرقية التابعة لقضاء الخالدية لدراسة ( استجابة نمو وحاصل الباقالء صنف أسبانيLuz De Otono ) للرش بعنصر النحاس والتسميد البوتاسي. أستخدم في تنفيذ التجربة ترتيب األلواح ( المنشقةSplit-Plots ) وفق تصميم القطاعات ( العشوائية الكاملةRCBD ) وبأربعة،مكررات أذ احتلت مستويات السماد البوتاسي األلواح الرئيسة ( Main Plot ) وهي1 و01 و011 و001 كغم K هـ- 0، بينما احتلت مستويات الرش بالنحاس األلواح ( الثانويةSub Plot ) وهي1 و01 و41 و41 ملغمCu لتر- 0 . تم إضافة مستويات السماد البوتاسي دفعة واحدة عند الزراعة على شكل كبريتات البوتا سيوم4 SO 2 K ( 00 % K )، إما مستويات الرش بالنحاس فقد أضيفت على دفعتين األولى بعد21 يوماً من الزراعة والثانية في بداية مرحلة األزهار وعلى شكل كبريتات النحاس المائيةO 2 .5H 4 CuSO ( 94.02 % Cu ) لكل وحدة تجريبية وحتى البلل التام ألوراق النباتات ، أجريت عملية الرش في وق ت الصباح الباكر بواسطة مرشة ظهرية سعة41 لتر ( وبضغط2) بار، تمت إضافة مادة ناشرة وهي محلول التنظيف الزاهي لمحلول الرش وبكمية00 سم4 لكل011 لتر ماء لتقليل الشد السطحي للماء وضمان البلل التام لألوراق بهدف زيادة كفاءة امتصاص محلول الرش ، حرثت ارض التجربة حراث تين متعامدتين ثم نعمت وسويت وبعد ذلك أخذت عينات عشوائية من حقل التجربة وعلى عمق 1 - 41 سم لدراسة بعض الخصائص الفيزيائية والكيميائية ألرض التجربة (الجدول0 ). قسمت أرض التجربة إلى وحدات،تجريبية كل واحده منها احتوت على أربعة،خطوط طول الخط 4 م والمسافة بين خط و أخر01 سم وبين جورة وأخرى40 سم لتصبح مساحة الوحدة التجريبية9 م4 وكانت المسافة 355 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 بين كل وحدة تجريبية0 م والمسافة بين المكررات4 .م سمدت التجربة بالسماد الفوسفاتي وبمستوى001 كغم 5 O 2 P ه- 0 ( وعلى دفعه واحده أثناء أعداد األرض للزراعة4 ) تمت الزراعة في00 \ 00 \ 02 41 وذلك بحفر شق على طول الوحدة التجريبية وبعمق تراوح بين0 - 9 سم ثم وضعت بذرتين في الجورة الواحدة وبعد ذلك غطيت البذور بطبقة مناسبة من التربة. بعد اكتمال اإلنبات أجريت عملية الخف إلبقاء نبات واحد في الجورة لتصبح الكثافة النباتية01111 نبات هـ- 0. المواد وطرائق العمل نبات- 0 ) والتي تم قياسها بطريقة األقراص، أذ اخذ91 قرص بقطر0.0 سم من91 ورقة من أوراق النباتات الستة ثم جففت األوراق :واألقراص كالً على حده واخذ وزنها الجاف ثم قدرت المساحة الورقية باستخدام المعادلة التالية الوزن الجاف ألورا ق النبات المساحة الورقية للنبات = ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـــــــــــــــــــــــــــــــــــــــــــــــــ ــــــ ـــــ × مساحة91 قرص ( 31 ) الوزن الجاف لـ91 قرص ( 31 ) ( 31 ) كما تم تقدير تركيز النحاس في األوراق (ملغم كغم- 0 مادة جافة) وذلك بأخذ0 غم من العينة الجافة لألوراق ومن ثم تم تقدير نسبة النحاس باستعمال جهازSpectrophotometer (وفقا للطريقة التي ذكرها49 .) كما قدرت نسبة البوتاسيوم في األوراق (ملغم كغم- 0 )مادة جافة باستخدام جهازphotometer Flame نوع PGl 4111 Automatic إنكليزي ( المنشأ بالطريقة التي ذكرها40 ) . وعند الحصاد أخذت جميع نباتات المرزين الوسطيين لكل وحدة تجريبية بعد استبعاد النباتات الطرفية منها لدراسة الصفات األتية: ارتفاع النبات )(سم الذي قيس من قاعدة النبات عند مستوى سطح األر.ض وحتى نهاية الساق الرئيس ثم اخذ معدلها )طول الجذر الرئيس (سم تم حفر حفرة دائرية حول جذور النباتات المأخوذة من المرزين الوسطيين وعلى عمق أكثر من 41 سم وبعد ذلك تم قلعها من التربة وغسلها جيدا بالماء ثم قيس طول الجذر الرئيس لكل منها ثم اخذ معدلها. عدد القر نات نبات- 0 أخذت .كمعدل لعدد القرنات في نباتات الخطوط الوسطية وزن011 )بذرة (غم أخذت 011 بذرة بصورة عشوائية من بذور كل وحدة تجريبية وتم وزنها بالميزان اإللكتروني .الحساس 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 حاصل البذور للنبات الواحد (غم) تم حساب ة .)كمعدل لوزن البذور في نباتات الخطوط الوسطية نس بة البروتين في البذور( % ) تم تقدير نسبة النتروجين في البذور باستخدام جهازSemi-micro kjeldal المذكورة في الطريقة ( الرسمية للمحللين الكيميائيين40 ) وبعد ذلك تم حساب نسبة البروتين في البذور:وكاالتي %( نسبة البروتين = نسبة النتروجين × ) 9.40 التحليل اإلحصائي: حللت البيانات للصفات المدروسة وفقاً للتصميم المستخدم في التجربة و استخدم اختبار أقل فرق معنويL.S.D للمقارنة بين المتوسطات الحسابية عند مستوى احتمال1.10 ( 9 )وباستعمال البرنامج اإلحصائي Genstat الجاهز بالحاسبة اإللكترونية. النتائج والمناقشة ارتفاع النبات (سم) اتفقت هذه النتيجة مع نتائج بحوث أخرى أظهرت التأثير المعنوي إلضافة البوتاسيوم في زيادة ارتفاع النبات ولمحاصيل بقولية ( متعددة2 و5 و15 و44 و41 و81 .) يوضح الجدول4 معنوية التداخل بين تراكيز النحاس ومستويات البوتاسيوم، أذ حققت النباتات المسمدة بالمستوى001 كغمK ه- 0 والمرشوشة بالتركيز41 ملغمCu لتر- 0 أعلى متوسط الرتفاع النبات بلغ09.14 سم مقارنة بمعامالت التداخل األخرى وبنسبة زيادة بلغت41.44 % مقارنة بال نباتات المسمدة بالمستوى01 كغمK ه- 0 غير المرشوشة بالنحاس التي أعطت أقل متوسط لصفة ارتفاع النبات بلغ90.00 .سم بلغ90.00 .سم 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 جدول2 تأثير الرش بعنصر النحاس والتسميد البوتاسي )والتداخل بينهما في متوسط ارتفاع النبات (سم لمحصول الباقالء جدول2 تأثير الرش بعنصر النحاس والتسميد البوتاسي )والتداخل بينهما في متوسط ارتفاع النبات (سم لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراك يز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 00 00 00 1 71.32 34.50 30.01 31.07 32.51 01 74.51 34.24 50.55 54.53 37.54 011 30.02 37.20 51.25 52.50 35.02 001 31.01 54.40 57.00 55.20 51.31 متوسط النحاس 30.07 35.15 30.35 51.01 L.S.D 0.05 K Cu KCu 7.25 2.42 3.23 ( المساحة الورقية للنبات دسم2. نبات- 1) ( المساحة الورقية للنبات دسم2. نبات- 1) تشير نتائج الجدول4 إلى تفوق نباتات التركيز01 ملغمCu لتر- 0 معنوياً بأعلى متوسط للصفة بلغ 444.9 دسم4. نبات- 0 ولم تختلف معنوياً مع نباتات التركيز41 ملغمCu لتر- 0 والتي بلغت مساحتها الورقية 440.4 دسم4. نبات- 0 بينما كان االختالف معنوياً مع النباتات المعاملة بالتركيز41 ملغمCu لتر- 0 والتي بلغت 406.6 دسم4. نبات- 0 والتي بدورها اختلفت معنوياً عن نباتات المقارنة التي أعطت أقل متوسط للمساحة الورقية بلغ410.4 دسم4. نبات- 0 . أن سبب زيادة المساحة الورقية عند التر كيز01 ملغمCu لتر- 0 ربما يعود إلى تفوقه في عدد التفرعات الرئيسة بالنبات وهذا ينعكس في زيادة عدد البراعم بالنبات ومن ثم زيادة عدد األوراق وبالتالي زيادة المساحة الورقية بالنبات. واتفقت هذه النتيجة مع نتائج دراسات أخرى بينت التأثير المعنوي إلضافة النحاس في ا( لمساحة الورقية01 و44 .) يتبين من نتائج الجدول4 ان النباتات المسمدة بالمستوى011 كغمK ه- 0 قد أ عطت أعلى متوسط للصفة بلغ424.2 دسم4. نبات- 0 ولم تختلف معنوياً عن نباتات المستوى01 كغمK ه- 0 بما يعادل449.6 دسم4. نبات- 0 بينما كان االختالف معنوياً وبنسبة زيادة بلغت6.40 و6.60 % ًبالتتابع قياسا بالمستوى001 كغمK ه- 0 ومعاملة المقارنة التي سجلت أوطأ متوسط للمساحة الورقية بلغ400.0 دسم4. نبات- 0 . ارتفاع النبات (سم) يتضح من النتائج في الجدول4 وجود زيادة في ارتفاع النبات مع زيادة تركيز النحاس، أذ أعطت ( النباتات المرشوشة بالتركيز العالي للنحاس41 ملغمCu لتر- 0 ) أعلى متوسط الرتفاع النبات بلغ00.10 سم ولم تختلف معنوياً عن نباتات التركيز41 لغم مCu لتر- 0 إذ بلغت46.40 ،سم بينما كان االختالف معنوياً مع التركيز01 ملغمCu لتر- 0 والتي بلغت 40.02سم ومعاملة المقارنة التي أعطت أقل معدل الرتفاع النبات بلغ 41.49 س م. ان سبب تفوق التركيز العالي للنحاس في ارتفاع النبات يعود إلى دورة في زيادة نسبة البوتاسيو م في األوراق (الجدول0 ) ومن المعروف أن للبوتاسيوم دور إيجابي ومباشر في تحفيز الخاليا على االنقسام واالستطالة .وخصوصاً الخاليا المرستيمية في القمم النامية للسيقان التي تنعكس في زيادة ارتفاع النبات جاءت هذه النتيجة متماشية مع نتائج دراسات أخرى بينت التأثير المعنوي إلضافة النحاس في زيادة ارتفاع النبات) 01 و44 و44 ). يتبين من نتائج الجدول4 وجود زيادة في متوسط ارتفاع النبات مع زيادة مستوى أضافة البوتاسيوم، أذ أعطت النباتات المسمدة بالمستوى001 كغم K ه- 0 أعلى متوسط للصفة بلغ00.40 سم ولم تختلف معنوياً عن نباتات ا لمستويين011 و01 كغمK ه- 0 إذ بلغتا40.44 و49.00 سم بالتتابع، بينما كان االختالف معنوياً مع معاملة المقارنة التي سجلت أقل متوسط لصفة ارتفاع النبات بلغ44.20 سم. أن هذه الزيادة في ارتفاع النبات ترجع إلى دور البوتاسيوم اإليجابي والمباشر في تحفيز الخاليا على ا النقسام واالستطالة والسيما الخاليا المرستيمية في القمم النامية للسيقان مما ينعكس ذلك على زيادة ارتفاع النبات. ارتفاع النبات (سم) أن سبب زيادة المساحة الورقية عند المستوى011 كغمK ه- 0 يعود إلى دور البوتاسيوم في زيادة انقسام واستطالة خاليا النب ات ومنها خاليا الورقة. بينت دراسات أخرى وجود تأثير معنوي للبوتاسيوم في زيادة المساحة ( الورقية للنبات0 و02 و41 .)إما ًبالنسبة للتداخل بين مستويات البوتاسيوم وتراكيز النحاس فكان تأثيره معنويا .في المساحة الورقية أذ حققت النباتات المسمدة بالمستوى011 كغمK ه- 0 والمرشوشة بالتركيز41 ملغمCu لتر- 0 أعلى متوسط للمساحة الورقية بلغ406.4 دسم4. نبات- 0 مقارنة بمعامالت التداخل األخرى وبزيادة معنوية بلغت نسبتها49.04 % عن النباتات غير المسمدة بالبوتاسيوم والمرشوشة بالتركيز41 ملغمCu لتر- 0 التي أعطت أوطأ متوسط للمساحة الور قية بلغ402.0 دسم4. نبات- 0 . 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 جدول0 تأثير الرش بعنصر النحاس والتسميد البوتاسي( والتداخل بينهما في متوسط المساحة الورقية دسم2 . نبات- 1 ) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 306.3 356.6 298.1 284.8 011.4 01 296.7 339.8 325.5 345.6 027.0 011 341.8 331.7 359.2 336.9 052.5 001 288.4 322.2 330.4 312.4 010.5 متوسط النحاس 005.0 003.7 025.0 010.0 L.S.D 0.05 K Cu KCu 10.23 10.10 25.40 تركيز النحاس في األوراق (ملغمCu كغم- 1 مادة جا)فة مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 جدول0 تأثير الرش بعنصر النحاس والتسميد البوتاسي( والتداخل بينهما في متوسط المساحة الورقية دسم2 . نبات- 1 ) لمحصول الباقالء جدول0 تأثير الرش بعنصر النحاس والتسميد البوتاسي( والتداخل بينهما في متوسط المساحة الورقية دسم2 . نبات- 1 ) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 306.3 356.6 298.1 284.8 011.4 01 296.7 339.8 325.5 345.6 027.0 011 341.8 331.7 359.2 336.9 052.5 001 288.4 322.2 330.4 312.4 010.5 متوسط النحاس 005.0 003.7 025.0 010.0 L.S.D 0.05 K Cu KCu 10.23 10.10 25.40 تركيز النحاس في األوراق (ملغمCu كغم- 1 مادة جا)فة تركيز النحاس في األوراق (ملغمCu كغم- 1 مادة جا)فة تركيز النحاس في األوراق (ملغمCu كغم- 1 مادة جا)فة تشير نتائج الجدول2 إلى تفوق النباتات المرشوشة بالتركيز41 ملغمCu لتر- 0 بإعطائها أعلى متوسط للصفة بلغ04.40 ملغمCu لتر- 0 مادة جافة ولم تختلف معنوياً عن النباتات المرشوشة بالتركيزين41 و01 ملغمCu لتر- 0 والتي هي بحدود04.96 و04.40 ملغمCu لتر- 0 بالتتابع في حين كان االختالف معنوياً وبزيادة بلغت نسبتها4.62 % عن معاملة المقارنة التي أعطت أقل متوسط للصفة بلغ01.40 ملغمCu لتر- 0. ارتفاع النبات (سم) أن سبب تفوق التركيز41 ملغمCu لتر- 0 في تركيز النحاس في أوراقها ربما يعود إلى زيادة في نسبة الكلوروفيل في األوراق وبالتالي زيادة كفاءه ( األوراق في امتصاص الضوء وبالتالي زيادة القوة االختزاليةNADPH ) والطاقة ( ATP) الالزمة المتصاص العناصر بصورة نشطة والسيما عنصر النحاس. تماشت ( هذه النتيجة مع4 )الذي .وجد زيادة في محتوى النحاس في النبات بزيادة مستوى أضافته أظهرت نتائج الجدول أن هناك زيادة ف ي تركيز النحاس في األوراق مع زيادة مستويات السماد البوتاسي ليصل إلى أعلى متوسط له عند المستوى العالي للبوتاسيوم( 001 كغمK ه- 0 ) والذي بلغ00.19 ملغم كغم- 0 مادة جافة، جدول5 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط تركيز النحاس في ا ألوراق (ملغم كغم- 1 مادة جافة) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 57.00 40.34 50.40 55.24 55.72 01 40.00 55.40 41.34 41.34 41.24 011 40.40 45.40 42.40 40.34 40.47 001 40.40 44.40 43.24 43.00 44.07 متوسط النحاس 40.34 42.01 42.34 42.70 L.S.D 0.05 K Cu KCu 0.23 1.00 5.57 351 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 ولم يختلف هذا المستوى معنوياً عن المستوى011 كغمK ه- 0 البالغ04.09 ملغم كغم- 0 غير أن االختالف كان معنوياً مع المعامالت األخرى وبنسبة زيادة بلغت4.24 و2 04.4 % قياساً بالمستوى01 كغمK ه- 0 ومعاملة المقارنة التي سجلت أوطأ متوسط للصفة بلغ20.94 ملغم.كغم- 0 .مادة جافة أن زيادة تركيز النحاس في األوراق بزيادة مستوى إضافة البوتاسيوم يعود إلى أن نباتات المستوى ( العالي للبوتاسيوم001 كغمK ه- 0 ) كانت تمتلك أعلى نسبة للبو تاسيوم في أوراقها (الجدول0 ) وهذا ربما يحفز هذه النباتات لزيادة امتصاص النحاس للوصول إلى حالة التوازن الغذائي األمثل بين العنصرين باإلضافة إلى دور البوتاسيوم في زيادة نفاذية أغشية خاليا نسيج الورقة من خالل تنظيم األس الهيدروجيني والبيئة التنافذية داخل الخال( يا مما ينعكس في زيادة انتقال المغذيات0 و41 و44 و46 ). بينت نتائج الجدول2 معنوية التداخل بين التسميد البوتاسي والتغذية الورقية بالنحاس، أذ أعطت النباتات المسمدة بالمستوى001 كغمK ه- 0 والمرشوشة بالتركيز41 ملغمCu لتر- 0 أعلى متوسط للصفة بلغ04.40 ملغم كغم- 0 مادة جافة ولم تختلف معنوياً عن النباتات المسمدة بنفس المستوى من البوتاسيوم والمرشوشة بالتركيز41 ملغمCu لتر- 0 فقد بلغ04.11 ملغم كغم- 0 في حين كان االختالف معنوياً مع بقية معامالت التداخل األخرى وبزيادة بلغت نسبتها42.20 % ًقياسا بمعاملة المقارنة لكال العن صرين التي أعطت أوطأ متوسط لتركيز النحاس في األوراق بلغت29.11 ملغم كغم- 0 .مادة جافة نسبة البوتاسيوم في األوراق (ملغم كغم- 1) تشير نتائج الجدول0 ( إلى تفوق النباتات المرشوشة بالتركيز العالي للنحاس41 ملغمCu لتر- 0 ) بإعطائها أعلى متوسط لنسبة البوتاسيوم في األ وراق بلغ4.46 ملغم كغم- 0 ولم تختلف معنوياً عن نباتات التركيزين41 و01 ملغمCu لتر- 0 والذي بلغ حوالي 4.49 و4.41 ملغم كغم- 0 غير أن جميع التراكيز اختلفت معنوياً عن معاملة المقارنة التي أعطت أقل متوسط للصفة بلغ0.40 ملغمCu لتر- 0 . اتفقت هذه النتيجة مع ( 44 ) الذي بينوا الدور اإليجابي .للنحاس في زيادة نسبة البوتاسيوم باألوراق كما تشير نتائج الجدول0 إلى تفوق النباتات المسمدة بالمستوى001 كغمK ه- 0 معنوياً بأعلى متوسط للصفة بلغ4.09 ملغم كغم- 0 ولم تختلف معنوياً عن النباتات المسمدة بالمستوى011 كغمK ه0 إذ بلغ4.49 ملغم كغم- 0 ًغير أن االختالف كان معنويا مع المستوى01 كغمK ه- 0 فقد بلغ4.14 ملغم كغم- 0 ونباتات المقارنة التي أعطت أقل متوسط للصفة بلغ 0.49 ملغم كغم- 0. أن زيادة نسبة البوتاسيوم في أوراق النباتات بزيادة مستوى أضافته يرجع إلى زيادة جاهزيته بمحلول التربة في منطقة ا لجذور وبالتالي زيادة امتصاصه من قبل النبات والتي انعكست في رفع نسبته في األوراق بما يتالءم مع حاجة النبات الية. جاءت هذه النتيجة متماشية مع نتائج بحوث أخرى بينت وجود تأثير معنوي للسماد ( البوتاسي في زيادة نسبة البوتاسيوم في األوراق ولمحاصيل بقولية مختلفة8 و2 و5 و11 و84 ). يوضح الجدول 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 0 معنوية التداخل بين مستويات البوتاسيوم وتراكيز النحاس في هذه الصفة. أعطت النباتات المسمدة بالمستوى 001 كغمK ه- 0 ذات التغذية بالتركيز41 ملغمCu لتر- 0 أعلى متوسط لنسبة البوتاسيوم في أوراقها بلغ4.14 ملغم كغم- 0 ولم تختلف معنوياً ع ن النباتات المسمدة بالمستويين001 و011 كغمK ه- 0 والمرشوشة بالتركيز41 ملغمCu لتر- 0 والتين بلغتا4.61 و4.09 ملغم كغم- 0 بالتتابع، بينما كان االختالف معنوياً مع معامالت التداخل األخرى وبنسبة زيادة بلغت004.06 % ًقياسا بمعاملة المقارنة لكال العنصرين التي أعطت أ قل متوسط للصفة بلغ 0.22 ملغم كغم- 0. ب أرى وب ب زي ي ارين ر ب ي ب غ و ل 0.22 ملغم كغم- 0. نسبة البوتاسيوم في األوراق (ملغم كغم- 1) جدول4 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط نسبة البوتاسيوم في األوراق (ملغم كغم- 1 ) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 1.55 2.10 1.53 1.70 1.37 01 2.04 2.25 1.74 2.14 2.02 011 1.34 2.00 2.55 2.57 2.27 001 1.30 2.53 0.03 2.00 2.47 متوسط النحاس 1.34 2.20 2.27 2.00 L.S.D 0.05 K Cu KCu 0.55 0.25 0.45 )طول الجذر الرئيس (سم جدول4 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط نسبة البوتاسيوم في األوراق (ملغم كغم- 1 ) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 1.55 2.10 1.53 1.70 1.37 01 2.04 2.25 1.74 2.14 2.02 011 1.34 2.00 2.55 2.57 2.27 001 1.30 2.53 0.03 2.00 2.47 متوسط النحاس 1.34 2.20 2.27 2.00 L.S.D 0.05 K Cu KCu 0.55 0.25 0.45 )طول الجذر الرئيس (سم يتضح من الجدول9 وجود زيادة في طول الجذر مع زيادة تركيز الرش بالنحاس، أذ أعطت النباتات المرشوشة بالتركيز41 ملغمCu لتر- 0 أعلى متوسط للصفة بلغ09.90 سم ولم تختلف معنوياً عن التركيز41 ملغمCu لتر- 0 إذ بلغ09.20 سم بينما كان االختالف معنوياً مع التركيز01 ملغمCu لتر- 0 فقد بلغ09.49 سم ومعاملة المقارنة التي أعطت أقل متوسط لطول الجذر بلغ00.90 ( سم. اتفقت هذه النتيجة مع44 ) الذين .اثبتوا دور النحاس المهم في زيادة طول الجذر من خالل دوره في زيادة انقسام خاليا الجذر يتبين من الجدول أن النباتات المسمدة بالمستوى011 كغمK ه- 0 قد أعطت أ على متوسط لطول الجذر بلغ04.40 سم متفوقة بذلك معنوياً على جميع المعامالت األخرى وبنسبة زيادة بلغت01.24 و9.06 و44.06 % قياساً بالمستويين001 و01 كغمK ه- 0 ومعاملة المقارنة التي سجلت أوطأ متوسط لطول الجذر بلغ 02.00 سم بالتتابع. أن هذه الزيادة تعزى إلى دور الب وتاسيوم في زيادة المساحة الورقية (الجدول4 ) والتي قد ينعكس عنها زيادة في منتجات عملية التمثيل الكربوني وبالتالي مساهمة المجموع الخضري بقدر أكبر من الغذاء المصنع لتغذية الجذر لزيادة انقسام واستطالة خالياه، أذ يعتمد الجذر في غذاءه الالزم لعملية التنفس وبناء الخاليا (على منتجات عملية التمثيل الضوئي في الجزء الخضري للنبات4 ). اتفقت هذه النتيجة مع( 0 و04 ) الذين 313 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 وجدوا أن إضافة البوتاسيوم إلى التربة أثرت معنوياً في زيادة طول الجذر. نسبة البوتاسيوم في األوراق (ملغم كغم- 1) يتبين من الجدول أن النباتات المسمدة بالمستوى011 كغمK ه- 0 والمرشوشة بالتركيزين41 و1 4 ملغمCu لتر- 0 قد أعطت أعلى متوسط لطول الجذر بلغ00.40 و00.41 سم بالتتابع وتفوقتا معنوياً على معامالت التداخل األخرى وبنسبة زيادة بلغت40.04 و40.29 % بالتتابع قياساً بالنباتات غير المسمدة بالبوتاسيوم والمرشوشة بالتركيز41 ملغمCu لتر- 0 التي أعطت أوطأ متوسط لل صفة بلغ04.64 .سم جدول7 تأثير الرش بعنصر النحاس والتسميد البوتاسي )والتداخل بينهما في متوسط طول الجذر الرئيس(سم لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 15.02 15.52 10.02 14.54 15.44 01 17.00 17.40 13.20 17.34 17.75 011 13.24 13.23 15.00 15.01 13.35 001 14.13 17.53 17.40 17.24 17.10 متوسط النحاس 14.75 17.27 17.55 17.75 L.S.D 0.05 K Cu KCu 0.40 0.52 0.54 عدد القرنات بالنبات يتضح من الجدول4 أن النباتات ذات التغذية بالتركيز العالي للنحاس( 41 ملغمCu لتر- 0 ) قد أعطت أعلى متوسط للصفة بلغ02.60 قرنة. نبات- 0 ولم تختلف معنوياً مع نباتات التركيزين41 و01 ملغمCu لتر- 0 والتي بلغت04.00 و04.60 قرنة. نبات- 0 بالتتابع غير أن جميعها تفوق معنوياً على نباتات المقارنة التي أعطت أقل معدل بلغ.49 01 قرنة. نبات- 0 . أن تفوق التركيز41 ملغمCu لتر- 0 في عدد القرنات يرجع إلى تفوقه في نسبة البوتاسيوم في األوراق (الجدول0) وفي ارتفا ع النبات وطول الجذر (الجدول ين4 و4 ) ومن المعلوم أن البوتاسيوم له دور واضح في زيادة كفاءة النبات على نقل منتجات عملية التمثيل ا لضوئي من أجزاء النبات لصالح األزهار الناشئة والتي انعكس ت ًإيجابا في زيادة نسبة الخصب في األزهار ومن ثم زيادة عدد .القرنات بالنبات ( اتفقت هذه النتيجة مع01 و42 ) الذين بينوا وجود تأثير معنوي إلضافة النحاس في مكونات الحاصل. يتضح من الجدول نفسه وجود زيادة في عدد القرنات بالنبات مع زيادة مستوى أضافة البوتاسيوم وقد أعطت النباتات المسمدة بالمستوى العالي للعنصر( 001 كغمK ه- 0 ) أعلى متوسط للصفة بلغ00.44 قرنة. نبات- 0 ولم تختلف معنوياً عن النباتات المسمدة بالمستوى011 كغمK ه- 0 والتي هي بحدود04.44 قرنة. نبات- 0 في حين اختلفت معنوياً مع نباتات المستوى01 كغمK ه- 0 ومعاملة المقارنة التي سجلت أوطأ متوسط 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 لعدد القرنات بالنبات بلغ00.20 ،قرنة ومن هذه النتائج يتضح أن المعاملة األولى تفوقت بنسبة بلغت42.44 % عن معاملة المقارنة. نسبة البوتاسيوم في األوراق (ملغم كغم- 1) أن تفوق المستوى001 كغمK ه- 0 في هذه الصفة يعود إلى تفوقه في ارتفاع النبات (الجدول4) وتركيز النحاس والبوتاسيوم في األوراق (الجدولين 2 و0 ) أذ أن العنصرين لهما دور إيجابي في تأخير شيخوخة األوراق وبالتالي أطاله فتره التمثيل وانتقال نواتج التمثيل إلى مواقع النشوء الجديدة في المرحلة ( التكاثرية للنبات6 و46) وال .ذي ينعكس في زيادة نسبة الخصب في األزهار ومن ثم زيادة عدد القرنات بالنبات ( تماشت هذه النتيجة مع نتائج دراسات أخرى بينت التأثير اإليجابي للبوتاسيوم في زيادة عدد القرنات بالنبات2 و5 و12 و44 و41 .) تشير نتائج الجدول إلى معنوية التداخل بين مستويات البوتاسيوم وتراكيز النحاس في هذه الصفة. أذ حققت النباتات المسمدة بالمستوى العالي لكال العنصرين( 001 كغمK ه- 0 + 41 ملغمCu لتر- 0 ) أعلى متوسط لعدد القرنات بالنبات بلغ06.16 قرنة. نبات- 0 ولم تختلف معنوياً عن النباتات المسمدة بنفس المستوى من البوتاسيوم والمرشوشة بالتركيز1 4 ملغمCu.لتر- 0 والبالغة09.44 قرنة. نبات- 0 بينما كان االختالف معنوياً مع جميع معامالت التداخل األخرى وبنسبة زيادة بلغت69.46 % قياساً بالنباتات المسمدة بالمستوى011 كغمK ه- 0 غير المرشوشة بالنحاس التي أعطت أوطأ متوسط لعدد القرنات بالنبات بلغ6.44 .قرنة نبات- 0. جدول3 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط عدد القرنات بالنبات لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 10.28 12.24 10.77 12.51 11.54 01 9.93 15.08 12.36 13.10 12.72 011 9.72 14.31 14.36 15.10 10.03 001 11.49 14.18 16.72 19.09 14.03 متوسط النحاس 10.07 10.04 10.44 15.04 L.S.D 0.05 K Cu KCu 2.51 1.52 0.25 وزن100 )بذرة (غم جدول3 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط عدد القرنات بالنبات لمحصول الباقالء جدول3 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط عدد القرنات بالنبات لمحصول الباقالء وزن100 )بذرة (غم يتضح من الجدول0 وجود زيادة في وزن011 بذرة مع زيادة مستوى الر ش بالنحاس، أذ أعطت ( النباتات المرشوشة بالتركيز العالي للنحاس41 ملغمCu لتر- 0 ) أعلى متوسط للصفة بلغ021.40 غم متفوقة بذلك معنوياً على بقية التراكيز وبزيادة بلغت نسبتها4.00 و0.42 و6.29 % قياساً بالتركيزين41 و01 ملغمCu لتر- 0 ومعاملة المقارنة التي أعطت أقل مت وسط لوزن011 بذرة بلغ040.00 غم. نسبة البوتاسيوم في األوراق (ملغم كغم- 1) أن تفوق التركيز العالي للنحاس في هذه الصفة يعود إلى تفوقه في ارتفاع النبات (الجدول4 ) وهذا يعني توفر خزين غذائي أكثر 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 في النبات الذي ينتقل الحقاً بعد نشوء البذور ليزيد من امتالئها إضافة إلى ذلك فأن هذه المعاملة قد تفوقت في ط ول الجذر (الجدول9 ) وهذا ينعكس في زيادة معدل امتصاص الماء والعناصر الغذائية التي لها دور في تحفيز نشاط األنزيمات وعملية انقسام واستطالة خاليا البذرة وبالتالي زيادة وزن البذرة. اتفقت هذه النتيجة مع نتائج دراسات أخرى بينت الدور اإليجابي إلضافة النحاس في زياد( ة وزن البذرة4 و01 و42 .) يتضح من الجدول0 أن النباتات المسمدة بالمستوى011 كغمK ه- 0 قد أعطت أعلى متوسط للصفة بلغ040.90 غم ولم تختلف معنوياً عن نباتات المستويين 001 و01 كغمK ه- 0 فقد بلغتا040.42 و042.60 غم بالتتابع، في حين اختلفت معنوياً مع معاملة المقار نة التي سجلت أقل متوسط لوزن011 بذرة بلغ 046.20 غم، كما يتبين أن جميع مستويات البوتاسيوم01 و011 و001 كغمK ه- 0 تفوقت معنوياً على معاملة المقارنةK0 وبنسب زيادة بلغت2.44 و4.10 و2.04 % بالتتابع. أن تفوق المستوى011 كغمK ه- 0 في هذه الصفة يعود إلى تفوقه في الم ساحة الورقية (الجدول4 ) وبالتالي زيادة انتقال نواتج التمثيل الضوئي إلى البذور النامية ليزيد من معدل امتالئها ومن ثم زيادة وزنها فضالً عن تفوق هذا المستوى في طول الجذر (الجدول9 ) وهذا يعني زيادة كفاءه النبات في امتصاص الماء والمغذيات التي لها دور في زيادة ا نقسام الخاليا واستطالتها .وامتالئها ومن ثم زيادة حجمها ( وفي هذا المجال أشار41 .) إلى أن وزن البذرة عبارة عن داله لمعدل التمثيل الضوئي وانتقال نواتجه كما اتفقت هذه النتيجة مع نتائج بحوث أخرى ( وجدت تأثيراً معنوياً ألضافه البوتاسيوم في زيادة وزن البذرة02 و00 و44 و40 ). بينت نتائج الجدول معنوية التداخل بين التسميد البوتاسي والتغذية الورقية بالنحاس. حاصل بذور النبات(غم. نبات- 1) يتضح من الجدول6 وجود زي ادة في حاصل بذور النبات مع زيادة تركيز الرش بالنحاس حتى أعطت النباتات التي رشت بالتركيز41 ملغمCu لتر- 0 أعلى متوسط لحاصل البذور بلغ90.16 غم. نبات- 0 وقد تفوقت معنوياً على جميع المعامالت األخرى وبنسبة زيادة بلغت0.00 و4.09 و40.90 % قياساً بالتركيزين41 و01 لغم مu C لتر- 0 ومعاملة المقارنة التي سجلت أقل متوسط للصفة بلغ00.66غم. نبات- 0 بالتتابع، فضالً عن ذلك فأن جميع تراكيز النحاس تفوقت معنوياً في هذه الصفة على معاملة المقارنة. أن سبب تفوق التركيز41 ملغمCu لتر- 0 في هذه الصفة يعود إلى تفوقه في عدد القرنات بالنبات ووزن011 بذرة (الجدولين 4 و0 ) فضالً عن تفوقه في ارتفاع النبات ونسبة البوتاسيوم في األوراق وطول الجذر (الجداول4 و0 و9 ) كل ذلك انعكس ًإيجابا في زيادة حاصل بذور النبات. اتفقت هذه النتيجة مع نتائج دراسات أخرى بينت الدور اإليجابي للنحاس في زيادة حاصل بذور النب( ات ولمحاصيل حقلية مختلفة7 و14 و82 و85 و87 ). يبين الجدول6 ان النباتات المسمدة بالمستوى011 كغمK ه- 0 قد أعطت أعلى متوسط لحاصل بذور النبات بلغ40.49 غم. نبات- 0 ولم تختلف معنوياً عن نباتات المستوى001 كغمK ه- 0 البالغة94.42 غم. نبات- 0 بينما كان االختالف معن وياً وبنسبة زيادة بلغت00.00 و44.44 % قياساً بنباتات المستوى01 كغمK ه- 0 ومعاملة المقارنة التي سجلت أقل متوسط للصفة بلغ04.01 غم. نبات- 0 .بالتتابع أن سبب زيادة حاصل بذور النبات بتأثير المستوى011كغمK ه- 0 يعود إلى تفوقه في ووزن011 بذرة (الجدول 0) وتميزه في عدد القرنات بالنبات (الجدول 4 ) فضالً عن تفوقه في المساحة الورقية وطول الجذر (الجدولين4 و9 ) كل ذلك ساهم بشكل مباشر وغير مباشر في زيادة حاصل بذور النبات. جاءت هذه النتيجة متماشية مع نتائج دراسات أخرى بينت التأثير اإليجابي للبوتاسيوم في حاصل البذور ولمحاصيل ( بقولية مختلفة4 و0 و00 و40 ). يتضح من نتائج الجدول معنوية التداخل بين مستويات البوتاسيوم وتراكيز النحاس في هذه ( الصفة. أذ حققت النباتات المسمدة بالمستوى العالي لكال العنصرين001 كغمK ه- 0 + 41 ملغمCu لتر- 0 ) أعلى متوسط لحاصل بذور النبات بلغ04.20 غم. نبات- 0 ولم تختلف معنوياً عن النباتات المسمدة بالمستوى 011 كغمK ه- 0 والمرشوشة بالتركيزين41 و41 ملغمCu لتر- 0 والبالغتين01.44 و00.40 غم . نبات- 0 ،بالتتابع غير إنها اختلفت معنوياً مع جميع معامالت التداخل األخرى وبزيادة مقدارها 42.40 غم( 40.00 % ) قياساً بالنباتات غير المسمدة بالبوتاسيوم والمرشوشة بالتركيز41 ملغمCu لتر- 0 التي أعطت أقل متوسط لحاصل البذور بلغ20.94 غم. نبات- 0 . نسبة البوتاسيوم في األوراق (ملغم كغم- 1) أذ أعطت النباتات المسمدة بالمستوى01 كغمK ه- 0 والمرشوشة بالتركيز41 ملغمCu لتر- 0 أعلى متوسط لوزن011 بذرة بلغ024.41 غم ولم تختلف معنوياً مع النباتات المسمدة بالمستو ى 011 كغمK ه- 0 والمرشوشة بالتركيزين 41 و41 ملغمCu لتر- 0 اللتين بلغتا024.00 و021.60 غم بالتتابع، جدول5 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط وزن100 )بذرة(غم لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغم Cu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 115.71 104.50 124.30 105.21 120.55 01 125.05 127.07 152.03 150.00 105.04 011 102.17 105.75 150.01 152.54 105.74 001 104.50 105.01 105.40 105.74 104.35 متوسط النحاس 125.45 100.71 104.55 150.34 L.S.D 0.05 K Cu KCu 5.10 2.33 7.00 جدول5 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط وزن100 )بذرة(غم لمحصول الباقالء جدول5 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط وزن100 )بذرة(غم لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغم Cu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 115.71 104.50 124.30 105.21 120.55 01 125.05 127.07 152.03 150.00 105.04 011 102.17 105.75 150.01 152.54 105.74 001 104.50 105.01 105.40 105.74 104.35 متوسط النحاس 125.45 100.71 104.55 150.34 L.S.D 0.05 K Cu KCu 5.10 2.33 7.00 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 غير أن االختالف كان معنوياً مع بقية معامالت التداخل األخرى وبزيادة معنوية بلغت نسبتها 41.00 % ًقياسا بمعاملة المقارنة لكال العنصرين التي سجل ت أقل متوسط للصفة بلغ000.90 .غم حاصل بذور النبات(غم. نبات- 1) أن معنوية التداخل تشير إلى حصول توازن غذائي أمثل بين العنصرين فأنعكس ذلك في زيادة نشاط جميع العمليات الحيوية الجارية في النبات والتي انعكست في زيادة حاصل بذور النبات. 315 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 جدول0 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط حاصل بذور النبات )(غم لمحصول الباقالء مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 جدول0 تأثير الرش بعنصر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط حاصل بذور النبات )(غم لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 52.44 55.57 48.63 51.78 42.10 01 56.66 58.97 70.03 56.44 70.40 011 61.35 63.72 81.25 80.72 31.37 001 53.50 74.94 57.50 83.41 73.05 متوسط النحاس 44.00 70.00 75.04 75.00 L.S.D 0.05 K Cu KCu 8.00 0.70 0.73 )%( نسبة البروتين في البذور تشير نتائج الجدول01 إلى وجود زيا دة في نسبة البروتين في البذور مع زيادة تركيز الرش بالنحاس، أذ ( أعطت النباتات المرشوشة بالتركيز العالي للنحاس41 ملغمCu لتر- 0 ) أعلى متوسط لنسبة البروتين في بذورها بلغ40.64 % وبزيادة معنوية بلغت نسبتها4.14 و2.04 و02.94 % قياساً بالتركيزين41 و01 ملغمu C لتر- 0 ومعاملة المقارنة التي أعطت أقل متوسط للصفة بلغ40.44 % .بالتتابع للنحاس أهمية في عملية تكوين البروتين من خالل دوره في زيادة تثبيت النتروجين الجوي وكذلك من خالل رفع قدرة النبات على زيادة تكوين البروتين واألحماض النوويةDNA وRNA المهمة في عملية تكوين البروتين أذ لوحظ تجمع لألمونيوم وكذلك انخفاض مستويات الـ DNA في األجزاء النباتية التي تعاني من نقص النحاس كما انه ضروري في عملية ( اختزال النترات04 و41 ). حاصل بذور النبات(غم. نبات- 1) أن هذه النتيجة جاءت متماشية مع نتائج دراسات أخرى بينت دور النحاس اإليجابي في زيادة نسبة البروتين في ال بذور( 4و44و42و44 .) تشير نتائج الجدول01 إلى وجود زيا دة في نسبة البروتين في البذور مع زيادة تركيز الرش بالنحاس، أذ ( أعطت النباتات المرشوشة بالتركيز العالي للنحاس41 ملغمCu لتر- 0 ) أعلى متوسط لنسبة البروتين في بذورها بلغ40.64 % وبزيادة معنوية بلغت نسبتها4.14 و2.04 و02.94 % قياساً بالتركيزين41 و01 ملغمu C لتر- 0 ومعاملة المقارنة التي أعطت أقل متوسط للصفة بلغ40.44 % .بالتتابع للنحاس أهمية في عملية تكوين البروتين من خالل دوره في زيادة تثبيت النتروجين الجوي وكذلك من خالل رفع قدرة النبات على زيادة تكوين البروتين واألحماض النوويةDNA وRNA المهمة في عملية تكوين البروتين أذ لوحظ تجمع لألمونيوم وكذلك انخفاض مستويات الـ DNA في األجزاء النباتية التي تعاني من نقص النحاس كما انه ضروري في عملية ( اختزال النترات04 و41 ). أن هذه النتيجة جاءت متماشية مع نتائج دراسات أخرى بينت دور النحاس اإليجابي في زيادة نسبة البروتين في ال بذور ( 4 و44 و42 و44 .) يتضح من الجدول01 وجود زيادة في نسبة البروتين في البذور مع زيادة مستوى أضافة البوتاسيوم حتى أعطت النباتات المسمدة بالمستوى001 كغمK ه- 0 أعلى متوسط لنسبة البروتين في بذورها بلغ46.96 % ولم تختلف معنوياً مع نباتات المستوى011 كغمK ه- 0 و بنسبة 40.99 % غير ان االختالف كان معنوياً مع المستوى01 كغمK ه- 0 بنسبة49.06 % ومعاملة المقارنة التي أعطت أقل متوسط للصفة بلغ42.06 % . أن زيادة نسبة البروتين بإضافة البوتاسيوم قد يرجع إلى تأثير العنصر اإليجابي في تحفيز قدرة خاليا النباتات المتصاص النتروجين ال( ذي يشترك في بناء األحماض األمينية ومن ثم تكوين البروتين1 و9 و44 ) إضافة إلى ذلك فأن للبوتاسيوم تأثير معنوي في زيادة المساحة الورقية (الجدول4 ) وبالتالي زيادة منتجات عملية التمثيل الضوئي والتي تدخل في مجرى عملية التنفس ومن خالل المركبات الوسطية الناتجة عن عملية األكسدة لهذه المنتجات تتكون األحماض األمينية التي تساهم في بناء البروتين. جاءت هذه النتيجة متماشية مع نتائج بحوث 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 أخرى بينت التأثير اإليجابي ( للبوتاسيوم في زيادة نسبة البروتين في بذور الماش واللوبياء وفول الصويا5 و11 و44 و41 ). أخرى بينت التأثير اإليجابي ( للبوتاسيوم في زيادة نسبة البروتين في بذور الماش واللوبياء وفول الصويا5 و11 و44 و41 ). حاصل بذور النبات(غم. نبات- 1) جدول10 تأثير الرش بعنص ر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط نسبة البروتين في البذور( % ) لمحصول الباقالء جدول10 تأثير الرش بعنص ر النحاس والتسميد البوتاسي والتداخل بينهما في متوسط نسبة البروتين في البذور( % ) لمحصول الباقالء مستويات البوتاسيوم (كغمK هـ- 0) تراكيز النحاس (ملغمCu لتر- 0) متوسط البوتاسيوم 0 10 20 00 1 22.17 24.25 24.77 27.57 25.50 01 20.15 23.42 25.20 25.74 27.50 011 13.00 25.40 25.52 00.00 25.77 001 25.15 20.01 00.20 00.55 20.70 متوسط النحاس 24.22 23.51 25.12 25.03 L.S.D 0.05 K Cu KCu 1.10 0.50 N.S المصادر 0 -ابو ضاحي، يوسف محمد ومؤيد أحمد اليونس، 0600. دليل تغذية النبات. وزارة التعليم العالي والبحث ال علمي 0 -ابو ضاحي، يوسف محمد ومؤيد أحمد اليونس، 0600. دليل تغذية النبات. وزارة التعليم العالي والبحث ال علمي – .جامعة بغداد 4 - ،احمد رياض عبد اللطيف، 0602 . الماء في حياة النبات. مديرية دار الكتب للطباعة،والنشر جامعة ،الموصل :العراق.ع.ص004. 4 - ،الحلبوسي أسامة حسين مهيدي محمد، 4110 . تأثير التسميد النتروجيني والفوسفاتي والرش بالبوتاسيوم في .صفات محصول فول الصوياGlycine max L. رسالة ماجستير. قسم المحاصيل الحقلية- كلية الزراعة- جامعة األنبار. 2 - ،الحلبوسي أسامة حسين مهيدي، 4104 . تأثير مستويات من السماد الفوسفاتي والبوتاسي والتغذية الورقية .بالحديد والبورون في النمو والحاصل والمكونات الفعالة في الحلبة أطروحة دكتوراه. قسم المحاصيل،الحقلية كلية الزراعة- جامعة األنبار. 0 - ،الدليمي محمد علي احمد درج، 4100. استجابة نمو فول الصويا وحاصلة والصفات النوعية للسماد البوتاس والتغذية الورقية بالزنك. رسالة،ماجستير قسم المحاصيل الحقلية. كلية الزراعة- جامعة األنبار. 9 - ،الراوي خاشع محمود وع بد العزيز خلف الله، 0601. تصميم وتحليل التجارب الزراعية. مديرية دار الكتب :للطباعة والنشر. جامعة الموصل. ع.ص204. 4 - ،الزوبعي عبد الرزاق علي حمادي، 4110 . تأثير التسميد بالبوتاسيوم والرش بالنحاس في امتصاص بعض المغذيات ونمو وحاصل الذرة الصف L.) (Zea mays . رسالة .ماجستير كلية.الزراعة جامعة .بغداد 311 ISSN: 1992-7479 0 - ،الشبيني جمال محمد، 4114 . البوتاسيوم في األرض والنبات. معهد بحوث األرضي .والمياه والبيئة 6 - ،الصحاف فاضل حسين، 0606. تغذية النبات التطبيقي. مطبعة دار الحكمة للطباعة والنشر- جامعة.بغداد 01 - ،المحمدي شامل إسماعيل نعمة، 4100 . .استجابة نمو وحاصل بعض أصناف حنطة الخبزTriticum astivum L. للتغذية الورقية بالنحاس. مجلة األنبار للعلوم،الزراعية :المجلد0 (العدد2 ) عدد 00 - ،المحمدي مروة سلمان هالل ، 4104. تأثير مستويات من السماد النتروجيني والبوتاسي في نمو وحاصل تركيبين وراثيين لمحصول الماشVigna radiate L . رسالة ماجستير. كلية الزراعة. جامعة األنبار. حاصل بذور النبات(غم. نبات- 1) 04 -النعيمي ، سعد الله نجم عبد الله ، 0666 .األسمدة وخصوبة.التربة وزارة التعليم العالي والبحث العلمي– جامعة الموصل-دار .الكتب للطباعة 04 - ،حسين لينا على وكري مة محمد وهيب ، 4101 . العالقة بين نمو الجذر وحاصل العصفر بتأثير فترات الري ،ومستويات البوتاسيوم. مجلة العلوم الزراعية العراقية20 ( 4 :) 41 - 20. 02 - ،حنشل .ماجد علي4112 .. تأثير مستويات التسميد البوتاسي في حاصل ونوعية بذور البزاليا صنف اينورد مجلة األنبار للعلوم ال زراعية4 ( 4 : ) 402 - 406 . 00 - ،خيرو أوس ممدوح ، 4116 . تأثير التسميد األراضي والورقي بالبوتاسيوم في نمو وحاصل اللوبياء Vigna sinesis . مجلة ديالى للعلوم الزراعي ة 0 ( 4 :) 01 - 24. 09 -عبد ،الهادي ،سعدون جمال احمد عباس وكاظم محمد عبد الله ، 4101. تأثير رش المحلول المغ ذي .والتسميد البوتاسي في نمو وحاصل الصنف المحلي لنبات البزاليا الخضراءPisium Sativum L ،. مجلة الكوفة للعلوم الزراعية4 ( 0 :) 04 - 42. 00 - ،خيرو أوس ممدوح ، 4116 . تأثير التسميد األراضي والورقي بالبوتاسيوم في نمو وحاصل اللوبياء Vigna sinesis . مجلة ديالى للعلوم الزراعي ة 0 ( 4 :) 01 - 24. 41 - ،عيسى طالب احمد ، 0661 .. فسيولوجيا نباتات المحاصيل(مترجم). وزارة التعليم العالي والبحث العلمي .جامعة بغداد.ع.ص269. 41 - ،عيسى طالب احمد ، 0661 .. فسيولوجيا نباتات المحاصيل(مترجم). وزارة التعليم العالي والبحث العلمي .جامعة بغداد.ع.ص269. 41 - ،عيسى طالب احمد ، 0661 .. فسيولوجيا نباتات المحاصيل(مترجم). وزارة التعليم العالي والبحث العلمي .جامعة بغداد.ع.ص269. 21- A.O.A.C., 1980. 0fficial Methods of Analysis of the Association of official Analysis chemist's .PP. 211-223. 21- A.O.A.C., 1980. 0fficial Methods of Analysis of the Association of official Analysis chemist's .PP. 211-223. 22- Ali, A. ;M. A. Nadeem, A. T. M. Tahir and M. A. Hussain, 2007. Effect of different Potash levels on the growth, yield and Protein contents chickpea (cicer arietinum L) .pak.J.Bot.39 ( 2):523-527. 22- Ali, A. ;M. A. Nadeem, A. T. M. Tahir and M. A. Hussain, 2007. Effect of different Potash levels on the growth, yield and Protein contents chickpea (cicer arietinum L) .pak.J.Bot.39 ( 2):523-527. 311 مجلة األنبار للعلوم الزراعية مجلد31 العدد2 ، 2132 ISSN: 1992-7479 23- Anderson, L. L. and D. G. Bullock, 1998. Variable rate fertilizer application for corn and soybean. J. of plant-nutrition (USA) .V. 21(7) P. 1355- 1361. 24- Arnon, D. I., 1950. Functional aspects of copper in plants .P: 89- 112 . حاصل بذور النبات(غم. نبات- 1) In copper metabolism .The Jone Hopkins press, Baltimore. 25- Black, C. A., 1965. Methods of Soil analysis .Amer. Soc. Of Agron .Inc .USA. 26- Boardman, N. K., 1975. Trace elements in Photosynthesis .P: 199-212. In: Trace elements in Soil – Plant – Animal System .Nicholas, E. D,. Egan, D. J. D. and Egan, A. R. Academic press . 27- Henryka, S., 1997. Physiological aspects of nitrogen fixation response to copper nutrition in several grain legume species .Developments in plant and Soil Science. Vol, 71 : 233-237. 28- Hussain, F., A. U. Malik, M. A. Huji and A. L. Malghani, 2011. Growth and yield response of two Cultivars of Mung been (Vigna radiate L) to different potassium levels .The J. of Animal and plant science. 21 (3): 622-625. 29- LPl, international potash institute, 2000. Potassium increases Salinity tolerance file: A: / lPl Serves the word. 30- Jaspinder , S. K. and H. S. Grewal, 2005. Effect of split application of potassium on growth, yield and potassium accumulation by soybean .Agroeco system J. 39(30:)217-222. 31- Johnson, H., 1967. Alkaloids. Encyclopedia of food sources. 3.450-455. 32.Kalaikandhan,P.,P.V.Jayareng,R.sivasankarandS.Mathivanan .2014. 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https://openalex.org/W2565855371	https://www.nature.com/articles/srep39150.pdf	English	null	Declining Prevalence of Disease Vectors Under Climate Change	Scientific reports	2,016	cc-by	7,070	Declining Prevalence of Disease Vectors Under Climate Change Luis E. Escobar1,2,3, Daniel Romero-Alvarez4, Renato Leon5, Manuel A. Lepe-Lopez3, Meggan E. Craft2, Mercy J. Borbor-Cordova6 & Jens-Christian Svenning7 received: 26 May 2016 accepted: 18 November 2016 Published: 16 December 2016 received: 26 May 2016 accepted: 18 November 2016 Published: 16 December 2016 More than half of the world population is at risk of vector-borne diseases including dengue fever, chikungunya, zika, yellow fever, leishmaniasis, chagas disease, and malaria, with highest incidences in tropical regions. In Ecuador, vector-borne diseases are present from coastal and Amazonian regions to the Andes Mountains; however, a detailed characterization of the distribution of their vectors has never been carried out. We estimate the distribution of 14 vectors of the above vector-borne diseases under present-day and future climates. Our results consistently suggest that climate warming is likely threatening some vector species with extinction, locally or completely. These results suggest that climate change could reduce the burden of specific vector species. Other vector species are likely to shift and constrain their geographic range to the highlands in Ecuador potentially affecting novel areas and populations. These forecasts show the need for development of early prevention strategies for vector species currently absent in areas projected as suitable under future climate conditions. Informed interventions could reduce the risk of human exposure to vector species with distributional shifts, in response to current and future climate changes. Based on the mixed effects of future climate on human exposure to disease vectors, we argue that research on vector-borne diseases should be cross-scale and include climatic, demographic, and landscape factors, as well as forces facilitating disease transmission at fine scales. More than half of the world’s population is at risk of vector-borne diseases1, causing public health concerns due to elevated mortality and high levels of disability-adjusted life-years (DALYs)2. Vector-borne diseases are currently a major problem in tropical developing countries1. Climate variability influences vector population dynamics, distribution, and vector-borne disease transmission3. Dengue transmission is associated in space and time with local climate effects on survival of its vector Aedes aegypti—more rain and higher temperature generates more transmission4,5. Changes in inter-annual climate variability such as the El Niño Southern Oscillation have been shown to be important drivers for malaria transmission6. Further, vectors also show distributional shifts from low to high altitudes during warm years7. g g y It has been estimated that by 2100 the average global temperature will have risen between 1.0 and 3.5 °C, radically increasing the burden of vector-borne diseases8. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results V Vectors species distribution forecasts. Our ecological niche models were able to accurately characterize environmental conditions for each vector (Supplementary Fig. S1). The results show that present-day predictions based on remote sensing data reveal areas of high risk for exposure to arboviruses vectors across the coastal region of western Ecuador and in the Amazonian lowlands in the northeast. Additional areas of exposure risk to vec- tors were found across valleys of the western region of the Andes mountains (Fig. 2), matching dengue endemic areas of Ecuador and reports of the recent chikungunya epidemic14. The highlands exhibited reduced suitability to arbovirus vectors under present-day environmental conditions (Fig. 2). Climate-based predictions suggest an increased risk in southern areas in the short term (i.e., 2030). Mid-term predictions suggest that by 2050 high risk will be distributed in the coast, southern lowlands and in northeastern areas neighboring Colombia and Peru, with reduced suitability in central areas across the Andes. Models for 2100 for the arbovirus vectors Ae. aegypti and Ae. albopictus, showed an evident geographic shift in the suitable areas, with increased risk for exposure in the Andes valleys. Ae. aegypti should experience a reduction in its potential area of distribution by 69%, 43%, and 48% and population at risk by 84%, 47%, 40% by 2030, 2050, and 2100, respectively. For Ae. albopictus, the poten- tial area of distribution should be reduced by 45%, 35%, and 53% and the number of people potentially exposed by 58%, 46%, and 52% in 2030, 2050, and 2100, respectively (Figs 3 and 4). y p y g Under present-day climate conditions, malaria vectors showed two foci of risk for vectors exposure in west central and northeastern Ecuador. However, under future climates these vectors may find wide suitable areas in western Ecuador across the coast with an impressive shift to the highlands by 2100, resulting in a rise in exposure risk across the Andes Mountains (Fig. 2). The potential area for distribution for An. albimanus is forecasted to be reduced by 43%, 46%, and 55% and the estimated people exposed to be reduced by 93%, 92%, and 58% in 2030, 2050, and 2100, respectively (Figs 3 and 4). p y g Leishmaniasis vectors showed a broad distribution across all the biomes of Ecuador, with higher risk in the lowlands. Models predicted a rise in leishmaniasis exposure risk in the Andes Mountains by 2030 and 2050 (Fig. 2). Declining Prevalence of Disease Vectors Under Climate Change Luis E. Escobar1,2,3, Daniel Romero-Alvarez4, Renato Leon5, Manuel A. Lepe-Lopez3, Meggan E. Craft2, Mercy J. Borbor-Cordova6 & Jens-Christian Svenning7 Not reported officially in Ecuador and no active surveillance for its monitoring, but present in neighboring countries. Table 1. List of vector species by disease included in this study. Not reported officially in Ecuador and not active surveillance for its monitoring, but present in neighboring countries. Ecuador has a considerable burden of vector-borne diseases across a variety of climatic conditions, human population density, and biodiversity across the country (Supplementary Material). Here, we used ecological niche modeling to determine the present-day and future potential distribution of multiple important disease vector species in Ecuador (Table 1). Niche models were estimated using a coarse-grained macroecological approach focused on the broader-scale climate relations of the vectors (e.g., maximum temperature, annual precipitation)13. We searched for environmental overlap between niche models of vectors and environments present in Ecuador (Fig. 1 Risk A and B). Niche models were then transferred to future climates to identify future suitable areas for vector presence (Fig. 1 Risk A and C). The geographic areas suitable for the vectors in Ecuador are considered here to be of potential risk for human exposure to the vectors (Fig. 1). Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 Declining Prevalence of Disease Vectors Under Climate Change Luis E. Escobar1,2,3, Daniel Romero-Alvarez4, Renato Leon5, Manuel A. Lepe-Lopez3, Meggan E. Craft2, Mercy J. Borbor-Cordova6 & Jens-Christian Svenning7 As an example, simulations of sandfly vector ecology suggest that leishmaniasis will also shift its potential distribution in response to climate change9. Thus, analyzing vector distribution patterns is crucial for providing insights to estimate their present-day distribution in detail and to anticipate their distribution in face of future climate scenarios. Vectors species’ potential distribution can be explored via ecological niche modeling10,11. Ecological niche models aim to characterize the environmental conditions required by a species to maintain populations in the long term without need of immigration. Defining these conditions allows us to identify the potential distribu- tion of the species12. Models calibrated using available present-day data can also be projected into future climate scenarios12. 1Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, Minnesota, 55108, USA. 2Department of Veterinary Population Medicine, University of Minnesota, St Paul, Minnesota, 55108, USA. 3Facultad de Medicina Veterinaria y Zootecnia, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala. 4Unit of Molecular Parasitology & Tropical Medicine, Centro de Biomedicina, School of Medicine, Universidad Central del Ecuador, Quito, Ecuador. 5Laboratorio de Entomología Médica & Medicina Tropical, Universidad San Francisco de Quito, Quito, Ecuador. 6Faculty of Marine Sciences, Biology, Oceanic Sciences and Natural Resources, Escuela Superior Politécnica del Litoral, Guayaquil, Ecuador. 7Section for Ecoinformatics & Biodiversity, Department of Bioscience, Aarhus University, DK-8000 Aarhus C, Denmark. Correspondence and requests for materials should be addressed to L.E.E. (email: lescobar@umn.edu) Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 1 www.nature.com/scientificreports/ Disease Pathogen Vector Agent Genus Common name Order Vector species Chagas disease Protozoa Trypanosoma Kissing bug Hemiptera Rhodnius ecuadoriensis Triatoma carrioni T. dimidiata T. dispar Chikungunya Virus Alphavirus Yellow fever mosquito Diptera Aedes aegypti Asian tiger mosquito Ae. albopictus* Dengue Fever Virus Flavivirus Yellow fever mosquito Diptera Aedes aegypti Asian tiger mosquito Ae. albopictus Malaria Protozoa Plasmodium Anopheles mosquito Diptera Anopheles albimanus American malaria mosquito An. darlingi Mosquito An. neivai Mosquito An. pseudopunctipennis Mosquito An. punctimacula Leishmaniasis Protozoa Leishmania Phlebotomine sandfly Diptera Lutzomyia gomezi Lu. hartmanni Lu. trapidoi Yellow Fever Virus Flavivirus Yellow fever mosquito Diptera Ae. aegypti Asian tiger mosquito Ae. albopictus Zika Virus Flavivirus Yellow fever mosquito Diptera Ae. aegypti Table 1. List of vector species by disease included in this study. *Not reported officially in Ecuador and not active surveillance for its monitoring, but present in neighboring countries. Table 1. List of vector species by disease included in this study. Results V Ecological modeling approach used to assess disease vector species’ potential distributions. The ecological niche of each vector species was estimated (red ellipsoid) based on present-day environmental conditions (E). The environments available in Ecuador were determined under present-day (yellow square) and future (orange square) climate conditions. Areas with climatic conditions in Ecuador overlapping between the niche of vectors and both present-day and future climate conditions were identified as Risk A (risk under present-day and future scenario; red). Areas of Ecuador with climate under present-day overlapping with the niche of vectors were defined as Risk B (risk under present-day only; pink). Areas with overlap between the niche of vectors and future climate in Ecuador only were defined as Risk C (risk under future climate only; brown). (Figure created using Adobe Photoshop CC 2014 https://www.adobe.com). and Supplementary Fig. S1) in face of temperatures rising above those experienced across species current range (Supplementary Fig. S2). Triatoma dimidiata is forecasted to increase its potential distribution by 189% in 2030, 172% in 2050, and ~17% in 2100 (Fig. 3). Vector exposure under climate change. The general pattern revealed that human populations in the Andean highlands would be increasingly exposed to disease vectors as the future climate changes unfold due to likely upward vector species range shifts (Fig. 2 and Supplementary Fig. S3). While models in the Northern Hemisphere show a displacement of species from south to north and models in the Southern Hemisphere show displacements from north to south9,15, we found that the equatorial latitudes of Ecuador show just a slight north- ward range shift, with strong upward altitudinal displacements into the Andes Mountains. Importantly, across all 14 vector species we found consistent non-artefactual reductions in potential range area and people potentially exposed for nine vector species by 2030, 2050, or 2100, with the malaria vectors Anopheles darlingi and An. neivai as the main exceptions (Figs 3 and 4; and Supplementary Fig. S1). The synchrony between reduction of areas and populations at risk among the numerous vectors explored, revealed a pattern of potential adverse ecological effects of climate change on vector distributions (Fig. 3). Indeed, three species showed reduction of both area and people potentially exposed under all three future climate time steps (Ae. aegypti, Ae. albopictus, An. albimanus; Figs 3 and 4; and Supplementary Fig. S1). Lack of future suitable environments was found for two vector species, suggesting potential for extinction (i.e., T. Results V dispar, and An. pseudopunctipennis, Supplementary Fig. S2). Results V Surprisingly, by 2100, we found that future climate will provide suitability for leishmaniasis vectors across the Andes, but we were unable to assess future suitability in lowland ecosystems given the non-analogue high temperatures anticipated in such areas (Supplementary Fig. S2). p p ( pp y g ) Vectors of chagas disease commonly occupied highland biomes (e.g., T. carrioni and T. dispar); thus, under future climate models we forecast extirpation of endemic vector species across Ecuador (e.g., T. dispar; Fig. 3 Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 2 www.nature.com/scientificreports/ tificreports/ Figure 1. Ecological modeling approach used to assess disease vector species’ potential distributions. The ecological niche of each vector species was estimated (red ellipsoid) based on present-day environmental conditions (E). The environments available in Ecuador were determined under present-day (yellow square) and future (orange square) climate conditions. Areas with climatic conditions in Ecuador overlapping between the niche of vectors and both present-day and future climate conditions were identified as Risk A (risk under present-day and future scenario; red). Areas of Ecuador with climate under present-day overlapping with the niche of vectors were defined as Risk B (risk under present-day only; pink). Areas with overlap between the niche of vectors and future climate in Ecuador only were defined as Risk C (risk under future climate only; brown). (Figure created using Adobe Photoshop CC 2014 https://www.adobe.com). Figure 1. Ecological modeling approach used to assess disease vector species’ potential distributions. Th l i l i h f h i i d ( d lli id) b d d i Figure 1. Ecological modeling approach used to assess disease vector species’ potential distributions. The ecological niche of each vector species was estimated (red ellipsoid) based on present-day environmental conditions (E). The environments available in Ecuador were determined under present-day (yellow square) and future (orange square) climate conditions. Areas with climatic conditions in Ecuador overlapping between the niche of vectors and both present-day and future climate conditions were identified as Risk A (risk under present-day and future scenario; red). Areas of Ecuador with climate under present-day overlapping with the niche of vectors were defined as Risk B (risk under present-day only; pink). Areas with overlap between the niche of vectors and future climate in Ecuador only were defined as Risk C (risk under future climate only; brown). (Figure created using Adobe Photoshop CC 2014 https://www.adobe.com). Figure 1. Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 Discussion Controlling calibration area, novel climates, model complexity, and model transference to ensure robust predic- tions, we developed forecasts of geographic shifts in the potential range under future climates for 14 important disease vectors in Ecuador, finding consistent predictions of reductions in the vector’s potential ranges and the number of people exposed for most of the species studied. Our modeling experiments were developed based on a detailed design supported by ecological theories while outputs were evaluated carefully12. The overall patterns suggest that vectors of arboviruses and leishmaniasis will experience geographic range reductions by 2100 under future climate conditions, while chagas vectors had mixed results with some species increasing (T. dimidata) and others reducing (T. carrioni, T. dispar, R. ecuadoriensis) their geographic distribution (Fig. 3). A similar situation was observed for malaria vectors where one species (i.e., An. darlingi) was predicted to increase in the geographic range while the other species were forecasted to experience range reductions. Therefore, An. darlingi and T. dimidata are of particular public health concern as such vectors could expand their range under future climate conditions, which could impact the epidemiology of malaria and chagas disease in Ecuador respectively. Climate change was even forecasted to possibly extirpate important vectors such as Triatoma dispar, a vector of chagas disease. A reliable extinction would require ecological niche conservatism with no adaptation of vectors to warming climates; adaptation of species to warming climate is, however, restrained by high temperatures16,17. In Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 3 www.nature.com/scientificreports/ ture.com/scientificreports/ Figure 2. Maps of potential transmission risk in Ecuador. Vector species were grouped by disease. (a) arboviruses (chikungunya, dengue, yellow fever, and zika); (b) malaria; (c) leishmaniasis; and (d) chagas disease. Hotspots of vector prediction suggested areas of high (red) or low (yellow) disease transmission risk. (Figure done using the raster and rasterVis packages in R version 3.3.1: A Language and Environment for Statistical Computing, R Core Team, R Foundation for Statistical Computing, Vienna, Austria (2016) https:// www.r-project.org). Figure 2. Maps of potential transmission risk in Ecuador. Vector species were grouped by disease. (a) arboviruses (chikungunya, dengue, yellow fever, and zika); (b) malaria; (c) leishmaniasis; and (d) chagas disease. Hotspots of vector prediction suggested areas of high (red) or low (yellow) disease transmission risk. Discussion Studies showing the attenuated negative impacts on the epidemiology of some vector species may help to reduce focus on the effects of climate change on the burden of vector-borne diseases of likely reduced future importance, and may encourage scientists to instead focus on the effects of other equally or more important factors for future human health, such as land use change, to help in the design and implementation of effective public health policies. We argue that a mature understanding of climate change effects on health could be achieved if and only if the research communities moves to a broader interpreta- tion of climate effects, covering plausible negative and positive effects of such changes across ecosystems and taxa. other words, there is a physiological upper limit that cannot be exceeded easily by species, providing some confi- dence to our estimation. Our results show a benefit of climate change in terms of plausible extinction and range reduction for some vectors and consequent decrease in exposure risk for people, contrasting with the proposed negative effects of climate change on human health18. Our findings support other mathematical modeling and laboratory experiments proposing potential benefits of climate change in terms of limiting the burden of malaria and dengue vectors19–22, mitigating potential overinterpretation of other studies suffering incorrect study designs and thus proposing dramatic impacts of future climate on the epidemiology of infectious diseases which could fail to inform decision makers with realistic scenarios. Studies showing the attenuated negative impacts on the epidemiology of some vector species may help to reduce focus on the effects of climate change on the burden of vector-borne diseases of likely reduced future importance, and may encourage scientists to instead focus on the effects of other equally or more important factors for future human health, such as land use change, to help in the design and implementation of effective public health policies. We argue that a mature understanding of climate change effects on health could be achieved if and only if the research communities moves to a broader interpreta- tion of climate effects, covering plausible negative and positive effects of such changes across ecosystems and taxa. Discussion (Figure done using the raster and rasterVis packages in R version 3.3.1: A Language and Environment for Statistical Computing, R Core Team, R Foundation for Statistical Computing, Vienna, Austria (2016) https:// www.r-project.org). Figure 2. Maps of potential transmission risk in Ecuador. Vector species were grouped by disease. (a) arboviruses (chikungunya, dengue, yellow fever, and zika); (b) malaria; (c) leishmaniasis; and (d) chagas disease. Hotspots of vector prediction suggested areas of high (red) or low (yellow) disease transmission risk. (Figure done using the raster and rasterVis packages in R version 3.3.1: A Language and Environment for Statistical Computing, R Core Team, R Foundation for Statistical Computing, Vienna, Austria (2016) https:// www.r-project.org). Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 4 www.nature.com/scientificreports/ Figure 3. Potential vectors’ distribution under current and future climate scenarios. Number of pixel cells (1 km; y axis) predicted suitable by period (x axis) for vector species of arboviruses (a), malaria (b), leishmaniasis (c), and chagas disease (d) were estimated under current and future climate conditions by 2030, 2050, and 2100. Figure 3. Potential vectors’ distribution under current and future climate scenarios. Number of pixel cells (1 km; y axis) predicted suitable by period (x axis) for vector species of arboviruses (a), malaria (b), leishmaniasis (c), and chagas disease (d) were estimated under current and future climate conditions by 2030, 2050, and 2100. other words, there is a physiological upper limit that cannot be exceeded easily by species, providing some confi- dence to our estimation. Our results show a benefit of climate change in terms of plausible extinction and range reduction for some vectors and consequent decrease in exposure risk for people, contrasting with the proposed negative effects of climate change on human health18. Our findings support other mathematical modeling and laboratory experiments proposing potential benefits of climate change in terms of limiting the burden of malaria and dengue vectors19–22, mitigating potential overinterpretation of other studies suffering incorrect study designs and thus proposing dramatic impacts of future climate on the epidemiology of infectious diseases which could fail to inform decision makers with realistic scenarios. Discussion f , g p g pf g y We have assumed static patterns of human density in Ecuador in face of climate change, in other words, given that we based our estimation on values of human density, we assume that the present-day patterns of low and high will remain in the future. However, human displacement as a result of climate change is a complex factor that deserves further exploration. Further, topographic steepness limits the potential for upslope human migrations in the region, with most non-steep upland areas already densely occupied. While there is consensus that vectors are sensitive to weather and climate23, there is still uncertainty on the various impacts of climate variability on fine-scale transmission dynamics and socioeconomic variables23,24. We mitigated uncertainty in our forecasts by tuning model parameters for biologically realistic predictions by capturing the environmental tolerances of vec- tors from their entire geographic range and assessing model fit with available data. Our present-day predictions highlight areas of vector suitability at high spatial resolution for all the species included in this study. These areas suitable for vectors occurrence should be considered into account by public health authorities given the plausible disease underreporting in Ecuador (i.e., epidemiological silence14). The areas predicted to be of present-day risk may be of special interest for testing control measures and for identifying currently neglected human populations. The areas found suitable via remote sensing data could be also useful to guide local studies aiming to understand the distributional ecology of vectors, considering their interaction with other vector species, prey, variations in micro- climate, availability of breeding sites, and the effects of socio-economic factors facilitating vectors’ occurrence25. Such local-scale factors were not considered in this study, but would provide valuable information to bet- ter understand fine-scale patterns in the ecology of vectors across different geographic and temporal scales26. Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 5 www.nature.com/scientificreports/ Figure 4. Percent change of vector potential distribution and potential for human exposure to vectors under present-day and future climate. Present-day models were compared against future climate by 2030 (a), 2050 (b), and 2100 (c) in terms of area (x axis) and human population at risk of exposure (y axis). Top right quadrant (pink) denotes an increase in vectors’ range and human population at risk. Bottom-right (green) resembles an increase in range, but reduction in the number of humans exposed. Discussion Bottom-left (blue) denotes reduction in vectors’ range and reduction in population predicted at risk. Top-left (yellow) denotes increase in human populations at risk, but reduction in the vector’s range. Zero means no change. (Plots done using the raster package in R version 3.3.1: A Language and Environment for Statistical Computing, R Core Team, R Foundation for Statistical Computing, Vienna, Austria (2016) https://www.r-project.org). Figure 4. Percent change of vector potential distribution and potential for human exposure to vectors under present-day and future climate. Present-day models were compared against future climate by 2030 (a), 2050 (b), and 2100 (c) in terms of area (x axis) and human population at risk of exposure (y axis). Top right quadrant (pink) denotes an increase in vectors’ range and human population at risk. Bottom-right (green) resembles an increase in range, but reduction in the number of humans exposed. Bottom-left (blue) denotes reduction in vectors’ range and reduction in population predicted at risk. Top-left (yellow) denotes increase in human populations at risk, but reduction in the vector’s range. Zero means no change. (Plots done using the raster package in R version 3.3.1: A Language and Environment for Statistical Computing, R Core Team, R Foundation for Statistical Computing, Vienna, Austria (2016) https://www.r-project.org). An inter-sectoral approach should be implemented at the municipality level to address issues of sanitation, safe water availability, and preventive health care in areas predicted of present-day risk (Fig. 2). y y g Our models were based on a broad-scale biogeographic approach27, therefore we did not consider microcli- matic conditions at local scale and avoided the complexities and uncertainties of including factors such as biolog- ical interactions in the modeling12. We accounted for the environmental tolerances of vector species from vector records across the entire global distribution of each species. This framework should reduce the risk of incomplete niche estimation (niche truncation) due to failure to represent the full niche space for a species in the model calibration28, i.e., by including records from as wide areas in geographic and environmental space as possible (e.g., the entire species range). Thus, for species of broad distributions (e.g., global), making models with data from the areas of interest only (e.g., Ecuador) could capture a limited portion of the species’ ecological niche generating an incomplete characterization of the species’ environmental tolerances. Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 Methods We forecasted the distribution of disease vectors under present-day conditions and future climate in Ecuador using vector records across their entire global ranges from data of museum collections and literature. After vector occurrence data curation, we developed ecological niche models for each vector species in Maxent33,34, assuming that each species record originated from a stable vector population instead of migratory or accidentally trans- located individuals. We assessed regularization coefficients and model complexity in Maxent34,35. Our model evaluation was based on information theory through assessing Akaike information criterion (AIC) values to explore model fit with the data available35, which provides robustness to our model assessment procedure, a key factor when modeling infectious diseases10. Thus, AIC is a well-known method to discriminate among mod- els, especially when working with the Maxent algorithm employed in our study35. Additionally, using Maxent we estimated the Area Under the Curve of the Receiver Operating Characteristic values as this metric is com- monly employed to assess the specificity and sensitivity of models. The final models, calibrated on each species’ entire range, were projected to Ecuador under present-day and future climatic conditions using strict model transference36,37. Potential distribution of vectors were forecasted under present-day conditions using remote sensing imagery including EVI time series data and land surface temperature38, and precipitation values from field stations39, while future climate models where calibrated in present-day climate and then projected to future climate conditions, based on data of the CliMond repository39. We used the SRES A2 scenario (Special Report on Emissions Scenarios) of future climatic conditions given that Latin America has increasing populations, eco- nomic development, per capita economic growth, but also relatively fragmented and slow technological change and limited abilities to mitigate greenhouse effects40, and considering that the last IPPC assessment concluded that there are not important improvements to reduce emissions worldwide41. Indeed, since the first future climate scenarios, the more optimistic low-emission scenarios have become implausible given the ongoing empirical trends in emissions29,42,43. The A2 scenario is equivalent to the Representative Concentration Pathway (RCP) 8.5 proposed in the IPCC Fifth Assessment Report (AR5)41. Additionally, we developed non-analogous environment evaluations using the Mobility Oriented Parity test (MOP) script in R37 to ensure that future predictions were restricted to regions in Ecuador with analogous climates somewhere in range of each species, to avoid extrap- olation uncertainties. Discussion Models calibrated using a subset of the species’ range will generate sub-estimations of the species’ ecological niche and in turn sub-estimations of its potential distribution.h p The future climate forecasts used in this study (A2; 2030, 2050, 2100) are realistic scenarios of the environmen- tal conditions that may be present in Ecuador given the ongoing trends in greenhouse gas emissions29. Predictions in future climate scenarios showed a likely expansion of exposure risk of all pathogens to populations in the highlands of Ecuadorian Andean regions. The geomorphology and latitudinal position of Ecuador may suggest similar predictions for other tropical regions. Although potential range shifts and extinction have been reported for the future distribution of several plant and animal species30, we complement this information with the poten- tially negative effects of climate change on range contraction, displacement, and extirpation of vectors species, and an overview of changes in patterns of human populations at risk for Ecuador. These estimates were based on the assumption of niche conservatism of vector species31. By supporting this assumption, our findings provide information to anticipate strategies to reduce the burden of vector-borne diseases via increasing the awareness in areas currently disease-free, but predicted vulnerable to vectors displacement. An early warning scheme in Andean region should be formulated with inclusion of non-endemic vector species given risk predicted in high- lands and the limited understanding of the colonization capacity of vectors in face of climate change. Preventive measures can contribute globally to the early detection and prevention of vector-borne diseases in highlands worldwide in face of global warming. Despite the local benefits of reduced transmission due to the extinction of Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150 6 www.nature.com/scientificreports/ specific vector species, range shifts of other vectors could increase vector-borne diseases incidence and spread, resulting in epidemiological surprises32. Based on our mixed results of positive and negative effects of future climate on human exposure to disease vectors, we argue that research on vector-borne diseases in face of future environmental change should be cross-scale and include climatic, demographic, and landscape factors, as well as forces facilitating disease transmission acting at fine scales. Importantly, care should be take that vector-borne disease research on climate change effects will not discourage health agencies from research into understanding local transmission processes and implementing control measures for critical threats of disease transmission at the local level including human behavior and habitat destruction. Methods The final maps were used to identify the percentage of area predicted as suitable in each climate scenario. We used the LandScan human population estimates for year 2011 at 1-km spatial resolution44 to estimate the people living within the suitable areas for a given vector species. Estimation based on current cli- mates was compared with models of future climate conditions to establish the percent of change among climate scenarios. A detailed description of the modeling framework is available in the Supplementary Material. References 1. World Health Organization. A global brief on vector-borne diseases. 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Declining Prevalence of Disease Vectors Under Climate Change. ci. Rep. 6, 39150; doi: 10.1038/srep39150 (2016). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 8 Scientific Reports \| 6:39150 \| DOI: 10.1038/srep39150
https://openalex.org/W2163031497	https://repository.ubn.ru.nl/bitstream/handle/2066/139587/1/139587.pdf	English	null	Search for the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msub></mml:math> and other hidden-beauty states in the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si2.gif" overflow="scroll"><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>+</mml:mo></mml:mrow></mml:msup><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><…	Physics letters. B	2,015	cc-by	25,243	Search for the Xb and other hidden-beauty states in the +(1S)channel at ATLAS Aad, G.; Besjes, G.J.; Caron, S.; Croft, V.; Groot, N. de; Filthaut, F.; Galea, C.; Klok, P.F.; König, A.C; Salvucci, A.; Strubig, A.; Et, a. 2015, Article / Letter to editor (Physics Letters B, 740, January, (2015), pp. 199-217) Doi link to publisher: https://doi.org/10.1016/j.physletb.2014.11.055 Search for the Xb and other hidden-beauty states in the +(1S)channel at ATLAS Aad, G.; Besjes, G.J.; Caron, S.; Croft, V.; Groot, N. de; Filthaut, F.; Galea, C.; Klok, P Salvucci, A.; Strubig, A.; Et, a. Version of the following full text: Publisher’s version Downloaded from: http://hdl.handle.net/2066/139587 Download date: 2024-10-24 Note: To cite this publication please use the final published version (if applicable). Search for the X[subscript b] and other hidden-beauty states in the [superscript +] [superscript -] (1 S) channel at ATLAS ation G. Aad, et al. “Search for the X[subscript b] and Other Hidden- beauty States in the [superscript +] [superscript -] (1 S) Channel at ATLAS.” Physics Letters B (December 2014). Published http://dx.doi.org/10.1016/j.physletb.2014.11.055 blisher Elsevier rsion Final published version cessed Fri Feb 06 05:21:57 EST 2015 able Link http://hdl.handle.net/1721.1/92581 rms of Use Creative Commons Attribution tailed Terms http://creativecommons.org/licenses/by/4.0/ The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Search for the X[subscript b] and other hidden-beauty states in the [superscript +] [superscript -] (1 S) channel at ATLAS Search for the X[subscript b] and other hidden-beauty states in the [superscript +] [superscript -] (1 S) channel at ATLAS The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation G. Aad, et al. “Search for the X[subscript b] and Other Hidden- beauty States in the [superscript +] [superscript -] (1 S) Channel at ATLAS.” Physics Letters B (December 2014). As Published http://dx.doi.org/10.1016/j.physletb.2014.11.055 Publisher Elsevier Version Final published version Accessed Fri Feb 06 05:21:57 EST 2015 Citable Link http://hdl.handle.net/1721.1/92581 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/4.0/ 1. Introduction pends on the branching fraction for Xb →π+π−Υ (1S), which is unknown. The π+π−Υ (1S) channel also provides the opportunity to measure the production of the Υ (13D J) states. These have not been observed at the Tevatron; their production cross sections in pp collisions are also unknown, but an early colour-octet cal- culation [17] gives values comparable to that of the Υ (2S). The Υ (13D2) has been observed in radiative transitions by CLEO [18] and BaBar [19]. The X(3872) is the ﬁrst and the best-studied of the new hidden-charm states seen in the last decade. Observed by Belle in decays B± →K ± X(→π+π−J/ψ) [1], it was quickly conﬁrmed by BaBar [2], CDF [3], and DØ [4]. In particular, CDF and DØ found that the X(3872) is produced directly in pp collisions; recently CMS has measured the product of the pp production cross section and the π+π−J/ψ branching fraction to be (6.56 ± 0.29 ± 0.65)% of the value for the ψ(2S) [5]. The mass, narrow width, J PC = 1++ quantum number assignment [6–10], and decay characteristics of the X(3872) make it unlikely to be a conventional quarkonium state, and there is an extensive literature discussing its structure. Weakly bound D0D∗0 molecular models (for example, Refs. [11, 12]) have been popular due to the proximity of the X(3872) to the D0D∗0 threshold; various [qc][¯q¯c] tetraquark (for example, Refs. [13,14]) and other models have also been proposed. The production of Υ (10 860) and some other hidden-beauty states may also be studied using π+π−Υ (1S). The Υ (10 860) de- cay to π+π−Υ (1S) has a surprisingly large partial width [20–22]: current world average results are (0.29 ± 0.15) MeV for the Υ (10 860), to be compared with (0.89 ± 0.08) keV for the Υ (3S), and (1.7 ± 0.2) keV for the Υ (4S) [23]. Belle has also presented evidence of exotic substructure in this decay [24]. The natural widths of the Υ (10 860), Υ (11 020), and other states above the open-beauty threshold are larger than the detector resolution, and must be explicitly considered in any search. Heavy-quark symmetry suggests the existence of a hidden- beauty partner – a so-called Xb state – which should be produced in pp collisions [15]. The molecular model of Swanson [12,16] predicts an Xb mass of 10561 MeV, while tetraquark predictions vary: for example, Ref. a b s t r a c t Article history: Received 16 October 2014 Received in revised form 26 November 2014 Accepted 26 November 2014 Available online 2 December 2014 Editor: W.-D. Schlatter This Letter presents a search for a hidden-beauty counterpart of the X(3872) in the mass ranges of 10.05–10.31 GeV and 10.40–11.00 GeV, in the channel Xb →π+π−Υ (1S)(→μ+μ−), using 16.2 fb−1 of √ s = 8 TeV pp collision data collected by the ATLAS detector at the LHC. No evidence for new narrow states is found, and upper limits are set on the product of the Xb cross section and branching fraction, relative to those of the Υ (2S), at the 95% conﬁdence level using the CLS approach. These limits range from 0.8% to 4.0%, depending on mass. For masses above 10.1 GeV, the expected upper limits from this analysis are the most restrictive to date. Searches for production of the Υ (13D J), Υ (10 860), and Υ (11 020) states also reveal no signiﬁcant signals. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. Search for the Xb and other hidden-beauty states in the π+π−Υ (1S) channel at ATLAS .ATLAS Collaboration ⋆ ⋆E-mail address: atlas.publications@cern.ch. . .055 shed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by Physics Letters B 740 (2015) 199–217 ⋆E-mail address: atlas.publications@cern.ch. peaks are used to validate the measurement technique. Results are http://dx.doi.org/10.1016/j.physletb.2014.11.055 0370-2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by SCOAP3. dx.doi.org/10.1016/j.physletb.2014.11.055 2693/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Funded by 3 1 ATLAS uses a right-handed coordinate system with its origin at the nominal in- teraction point (IP) in the centre of the detector, the x-axis pointing to the centre of the LHC ring, and the z-axis along the beam pipe; the y-axis points upward. Cylindrical coordinates (r, φ) are used in the transverse plane; φ is the azimuthal angle around the beam pipe. Pseudorapidity and transverse momentum are de- ﬁned in terms of the polar angle θ as η = − ln(tanθ/2) and pT = p sinθ. The (η, φ) distance between two particles is deﬁned as R = ( η)2 + ( φ)2. For a particle with momentum ⃗p = (px, p y, pz) and energy E, the rapidity is deﬁned as y = 0.5 ln([E + pz]/[E −pz]). 2. The ATLAS detector In the remainder of the event, dipion candidates are formed from oppositely charged pions with \|ηπ\| < 2.5, each required to have ≥1 pixel hits, ≥6 silicon microstrip hits, and pπ T > 400 MeV; no other requirements (such as lepton vetoes) are imposed. The Υ (1S) candidate and the dipion system are combined by perform- ing a four-track common-vertex ﬁt, with the μ+μ−mass con- strained to m1S, and tracks assigned μ or π masses as appropri- ate. This signiﬁcantly improves the mass resolution: for example, in the Υ (2S) simulation, the RMS improves from 142 MeV to 9.7 MeV. The π+π−Υ (1S) vertex ﬁt is required to have a chi- square less than 20; this is 95% eﬃcient for Υ (2S) decays but reduces background by a factor of ∼10. All remaining π+π−Υ (1S) candidates with invariant masses up to 11.2 GeV are retained. The ATLAS detector [26] is composed of an inner tracking sys- tem, calorimeters, and a muon spectrometer. The inner detector (ID) surrounds the pp interaction point and consists of silicon pixel and microstrip detectors, and a transition radiation tracker, all im- mersed in a 2 T axial magnetic ﬁeld. The ID spans the pseudora- pidity1 range \|η\| < 2.5 and is enclosed by a system of electromag- netic and hadronic calorimeters. Surrounding the calorimeters is the muon spectrometer (MS) consisting of three large air-core su- perconducting magnets (each with eight coils) providing a toroidal ﬁeld, a system of precision tracking chambers, and fast detectors for triggering. Monitored drift tubes and cathode-strip chambers provide precision measurements in the bending plane of muons within the pseudorapidity range \|η\| < 2.7. Resistive plate and thin gap chambers are used to make fast event data-recording decisions in the ranges \|η\| < 1.05 and 1.05 < \|η\| < 2.4 respectively, and also provide position measurements in the non-bending plane and im- prove pattern recognition and track reconstruction. MS momentum measurements are based on track segments formed in at least two of the three precision chamber planes. For a state decaying to π+π−Υ (1S), the acceptance A is de- ﬁned as the fraction of decays where both muons have pμ T > 4 GeV, both pions have pπ T > 400 MeV, and all four particles are within \|η\| < 2.5. 2 An un-prescaled trigger with a pμ T = 6 GeV threshold on the muons was also considered, but found to result in a lower sensitivity using the optimisation proce- dure described in Section 5. 1. Introduction [14] predicts masses of 10 492, 10 593, or 10 682 MeV, depending on the ﬂavour of the light quarks. In 2013, CMS reported [25] the results of their search for Xb → π+π−Υ (1S)(→μ+μ−), ﬁnding no evidence for narrow states in the 10.06–10.31 GeV and 10.40–10.99 GeV mass ranges. They set upper limits on the product of cross section and branching fraction at values between 0.9% and 5.4% of the Υ (2S) rate. The decay Xb →π+π−Υ (1S)(→μ+μ−), analogous to the decay mode in which the X(3872) was discovered, provides a straightforward way to reconstruct an Xb. Any resulting measure- ment or upper limit on the Xb production cross section then de- This Letter presents a search for the Xb and other hidden- beauty states at ATLAS, using a 16.2 fb−1 pp collision data sam- ple collected at √ s = 8 TeV during the 2012 run of the LHC. The analysis is performed simultaneously across eight kinematic bins of varying sensitivity; the Υ (2S) and Υ (3S) →π+π−Υ (1S) peaks are used to validate the measurement technique. Results are ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 200 presented in terms of the product of production cross section and π+π−Υ (1S) branching fraction, relative to that for the Υ (2S). 350 MeV of the Υ (1S) mass [23], m1S, is retained and consid- ered an Υ (1S) →μ+μ−candidate. To conﬁrm that this pair is the same as that used in the trigger, the reconstructed and trigger- level muons are required to match with R < 0.01. 2. The ATLAS detector States with pT < 5 GeV or rapidity \|y\| > 2.4 have very low acceptance, so candidates in these regions (which also suffer from high background) are excluded. 4. Data and simulation samples The ATLAS detector employs a three-level trigger [27] to re- duce the 20 MHz proton bunch collision rate to the few-hundred hertz transfer rate to mass storage. This analysis is based on a Level-1 muon trigger that searches for hit coincidences between muon trigger detector layers inside pre-programmed geometrical windows that bound the path of muon candidates above a given pμ T threshold, and provide a rough estimate of their position, for \|ημ\| < 2.4. There are two subsequent software-based trigger stages, in which muon candidates incorporate, with increasing pre- cision, information from both the MS and the ID, reaching position and momentum resolution close to that provided by the oﬄine re- construction. The techniques adopted in this analysis were developed us- ing √ s = 7 TeV data collected in 2011 and simulation samples generated under the same running conditions, with particular at- tention to Υ (2S) →π+π−Υ (1S) mass and (\|y\|, pT) distributions. Backgrounds due to inclusive Υ (1S) production and combinato- rial μ+μ−sources were studied using μ+μ−sideband and μ±μ± same-sign samples, and found to be featureless above 9.8 GeV. The results presented here are based on data from the 2012 √ s = 8 TeV pp run at the LHC; standard data-quality criteria are used to ensure eﬃcient detector performance. The trigger em- ployed in the event selection was subject to an instantaneous luminosity-dependent prescale factor,2 and provided an integrated luminosity of L = 16.2 fb−1. The resulting data sample includes over 10 million μ+μ−combinations: a ﬁt to the sample ﬁnds (6.00 ± 0.01) × 106 from Υ (1S) decays, (0.200 ± 0.002) × 106 from Υ (2S) decays, and the remainder from combinatorial back- ground. Given the reconstruction and event selection choices de- scribed in the previous section, each dimuon gives rise (on aver- age) to 19.5 π+π−Υ (1S) candidates with invariant mass below 11.2 GeV. A procedure to select at most one candidate per event was considered. However, after the adoption of the binning ap- proach described in Section 5, candidate selection was found to worsen the expected sensitivity to a hypothetical Xb signal. There- fore, all π+π−Υ (1S) candidates are retained for analysis. 3. Reconstruction and event selection Events are selected using a trigger requiring two muons of opposite charge, each with pμ T > 4 GeV, successfully ﬁtted to a common vertex. The μ+μ−mass range accepted by the trigger, 8–12 GeV, includes the Υ (1S), Υ (2S), and Υ (3S) signal peaks. In the oﬄine reconstruction for this analysis, muon reconstruc- tion relies on a statistical combination of an MS track and an ID track. The selected muons are restricted to \|ημ\| < 2.3, ensuring high-quality tracking and a reduction of fake muon candidates. This restriction also removes regions of strongly varying eﬃciency and acceptance. The reconstruction of π+π−Υ (1S) candidates begins with pairs of oppositely charged muon candidates that satisfy the same kine- matic conditions used by the trigger, and have ≥2 pixel and ≥6 silicon microstrip detector hits. Each pair is subjected to a common vertex ﬁt, and a loose chi-square selection is imposed to exclude very poor candidates. Any dimuon with an invariant mass within Simulated samples are used to optimise the selections, model signal decays, develop ﬁtting models, and calculate eﬃciencies. In- dividual samples are used for the Υ (2S), Υ (3S), Υ (13D J) triplet, Υ (10 860), and for two hypothetical Xb masses, 10 233 MeV and 10 561 MeV. Production is modelled with the AU2 [28] tune of Pythia 8.170 [29] and the CTEQ6L1 [30] parton distribution func- tions. In the decay to π+π−Υ (1S), the three-body phase space is uniformly sampled. Isotropic spin alignment of the parent is as- sumed: for Υ (2S) and Υ (3S) this is supported by a recent CMS measurement [31]. Passage of particles through the detector is ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 201 Fig. 1. The π+π−Υ (1S) invariant mass distribution in the kinematic bin most sen- sitive to an Xb signal: \|y\| < 1.2, pT > 20 GeV, and cosθ∗> 0. The only apparent peaks are at the masses of the Υ (2S) (10023 MeV) and Υ (3S) (10355 MeV). simulated with Atlfast II [32], supplementing Geant4 [33,34] with a parameterised calorimeter response. Simulated kinematic distributions of the ﬁnal-state pions and muons are sensitive to mismodelling of the pT and y distributions of the parent state. 5. Fitting strategy Due to the low pion momentum in the π+π−Υ (1S) rest frame, and the reconstruction threshold of pπ T = 400 MeV in the labo- ratory frame, true π+π−Υ (1S) decays are preferentially recon- structed if the parent has large pT or small θ∗(deﬁned as the angle, in the parent rest frame, between the dipion momentum and the lab-frame parent momentum). In background candidates, the dipion and dimuon systems are unrelated, and the dipions typically have low pππ T in the lab; boosting to the π+π−μ+μ− frame yields large values of θ∗, with a broad distribution around cosθ∗= 0. In the (pT, cosθ∗) plane, then, the ratio of signal to background candidates is largest in the upper-right region, and smallest in the lower-left. y pT cos θ The shape of the signal peaks reﬂects the detector resolution, which differs between the barrel and endcap, and varies as a func- tion of mass; in a given rapidity bin at a given mass, a single function can be used across the whole (pT, cosθ∗) range. In each rapidity bin, the signal is ﬁtted using two Gaussians with a com- mon mean, a narrow component fraction f , and a ratio r of broad to narrow widths. These parameters are found to be independent of mass, and are ﬁxed to the average values across the simula- tion samples. The remaining parameter, the width of the narrow component, σ , depends linearly on the mass of the parent state. Together with the splitting functions deﬁned above, this allows the signal shape and the fraction of the signal falling in each of the analysis bins to be determined for any Xb mass. The mass resolution and background shape differ at central and forward rapidities, so the analysis is performed in bins of \|y\| < 1.2 (barrel) and 1.2 < \|y\| < 2.4 (endcap). Several possible ways to ex- ploit the (pT, cosθ∗) discrimination were considered, including fur- ther binning, a diagonal cut in (pT, cosθ∗), and a requirement on the R between the Υ (1S) and each pion (as used by CMS [25]). The choice of method was based on optimising the expected sig- niﬁcance for a weak signal at a mass of 10 561 MeV. In the ﬁnal approach eight analysis bins of varying sensitivity are used, formed from combinations of high and low \|y\|, high and low pT, and high and low cosθ∗. 3. Reconstruction and event selection ATLAS has measured doubly differential pro- duction cross sections for Υ (2S) and Υ (3S) at 7 TeV in pT bins up to 70 GeV for \|y\| < 1.2 and 1.2 < \|y\| < 2.25 [35]. These results can be extended to \|y\| < 2.4 assuming ﬂat rapidity dependence, and up to pT = 100 GeV using CMS measurements [36]. The result- ing Υ (2S) cross section is compared to the production kinematics of a 7 TeV simulation using the 2011 ATLAS tune [37] of Pythia 6.4 [38]. The ratio of the two in (\|y\|, pT) bins deﬁnes production weights, which are applied to the 8 TeV Υ (2S) simulated sample, assuming that Pythia correctly models the increase in cross sec- tion with √ s. The same procedure is used for the Υ (3S); for other masses, linear extrapolation of the Υ (2S) and Υ (3S) weights is used. The simulated Υ (2S) and Υ (3S) →π+π−Υ (1S) samples are further reweighted to match dipion mass distributions observed by CLEO [39,40], to allow comparison of simulation and data. Fig. 1. The π+π−Υ (1S) invariant mass distribution in the kinematic bin most sen- sitive to an Xb signal: \|y\| < 1.2, pT > 20 GeV, and cosθ∗> 0. The only apparent peaks are at the masses of the Υ (2S) (10023 MeV) and Υ (3S) (10355 MeV). as splitting functions. At any speciﬁed mass, the signal yield frac- tion in any particular (\|y\|, pT, cosθ∗) bin can be calculated from an appropriately chosen product of three of these and their comple- ments, (1 −S). For example, the fraction in the bin (\|y\| < 1.2, pT > 20 GeV, cosθ∗< 0) is given by S\|y\| · (1 −Sb pT(m)) · S(3) cos θ∗(m). 5. Fitting strategy The optimal bin boundaries for pT and cosθ∗were determined to be 20 GeV and 0, respectively. Searches for the production of the Υ (10 860) and Υ (11 020), which have natural widths larger than the experimental resolu- tion, are also performed. Each of these states is modelled as a Breit–Wigner convolved with the mass-dependent signal shape de- scribed in the previous paragraph, representing the detector reso- lution. The fractions of the Υ (10 860) signal falling in the eight analysis bins are extracted from the simulation, while those for the Υ (11 020) are determined by extrapolation. All ﬁts presented here are binned, extended maximum-likeli- hood ﬁts in a local region around the mass of interest. The bin width is 2 MeV in all ﬁts. The background is described by a lin- ear combination of Chebychev polynomials up to second-order, with independent parameters in each analysis bin, unless other- wise speciﬁed. The π+π−Υ (1S) mass distribution for the most sensitive bin (\|y\| < 1.2, pT > 20 GeV, cosθ∗> 0) is shown in Fig. 1. The Υ (2S) and Υ (3S) peaks are clearly visible, but no other peaks are ap- parent. The background in this bin decreases with mass above the Υ (3S), whereas the fraction of signal events falling in this bin is constant above the Υ (3S) in the simulation. Thus higher sensitivity is expected at larger masses. 6. Results for the Υ (2S) and Υ (3S) To test kine- matic distributions in the Υ (2S) simulation, signal fractions in the eight analysis bins are checked against their expected values, and are found to be consistent within statistical uncertainties. where the product of the cross section and branching fraction, (σB)2S, is estimated from the extended cross-section measure- ment (see Section 4) using world-average values for the branching fractions B(Υ (1S) →μ+μ−) and B(Υ (2S) →π+π−Υ (1S)) [23]. The shape of the doubly differential cross section is also used to calculate the acceptance, A = (1.442 ± 0.004)%, assuming the CLEO dipion mass spectrum [39] and isotropic signal decays. The reconstruction eﬃciency for decays within the acceptance, ϵ = 0.283 ± 0.002, is taken from the Υ (2S) simulation. To test kine- matic distributions in the Υ (2S) simulation, signal fractions in the eight analysis bins are checked against their expected values, and are found to be consistent within statistical uncertainties. At each mass, the p-value is extracted using the asymptotic formula [41] for the q0 statistic, a modiﬁcation of the standard likelihood ratio (see Fig. 3). No evidence for new states with local signiﬁcance z ≥3 is found. The expected number of Xb events can be written as N = N2S · R · A A2S · ϵ ϵ2S , (2) N = N2S · R · A A2S · ϵ ϵ2S , N = N2S · R · A A2S · ϵ ϵ2S , (2) where R ≡(σB)/(σB)2S, the production rate relative to that of the Υ (2S). The eﬃciency, ϵ, as a function of mass is determined by ﬁtting a function a + b/(1 + e−c(m−d)) to the values from the simulated samples. The acceptance increases with the mass of the parent state due to the increased energy available to the pions and muons. Additionally, the measured production spectra of the Υ (1S), Υ (2S), and Υ (3S) states [35] are progressively harder in pT with increasing mass. The acceptance for a hypothetical Xb state of arbitrary mass is estimated here by linear extrapolation of calculations performed at the Υ (2S) and Υ (3S), using the mea- sured production spectra of these states. The ratio (Aϵ)/(Aϵ)2S rises from 1.1 at m = 10 GeV to 7.5 at m = 11 GeV. 6. Results for the Υ (2S) and Υ (3S) Based on the simulation samples, the fraction of signal in the barrel region \|y\| < 1.2 is independent of mass with an aver- age value of S\|y\| = 0.606 ± 0.004. Within the barrel, the frac- tion of signal with pT < 20 GeV develops smoothly with mass and can be characterised by an analytic turn-on curve Sb pT(m) = a/(1 + e−b(m−c)). In the endcap, the dependence is described by Sec pT(m), which has the same functional form as Sb pT(m) with dif- ferent values for the parameters. Similarly, within each (\|y\|, pT) bin the fraction of the signal with cosθ∗< 0 is modelled with a quadratic function, S(i) cos θ∗(m) = a + bm + cm2, where i = 1–4 labels the bin. These S functions, seven in total, are referred to below Fits to the π+π−Υ (1S) spectrum near the Υ (2S) are ﬁrst performed separately in barrel and endcap bins, across the full (pT, cosθ∗) range, with signal mass and width parameters free in the ﬁt (Figs. 2a and 2b). In both cases, the mass is consistent with the world average for the Υ (2S), and the σ parameters are within uncertainties of the values ﬁtted to the Υ (2S) simulation. Signal shape parameters are then ﬁxed to simulated values to reduce uncertainties, and a separate ﬁt is performed on each anal- ysis bin. The fraction of signal decays falling in the barrel is mea- sured to be 0.67 ± 0.04, compared to 0.606 ± 0.004 in the simula- ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 202 202 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 Fig. 2. The measured π+π−Υ (1S) invariant mass (data points), together with ﬁts (red solid curves) to the Υ (2S) peak in the (a) barrel and (b) endcap, with signal mass (m) and width parameters (σb and σec, respectively) free; and (c) a ﬁt to the Υ (3S) peak in the most sensitive bin, with zero suppressed on the vertical scale. Ns is the ﬁtted signal yield in each bin. The background component (green, long-dashed) and the signal component (peaked blue curve) are also shown separately. In each case, the lower panel shows background-subtracted data, with the total signal function (blue solid) and its two Gaussian components (pink, short-dashed curves) overlaid. 6. Results for the Υ (2S) and Υ (3S) A simultaneous ﬁt to the analysis bins is also performed at the Υ (3S) mass, with reduced χ 2 = 1.0 and statistical signiﬁcance (from the likelihood-ratio test statistic) z = 8.7. An individual ﬁt to the most sensitive bin (shown in Fig. 2(c), with larger binning to emphasise the peak) has signiﬁcance z = 6.5. The total Υ (3S) yield, 11 600 ± 1300, agrees with the prediction estimated analo- gously to Eq. (1), 11 400 ± 1500. 7.1. Hypothesis tests Using Eq. (2) and the formalism from Ref. [41], the expected signiﬁcance for a relative production rate R = 6.56% (the value of the analogous quantity for the X(3872) [5]) is calculated as a func- tion of mass, shown as the dashed blue line in Fig. 3; it exceeds 5σ for m ≳10.12 GeV. Expectations for a weaker signal with R = 3% are also shown (long-dashed red line). Given the null result, up- per limits are calculated on R after modifying the ﬁt to include systematic uncertainties. A hypothesis test for the presence of an Xb peak is performed every 10 MeV from 10 GeV to 11 GeV, assuming a narrow state3 that has a differential cross section with a (\|y\|, pT) distribution similar to that of the Υ (2S) or Υ (3S), decaying according to three- body phase space. The signal shape and bin splittings are treated as described in Section 5. At each mass, a simultaneous ﬁt to the analysis bins is performed in a range m ± 8σec, where σec(m) is the width of the narrow signal component in the endcap: the win- dow varies from ±72 MeV at 10 GeV to ±224 MeV at 10.9 GeV; 3 Here, a narrow state refers to one whose natural width is much smaller than the experimental resolution. 6. Results for the Υ (2S) and Υ (3S) (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 2. The measured π+π−Υ (1S) invariant mass (data points), together with ﬁts (red solid curves) to the Υ (2S) peak in the (a) barrel and (b) endcap, with signal mass (m) and width parameters (σb and σec, respectively) free; and (c) a ﬁt to the Υ (3S) peak in the most sensitive bin, with zero suppressed on the vertical scale. Ns is the ﬁtted signal yield in each bin. The background component (green, long-dashed) and the signal component (peaked blue curve) are also shown separately. In each case, the lower panel shows background-subtracted data, with the total signal function (blue solid) and its two Gaussian components (pink, short-dashed curves) overlaid. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.) tion; similar results were seen in 7 TeV data. The ratio 0.67/0.606 is used to rescale the barrel fraction in the simulation. near 11 GeV, m −8σec < m < 11.2 GeV is used. When ﬁtting near the Υ (2S) and Υ (3S) regions, the lineshapes for these signal components are added to the background model, with normali- sations governed by Gaussian constraints to the values obtained from the ﬁts given in Section 6. In the immediate vicinity of the peaks, m2S,3S ± 4σb, no search is performed; the width of the nar- row signal component in the barrel, σb, is 5.66 MeV (9.37 MeV) at the Υ (2S) (Υ (3S)) mass. This reduces the analysis range to 10.05–10.31 and 10.40–11.00 GeV. The total ﬁtted yield N2S = 34 300 ± 800 is consistent with (1) Nexpected 2 = (σB)2S · L · A · ϵ = 33 300 ± 2500, (1) where the product of the cross section and branching fraction, (σB)2S, is estimated from the extended cross-section measure- ment (see Section 4) using world-average values for the branching fractions B(Υ (1S) →μ+μ−) and B(Υ (2S) →π+π−Υ (1S)) [23]. The shape of the doubly differential cross section is also used to calculate the acceptance, A = (1.442 ± 0.004)%, assuming the CLEO dipion mass spectrum [39] and isotropic signal decays. The reconstruction eﬃciency for decays within the acceptance, ϵ = 0.283 ± 0.002, is taken from the Υ (2S) simulation. 7.3. Upper limit calculation Alterna- tive extrapolations between the Υ (1S) and Υ (2S), and between Υ (1S) and Υ (3S), are also tried; the greatest change in the accep- tance ratio, 12%, is assigned as the uncertainty. If an Xb state exists and lies within the range of masses to which this analysis is sensitive, its production cross section and/or its branching fraction must be lower, relative to the Υ (2S), than that of the X(3872) relative to the ψ(2S). There are arguments that the decay Xb →π+π−Υ (1S) should be suppressed, in the absence of the strong isospin-violating effects that are present for X(3872) →π+π−J/ψ [46,47]. In this case the Xb would have more prominent decays to π+π−χb1, π+π−π 0Υ (1S), and other ﬁnal states which are relatively diﬃcult to reconstruct. The parameters of the eﬃciency, the splitting functions, and the widths of the narrow signal components σb and σec as functions of mass, are varied by the uncertainties on their ﬁtted values; al- ternative functional forms are also tried. In each case, the largest deviation is assigned as the systematic uncertainty. The use of production weights (described in Section 4) relies on assumptions regarding rapidity dependence, and evolution from √ s = 7 TeV to 8 TeV. Removing these weights produces a ∼1% change in eﬃ- ciency ratio (most of the differences cancel), but changes the val- ues of the splitting functions by up to 8%. All results to this point assume that any hypothetical Xb pro- duction is unpolarised. Angular distributions of the Υ (1S, 2S, 3S) states in pp collisions are consistent with unpolarised produc- tion [31], but the Xb spin-alignment is unknown and can have a strong impact on the eﬃciency ratio, acceptance ratio, and bin splitting fractions. Rather than including this as a systematic un- certainty, upper limits are recalculated under longitudinal (‘LONG’) and three transverse (‘TRPP’, ‘TRP0’, ‘TRPM’) spin-alignment sce- narios [48]. Shifts in the upper limits (either up or down) depend only weakly on mass; the shift is smaller at large masses. In Fig. 4 the effect of each hypothesis is represented by a single number, Data versus simulation differences in the Υ (2S) width param- eters in the barrel and endcap (1.9% and 4.2%, respectively) are incorporated as a source of uncertainty, as is the statistical uncer- tainty on the averages used for signal shape parameters f and r (0.5–1.4%). 7.3. Upper limit calculation falling in each of the analysis bins. From Eq. (2), the upper limit on R is proportional to the inverse ﬁtted Υ (2S) yield, N−1 2S , and the ratios A2S/A and ϵ2S/ϵ. For each source of systematic uncertainty, the impact on these factors is quantiﬁed to ﬁnd the maximum shift across the mass range. These are then summed in quadrature and included in the ﬁt as Gaussian-constrained nuisance parameters. Upper limits are evaluated at the 95% conﬁdence level using the CLS method by implementing asymptotic formulae for the ˜qμ statistic [41]. The results (Fig. 4, solid line) range between R = 0.8% and 4.0%. Median expected upper limits assuming background only (dashed line), and corresponding ±1σ and ±2σ bands are also shown. These limits include the effect of systematic uncertainties: their inclusion increased the observed limits by up to 13% and in- ﬂated the ±1σ band by 9.5–25%, depending on the Xb mass. The X(3872) →π+π−J/ψ dipion mass distribution favours high mass [6,9]; for a potential hidden-beauty counterpart this distribution is unknown. For ψ(2S) →π+π−J/ψ [42], and both Υ (2S) [39] and Υ (4S) →π+π−Υ (1S) [43,44], the dipion mass distributions are concentrated near the upper boundary; those for Y(4260) →π+π−J/ψ [45] and Υ (3S) →π+π−Υ (1S) [40] are double-humped. The results quoted here assume decay according to three-body phase space; Υ (2S)- and Υ (3S)-like distributions change the splitting functions by up to 35%, decrease the eﬃciency ratio by up to 17%, and produce modest changes in other parame- ters. As a check, upper limits are recalculated with modiﬁed ﬁtting ranges (m ± 7σec and m ± 9σec) and doubled bin widths in the π+π−Υ (1S) mass distributions: shifts are small compared to the ±1σ bands. If an Υ (2S)-like mπ+π−distribution is assumed (cf. CMS [25]), expected upper limits increase: the fractional change is +17% at 10.1 GeV, and ∼+5% for m > 10.4 GeV. These results exclude Xb states with R = 6.56% for masses 10.05–10.31 GeV and 10.40–11.00 GeV. The expected upper limits are more restrictive than those from CMS above m ∼10.1 GeV, and improve as a function of mass; the discrimination in (pT, cosθ∗), exploited by the binning method, becomes increasingly important as mass increases. The next largest contribution is due to the linear extrapolation of the acceptance between the Υ (2S) and Υ (3S) values. 7.2. Systematic uncertainties The upper limit calculation depends indirectly on signal and background ﬁtting parameters, including the fraction of the signal ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 203 ATLAS Collaboration / Physi Fig. 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 4. Observed 95% CLS upper limits (solid line) on the relative production rate R = (σB)/(σB)2S of a hypothetical Xb parent state decaying isotropically to π+π−Υ (1S), as a function of mass. The median expectation (dashed) and the cor- responding ±1σ and ±2σ bands (green and yellow respectively) are also shown. The bar on the right shows typical shifts under alternative Xb spin-alignment sce- narios, relative to the isotropic (‘FLAT’) case shown with the solid point. Fig. 7.2. Systematic uncertainties 3. The solid curve shows the observed local p-value for the background-only hy- pothesis (left scale), and the corresponding signiﬁcance, z, of a peak in π+π−Υ (1S) (right scale), as a function of the mass of a hypothetical Xb parent state. Also shown are the expected values for the case of a signal with relative production rates (σB)/(σB)2S of 3% (red, long-dashed) and 6.56% (blue, dashed curve). 8. Results for the Υ (13D J ) triplet, Υ (10 860), and Υ (11 020) The search described above does not account for the closely spaced Υ (13D J) triplet or the broad Υ (10 860) and Υ (11 020). To ﬁt for the Υ (13D J), two extra peaks are added to the sig- nal model. CLEO [18] and BaBar [19] have measured the Υ (13D2) mass, with an average of (10 163.7 ± 1.4) MeV, but the mass split- ting within the triplet is unknown. Averaging over several mod- els [49] leads to triplet masses 10 156, 10 164, and 10 170 MeV (at 1 MeV precision). A ﬁt is performed using these values, as- suming independent normalisations but common signal shapes and bin splitting fractions. A signiﬁcance of z = 0.12 is found, with ﬁtted yields −1000 ± 3100, 600 ± 1800, and 800 ± 2300 for J = 1, 2, and 3. Reasonable changes to the mass splittings do not appreciably increase the signiﬁcance, so there appears to be no ev- idence for Υ (13D J) production. Assuming that J = 2 production dominates, or that the mass splitting is larger than the experi- mental resolution, the upper limit on R can be read from Fig. 4; combined with the measured Υ (13D2) →π+π−Υ (1S) branching fraction [19], this yields an upper limit on the relative cross section σ(pp →Υ (13D2))/σ(pp →Υ (2S)) ≤0.55. The crucial computing support from all WLCG partners is ac- knowledged gratefully, in particular from CERN and the ATLAS Tier- 1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Swe- den), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA) and in the Tier-2 facilities worldwide. (pp ( ))/ (pp ( )) The signal model for Υ (10 860) and Υ (11 020) is described in Section 5. Due to the large natural widths of these states, the ﬁtting range is extended to 10.498–11.198 GeV and the back- ground polynomial order increased to three. Signiﬁcances z = 0.6 and z = 0.3 are found for Υ (10 860) and Υ (11 020), for masses and widths ﬁxed to world-average values [23]. As these parame- ters have large uncertainties, the signiﬁcance is also calculated in a grid of m ± 20 MeV and Γ ± Γ , where Γ is the uncertainty on the world-average width [23]. 9. Conclusions A search for a hidden-beauty analogue of the X(3872) is conducted by reconstructing π+π−Υ (1S)(→μ+μ−) events in 16.2 fb−1 of pp collision data recorded at √ s = 8 TeV by ATLAS at the LHC. To optimise the sensitivity of the search, the analysis is performed in eight bins of rapidity, transverse momentum, and the angle (in the rest frame of the parent state) between the di- pion system and the laboratory-frame momentum of the parent. At each mass, the presence of a signal is tested by performing simultaneous ﬁts to the nearby π+π−Υ (1S) mass spectrum in these bins; no evidence for new narrow states is found for masses 10.05–10.31 GeV and 10.40–11.00 GeV. Upper limits are also set on the ratio R = [σ(pp →Xb)B(Xb →π+π−Υ (1S))]/[σ(pp → Υ (2S))B(Υ (2S) →π+π−Υ (1S))], with results ranging from 0.8% to 4.0% depending on the Xb mass. The analogous ratio for the X(3872) is 6.56%: a value this large is excluded for all Xb masses considered. Separate ﬁts to the Υ (13D J) triplet, Υ (10 860), and Υ (11 020) also reveal no signiﬁcant signals, and a CLS upper limit of 0.55 is set on σ(pp →Υ (13D2))/σ(pp →Υ (2S)). [5] CMS Collaboration, Measurement of the X(3872) production cross section via decays to J/ψπ+π−in pp collisions at √ s = 7 TeV, J. High Energy Phys. 1304 (2013) 154, http://dx.doi.org/10.1007/JHEP04(2013)154, arXiv:1302.3968. [6] A. Abulencia, et al., CDF Collaboration, Measurement of the di-pion mass spec- trum in X(3872) →J/ψπ+π−decays, Phys. Rev. Lett. 96 (2006) 102002, http://dx.doi.org/10.1103/PhysRevLett.96.102002, arXiv:hep-ex/0512074. [7] B. Aubert, et al., BaBar Collaboration, Search for B+ →X(3872)K +, X(3872) →J/ψγ , Phys. Rev. D 74 (2006) 071101, http://dx.doi.org/10.1103/ PhysRevD.74.071101, arXiv:hep-ex/0607050. J/ψγ y ( ) PhysRevD.74.071101, arXiv:hep-ex/0607050. [8] A. Abulencia, et al., CDF Collaboration, Analysis of the quantum numbers J PC of the X(3872), Phys. Rev. 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Finally, uncertainties on N2S and the bar- rel/endcap scaling factor are assigned based on uncertainties from the Υ (2S) ﬁts. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 204 chosen as the maximum difference in the median expected signif- icance from the unpolarised (‘FLAT’) case. chosen as the maximum difference in the median expected signif- icance from the unpolarised (‘FLAT’) case. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azer- baijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF, DNSRC and Lundbeck Foundation, Den- mark; EPLANET, ERC and NSRF, European Union; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, DFG, HGF, MPG and AvH Foundation, Germany; GSRT and NSRF, Greece; ISF, MINERVA, GIF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; BRF and RCN, Norway; MNiSW and NCN, Poland; GRICES and FCT, Portugal; MNE/IFA, Romania; MES of Russia and ROSATOM, Rus- sian Federation; JINR; MSTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SER, SNSF and Cantons of Bern and Geneva, Switzerland; NSC, Taiwan; TAEK, Turkey; STFC, the Royal Society and Leverhulme Trust, United Kingdom; DOE and NSF, United States of America. 9. 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Dimitrievska 13a, J. Dingfelder 21, C. Dionisi 133a,133b, P. Dita 26a, S. Dita 26a, F. Dittus 30, F. Djama 84, T. Djobava 51b, J.I. Djuvsland 58a, M.A.B. do Vale 24c, A. Do Valle Wemans 125a,125g, D. Dobos 30, C. Doglioni 49, T. Doherty 53, T. Dohmae 156, J. Dolejsi 128, Z. Dolezal 128, B.A. Dolgoshein 97,∗, M. Donadelli 24d, S. Donati 123a,123b, P. Dondero 120a,120b, J. Donini 34, J. Dopke 130, A. Doria 103a, M.T. Dova 70, A.T. Doyle 53, M. Dris 10, J. Dubbert 88, S. Dube 15, E. Dubreuil 34, E. Duchovni 173, G. Duckeck 99, O.A. Ducu 26a, D. Duda 176, A. Dudarev 30, F. Dudziak 63, L. Duﬂot 116, L. Duguid 76, M. Dührssen 30, M. Dunford 58a, H. Duran Yildiz 4a, M. Düren 52, A. Durglishvili 51b, M. Dwuznik 38a, M. Dyndal 38a, J. Ebke 99, W. Edson 2, N.C. Edwards 46, W. Ehrenfeld 21, T. Eifert 144, G. Eigen 14, K. Einsweiler 15, T. ATLAS Collaboration Ekelof 167, M. El Kacimi 136c, M. Ellert 167, S. Elles 5, F. Ellinghaus 82, N. Ellis 30, J. Elmsheuser 99, M. Elsing 30, D. Emeliyanov 130, Y. Enari 156, O.C. Endner 82, 117 149 177 17 147 176 2 L. Cerrito 75, F. Cerutti 15, M. Cerv 30, A. Cervelli 17, S.A. Cetin 19b, A. Chafaq 136a, D. Chakraborty 107, I. Chalupkova 128, P. Chang 166, B. Chapleau 86, J.D. Chapman 28, D. Charfeddine 116, D.G. Charlton 18, C.C. Chau 159, C.A. Chavez Barajas 150, S. Cheatham 86, A. Chegwidden 89, S. Chekanov 6, S.V. Chekulaev 160a, G.A. Chelkov 64,g, M.A. Chelstowska 88, C. Chen 63, H. Chen 25, K. Chen 149, L. Chen 33d,h, S. Chen 33c, X. Chen 146c, Y. Chen 66, Y. Chen 35, H.C. Cheng 88, Y. Cheng 31, A. Cheplakov 64, R. Cherkaoui El Moursli 136e, V. Chernyatin 25,∗, E. Cheu 7, L. Chevalier 137, V. Chiarella 47, G. Chiefari 103a,103b, J.T. Childers 6, A. Chilingarov 71, G. Chiodini 72a, A.S. Chisholm 18, R.T. Chislett 77, A. Chitan 26a, M.V. Chizhov 64, S. Chouridou 9, B.K.B. Chow 99, D. Chromek-Burckhart 30, M.L. Chu 152, J. Chudoba 126, J.J. Chwastowski 39, L. Chytka 114, G. Ciapetti 133a,133b, A.K. Ciftci 4a, R. Ciftci 4a, D. Cinca 53, V. Cindro 74, A. Ciocio 15, P. Cirkovic 13b, Z.H. Citron 173, M. Citterio 90a, M. Ciubancan 26a, A. Clark 49, P.J. Clark 46, R.N. Clarke 15, W. Cleland 124, J.C. Clemens 84, C. Clement 147a,147b, Y. Coadou 84, M. Cobal 165a,165c, A. Coccaro 139, J. Cochran 63, L. Coffey 23, J.G. Cogan 144, J. Coggeshall 166, B. Cole 35, S. Cole 107, A.P. Colijn 106, J. Collot 55, T. Colombo 58c, G. Colon 85, G. Compostella 100, P. Conde Muiño 125a,125b, E. Coniavitis 48, M.C. Conidi 12, S.H. Connell 146b, I.A. Connelly 76, S.M. Consonni 90a,90b, V. Consorti 48, S. Constantinescu 26a, C. Conta 120a,120b, G. Conti 57, F. Conventi 103a,i, M. Cooke 15, B.D. Cooper 77, A.M. Cooper-Sarkar 119, N.J. Cooper-Smith 76, K. Copic 15, T. Cornelissen 176, M. Corradi 20a, F. Corriveau 86,j, A. Corso-Radu 164, A. Cortes-Gonzalez 12, G. Cortiana 100, G. Costa 90a, M.J. Costa 168, D. Costanzo 140, D. Côté 8, G. Cottin 28, G. Cowan 76, B.E. Cox 83, K. Cranmer 109, G. Cree 29, S. Crépé-Renaudin 55, F. Crescioli 79, W.A. Cribbs 147a,147b, M. Crispin Ortuzar 119, M. Cristinziani 21, V. ATLAS Collaboration Croft 105, G. Crosetti 37a,37b, C.-M. Cuciuc 26a, T. Cuhadar Donszelmann 140, J. Cummings 177, M. Curatolo 47, C. Cuthbert 151, H. Czirr 142, P. Czodrowski 3, Z. Czyczula 177, S. D’Auria 53, M. D’Onofrio 73, M.J. Da Cunha Sargedas De Sousa 125a,125b, C. Da Via 83, W. Dabrowski 38a, A. Daﬁnca 119, T. Dai 88, O. Dale 14, F. Dallaire 94, C. Dallapiccola 85, M. Dam 36, A.C. Daniells 18, M. Dano Hoffmann 137, V. Dao 48, G. Darbo 50a, S. Darmora 8, J. Dassoulas 42, A. Dattagupta 60, W. Davey 21, C. David 170, T. Davidek 128, E. Davies 119,d, M. Davies 154, O. Davignon 79, A.R. Davison 77, P. Davison 77, Y. Davygora 58a, E. Dawe 143, I. Dawson 140, R.K. Daya-Ishmukhametova 85, K. De 8, R. de Asmundis 103a, S. De Castro 20a,20b, S. De Cecco 79, N. De Groot 105, P. de Jong 106, H. De la Torre 81, F. De Lorenzi 63, L. De Nooij 106, D. De Pedis 133a, A. De Salvo 133a, U. De Sanctis 150, A. De Santo 150, J.B. De Vivie De Regie 116, W.J. Dearnaley 71, R. Debbe 25, C. Debenedetti 138, B. Dechenaux 55, D.V. Dedovich 64, I. Deigaard 106, J. Del Peso 81, T. Del Prete 123a,123b, F. Deliot 137, C.M. Delitzsch 49, M. Deliyergiyev 74, A. Dell’Acqua 30, L. Dell’Asta 22, M. Dell’Orso 123a,123b, M. Della Pietra 103a,i, D. della Volpe 49, M. Delmastro 5, P.A. Delsart 55, C. Deluca 106, S. Demers 177, M. Demichev 64, A. Demilly 79, S.P. Denisov 129, D. Derendarz 39, J.E. Derkaoui 136d, F. Derue 79, P. Dervan 73, K. Desch 21, C. Deterre 42, P.O. Deviveiros 106, A. Dewhurst 130, S. Dhaliwal 106, A. Di Ciaccio 134a,134b, L. Di Ciaccio 5, A. Di Domenico 133a,133b, C. Di Donato 103a,103b, A. Di Girolamo 30, B. Di Girolamo 30, A. Di Mattia 153, B. Di Micco 135a,135b, R. Di Nardo 47, A. Di Simone 48, R. Di Sipio 20a,20b, D. Di Valentino 29, F.A. Dias 46, M.A. Diaz 32a, E.B. Diehl 88, J. Dietrich 42, T.A. Dietzsch 58a, S. Diglio 84, A. Dimitrievska 13a, J. Dingfelder 21, C. 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Endo 117, R. Engelmann 149, J. Erdmann 177, A. Ereditato 17, D. Eriksson 147a, G. Ernis 176, J. Ernst 2, M. Ernst 25, J. Ernwein 137, D. Errede 166, S. Errede 166, E. Ertel 82, M. Escalier 116, H. Esch 43, C. Escobar 124, ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 208 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 209 S. Hillert 21, S.J. Hillier 18, I. Hinchliffe 15, E. Hines 121, M. Hirose 158, D. Hirschbuehl 176, J. Hobbs 149, N. Hod 106, M.C. Hodgkinson 140, P. Hodgson 140, A. Hoecker 30, M.R. Hoeferkamp 104, F. Hoenig 99, J. Hoffman 40, D. Hoffmann 84, M. Hohlfeld 82, T.R. Holmes 15, T.M. Hong 121, L. Hooft van Huysduynen 10 W.H. Hopkins 115, Y. Horii 102, J-Y. Hostachy 55, S. Hou 152, A. Hoummada 136a, J. Howard 119, J. Howarth 4 M. Hrabovsky 114, I. Hristova 16, J. Hrivnac 116, T. Hryn’ova 5, C. Hsu 146c, P.J. Hsu 82, S.-C. Hsu 139, D. Hu 35 X. Hu 88, Y. Huang 42, Z. Hubacek 30, F. Hubaut 84, F. Huegging 21, T.B. Huffman 119, E.W. Hughes 35, G. 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Shi 152,ae, S. Shimizu 66, C.O. Shimmin 164, M. Shimojima 101, M. Shiyakova 64, A. Shmeleva 95, M.J. Shochet 31, D. Short 119, S. Shrestha 63, E. Shulga 97, M.A. Shupe 7, S. Shushkevich 42, P. Sicho 126, O. Sidiropoulou 155, D. Sidorov 113, A. Sidoti 133a, F. Siegert 44, Dj. Sijacki 13a, J. Silva 125a,125d, Y. Silver 154, D. Silverstein 144, S.B. Silverstein 147a, V. Simak 127, O. Simard 5, Lj. Simic 13a, S. Simion 116, E. Simioni 82, B. Simmons 77, R. Simoniello 90a,90b, M. Simonyan 36, P. Sinervo 159, N.B. Sinev 115, V. Sipica 142, G. Siragusa 175, A. Sircar 78, A.N. Sisakyan 64,∗, S.Yu. Sivoklokov 98, J. Sjölin 147a,147b, T.B. Sjursen 14, H.P. Skottowe 57, K.Yu. Skovpen 108, P. Skubic 112, M. Slater 18, T. Slavicek 127, K. Sliwa 162, V. Smakhtin 173, B.H. Smart 46, L. Smestad 14, S.Yu. Smirnov 97, Y. Smirnov 97, L.N. Smirnova 98,af , O. Smirnova 80, K.M. Smith 53, M. Smizanska 71, K. Smolek 127, A.A. Snesarev 95, G. Snidero 75, S. Snyder 25, R. Sobie 170,j, F. Socher 44, A. Soffer 154, D.A. Soh 152,ae, C.A. Solans 30, M. Solar 127, J. Solc 127, E.Yu. Soldatov 97, U. Soldevila 168, A.A. Solodkov 129, A. Soloshenko 64, O.V. Solovyanov 129, V. ATLAS Collaboration Solovyev 122, P. Sommer 48, H.Y. Song 33b, N. Soni 1, A. Sood 15, A. Sopczak 127, B. Sopko 127, V. Sopko 127, V. Sorin 12, M. Sosebee 8, R. Soualah 165a,165c, P. Soueid 94, A.M. Soukharev 108,c, D. South 42, S. Spagnolo 72a,72b, F. Spanò 76, W.R. Spearman 57, F. Spettel 100, R. Spighi 20a, G. Spigo 30, L.A. Spiller 87, M. Spousta 128, T. Spreitzer 159, B. Spurlock 8, R.D. St. Denis 53,∗, S. Staerz 44, J. Stahlman 121, R. Stamen 58a, S. Stamm 16, E. Stanecka 39, R.W. Stanek 6, C. Stanescu 135a, M. Stanescu-Bellu 42, M.M. Stanitzki 42, S. Stapnes 118, E.A. Starchenko 129, J. Stark 55, P. Staroba 126, P. Starovoitov 42, R. Staszewski 39, P. Stavina 145a,∗, P. Steinberg 25, B. Stelzer 143, H.J. Stelzer 30, O. Stelzer-Chilton 160a, H. Stenzel 52, S. Stern 100, G.A. Stewart 53, J.A. Stillings 21, J. Rieger 54, M. Rijssenbeek 149, A. Rimoldi 120a,120b, L. Rinaldi 20a, E. Ritsch 61, I. Riu 12, F. Rizatdinova 113, . Rizvi 75, S.H. Robertson 86,j, A. Robichaud-Veronneau 86, D. Robinson 28, J.E.M. Ro b . Robson 53, C. Roda 123a,123b, L. Rodrigues 30, S. Roe 30, O. Røhne 118, S. Rolli 162, A , , , , , E. Rossi 103a,103b, L.P. Rossi 50a, R. Rosten 139, M. Rotaru 26a, I. Roth 173, J. Rothber J C.R. Royon 137, A. Rozanov 84, Y. Rozen 153, X. Ruan 146c, F. Rubbo 12, I. Rubinskiy 44 159 48 30 48 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 213 K. Suruliz 150, G. Susinno 37a,37b, M.R. Sutton 150, Y. Suzuki 65, M. Svatos 126, S. Swedish 169, M. Swiatlowski 144, I. Sykora 145a, T. Sykora 128, D. Ta 89, C. Taccini 135a,135b, K. Tackmann 42, J. Taenzer 159, A. Taffard 164, R. Taﬁrout 160a, N. Taiblum 154, H. Takai 25, R. Takashima 68, H. Takeda 66, T. Takeshita 141, Y Takubo 65 M Talby 84 A A Talyshev 108,c J Y C Tam 175 K G Tan 87 J Tanaka 156 R Tanaka 116 J. Therhaag 21, T. Theveneaux-Pelzer 34, J.P. Thomas 18, J. Thomas-Wilsker 76, E.N. Thompson 35, , , y y , , , , S.A. Tupputi 20a,20b, S. Turchikhin 98,af , D. Turecek 127, I. Turk Cakir 4d, R. Turra 90a,90b, P.M. Tuts 35, A. Tykhonov 49, M. Tylmad 147a,147b, M. Tyndel 130, K. Uchida 21, I. ATLAS Collaboration Ueda 156, R. Ueno 29, M. Ughetto 84, M. Ugland 14, M. Uhlenbrock 21, F. Ukegawa 161, G. Unal 30, A. Undrus 25, G. Unel 164, F.C. Ungaro 48, Y. Unno 65, C. Unverdorben 99, D. Urbaniec 35, P. Urquijo 87, G. Usai 8, A. Usanova 61, L. Vacavant 84, V. Vacek 127, B. Vachon 86, N. Valencic 106, S. Valentinetti 20a,20b, A. Valero 168, L. Valery 34, S. Valkar 128, E. Valladolid Gallego 168, S. Vallecorsa 49, J.A. Valls Ferrer 168, W. Van Den Wollenberg 106, ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 214 C. Weiser 48, H. Weits 106, P.S. Wells 30, T. Wenaus 25, D. Wendland 16, Z. Weng 152,ae, T. Wengler 30, S. Wenig 30, N. Wermes 21, M. Werner 48, P. Werner 30, M. Wessels 58a, J. Wetter 162, K. Whalen 29, A. White 8, M.J. White 1, R. White 32b, S. White 123a,123b, D. Whiteson 164, D. Wicke 176, F.J. Wickens 130, W. Wiedenmann 174, M. Wielers 130, P. Wienemann 21, C. Wiglesworth 36, L.A.M. Wiik-Fuchs 21, P.A. Wijeratne 77, A. Wildauer 100, M.A. Wildt 42,ak, H.G. Wilkens 30, J.Z. Will 99, H.H. Williams 121, S. Williams 28, C. Willis 89, S. Willocq 85, A. Wilson 88, J.A. Wilson 18, I. Wingerter-Seez 5, F. Winklmeier 115, B.T. Winter 21, M. Wittgen 144, T. Wittig 43, J. Wittkowski 99, S.J. Wollstadt 82, M.W. Wolter 39, H. Wolters 125a,125c, B.K. Wosiek 39, J. Wotschack 30, M.J. Woudstra 83, K.W. Wozniak 39, M. Wright 53, M. Wu 55, S.L. Wu 174, X. Wu 49, Y. Wu 88, E. Wulf 35, T.R. Wyatt 83, B.M. Wynne 46, S. Xella 36, M. Xiao 137, D. Xu 33a, L. Xu 33b,al, B. Yabsley 151, S. Yacoob 146b,am, R. Yakabe 66, M. Yamada 65, H. Yamaguchi 156, Y. Yamaguchi 117, A. Yamamoto 65, K. Yamamoto 63, S. Yamamoto 156, T. Yamamura 156, T. Yamanaka 156, K. Yamauchi 102, Y. Yamazaki 66, Z. Yan 22, H. Yang 33e, H. Yang 174, U.K. Yang 83, Y. Yang 110, S. Yanush 92, L. Yao 33a, W-M. Yao 15, Y. Yasu 65, E. Yatsenko 42, K.H. Yau Wong 21, J. Ye 40, S. Ye 25, I. Yeletskikh 64, A.L. Yen 57, E. Yildirim 42, M. Yilmaz 4b, R. Yoosoofmiya 124, K. Yorita 172, R. Yoshida 6, K. Yoshihara 156, C. Young 144, C.J.S. Young 30, S. Youssef 22, D.R. Yu 15, J. Turkey 21 Physikalisches Institut, University of Bonn, Bonn, Germany 22 Department of Physics, Boston University, Boston, MA, United States 22 Department of Physics, Boston University, Boston, MA, United States 3 Department of Physics, Brandeis University, Waltham, MA, United Stat p f y , y, , , 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 25 p f y y 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (d) 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil 4 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; J / / J p Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica, Univers ysics Department, Brookhaven National Laboratory, Upton, NY, United St Physics Department, Brookhaven National Laboratory, Upton, NY, United States 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 27 onal Laboratory, Upton, NY, United States uclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, ucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania y p , y, p , , 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania , y, p , , nstitute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and Molecular Technolog University Politehnica Bucharest Bucharest; (d) West University in Timisoara Timisoara Romania 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research and Development of Isotopic and M ( ) (d) 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) National Institute for Research a 6 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b f y g g, ; f p f p g , y p , Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania luj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West Un Cluj Napoca; (c) University Politehnica Bucharest, Bucharest; (d) West University in Timisoara, Timisoara, Romania 7 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Ar 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 29 29 Department of Physics, Carleton University, Ottawa, ON, Canada 30 CERN, Geneva, Switzerland ATLAS Collaboration Yang 174, U.K. Yang 83, Y. Yang 110, S. Yanush 92, L. Yao 33a, W-M. Yao 15, Y. Yasu 65, E. Yatsenko 42, K.H. Yau Wong 21, J. Ye 40, S. Ye 25, I. Yeletskikh 64, A.L. Yen 57, E. Yildirim 42, M. Yilmaz 4b, R. Yoosoofmiya 124, K. Yorita 172, R. Yoshida 6, K. Yoshihara 156, C. Young 144, C.J.S. Young 30, S. Youssef 22, D.R. Yu 15, J. Yu 8, J.M. Yu 88, J. Yu 113, L. Yuan 66, A. Yurkewicz 107, I. Yusuff 28,an, B. Zabinski 39, R. Zaidan 62, A.M. Zaitsev 129,aa, A. Zaman 149, S. Zambito 23, L. Zanello 133a,133b, D. Zanzi 87, C. Zeitnitz 176, M. Zeman 127, A. Zemla 38a, K. Zengel 23, O. Zenin 129, T. Ženiš 145a, D. Zerwas 116, G. Zevi della Porta 57, D. Zhang 88, F. Zhang 174, H. Zhang 89, J. Zhang 6, L. Zhang 152, X. Zhang 33d, Z. Zhang 116, Z. Zhao 33b, A. Zhemchugov 64, J. Zhong 119, B. Zhou 88, L. Zhou 35, N. Zhou 164, C.G. Zhu 33d, H. Zhu 33a, J. Zhu 88, Y. Zhu 33b, X. Zhuang 33a K. Zhukov 95, A. Zibell 175, D. Zieminska 60, N.I. Zimine 64, C. Zimmermann 82, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. Zwalinski 30 S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. ATLAS Collaboration Zwalinski 30 1 Department of Physics, University of Adelaide, Adelaide, Australia 1 Department of Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, United States 3 Department of Physics, University of Alberta, Edmonton, AB, Canada , , , versity, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara ara, Turkey p f y , y f , , , 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Turkish Atomic Energy Authority, Ankara, Turkey p f y , y f , , , 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, TOBB University of Economics and Technology, Ankara; (d) Turkish Atomic Energy Authority, Ankara, Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara 4 (a) Department of Physics, Ankara University, Ankara; (b) Department of Physics, Gazi University, Ankara; (c) Division of Physics, T (d) Turkish Atomic Energy Authority, Ankara, Turkey 4 (a) Department of Physics, Ankara University, Ankara; (b) Departmen (d) Turkish Atomic Energy Authority, Ankara, Turkey (d) Turkish Atomic Energy Authority, Ankara, Turkey 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annec 5 LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United State 6 High Energy Physics Division, Argonne National Laboratory, Argonne, IL, United States 7 Department of Physics, University of Arizona, Tucson, AZ, United Stat 7 Department of Physics, University of Arizona, Tucson, AZ, United States p f y y f 8 Department of Physics, The University of Texas at Arlington, Arlington, TX, United States 8 Department of Physics, The University of Texas at Arlington, Arlington, TX, United States 8 Department of Physics, The University of Texas at Arlington, 9 Physics Department, University of Athens, Athens, Greece 10 Physics Department, National Technical University of Athens, Zograf 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan f y j y f j 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, Barcelona, Spain b 12 Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona, B 12 Institut de Física d’Altes Energies and Departament de Física de la U g p 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, U 3 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Insti 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 14 Department for Physics and Technology, University of Bergen, Bergen, Norway 14 Department for Physics and Technology, University of Bergen, Bergen, Norway 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 15 Physics Division, Lawrence Berkeley National Laboratory and U 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States 16 Department of Physics, Humboldt University, Berlin, Germany 16 Department of Physics, Humboldt University, Berlin, Germany 16 Department of Physics, Humboldt University, Berlin, Germany 17 p f y y y 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18 17 Albert Einstein Center for Fundamental Physics and Laboratory 17 Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, Univ 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics E Turkey 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Department of Physics, Dogus University, Istanbul; (c) Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey ATLAS Collaboration Yu 8, J.M. Yu 88, J. Yu 113, L. Yuan 66, A. Yurkewicz 107, I. Yusuff 28,an, B. Zabinski 39, R. Zaidan 62, A.M. Zaitsev 129,aa, A. Zaman 149, S. Zambito 23, L. Zanello 133a,133b, D. Zanzi 87, C. Zeitnitz 176, M. Zeman 127, A. Zemla 38a, K. Zengel 23, O. Zenin 129, T. Ženiš 145a, D. Zerwas 116, G. Zevi della Porta 57, D. Zhang 88, F. Zhang 174, H. Zhang 89, J. Zhang 6, L. Zhang 152, X. Zhang 33d, Z. Zhang 116, Z. Zhao 33b, A. Zhemchugov 64, J. Zhong 119, B. Zhou 88, L. Zhou 35, N. Zhou 164, C.G. Zhu 33d, H. Zhu 33a, J. Zhu 88, Y. Zhu 33b, X. Zhuang 33a, K. Zhukov 95, A. Zibell 175, D. Zieminska 60, N.I. Zimine 64, C. Zimmermann 82, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, Z. Zinonos 54, M. Ziolkowski 142, G. Zobernig 174, A. Zoccoli 20a,20b, M. zur Nedden 16, G. Zurzolo 103a,103b, V. Zutshi 107, L. Zwalinski 30 C. Weiser 48, H. Weits 106, P.S. Wells 30, T. Wenaus 25, D. Wendland 16, Z. Weng 152,ae, T. Wengler 30, S. Wenig 30, N. Wermes 21, M. Werner 48, P. Werner 30, M. Wessels 58a, J. Wetter 162, K. Whalen 29, A. White 8, M.J. White 1, R. White 32b, S. White 123a,123b, D. Whiteson 164, D. Wicke 176, F.J. Wickens 130, W. Wiedenmann 174, M. Wielers 130, P. Wienemann 21, C. Wiglesworth 36, L.A.M. Wiik-Fuchs 21, P.A. Wijeratne 77, A. Wildauer 100, M.A. Wildt 42,ak, H.G. Wilkens 30, J.Z. Will 99, H.H. Williams 121, S. Williams 28, C. Willis 89, S. Willocq 85, A. Wilson 88, J.A. Wilson 18, I. Wingerter-Seez 5, F. Winklmeier 115, B.T. Winter 21, M. Wittgen 144, T. Wittig 43, J. Wittkowski 99, S.J. Wollstadt 82, M.W. Wolter 39, H. Wolters 125a,125c, B.K. Wosiek 39, J. Wotschack 30, M.J. Woudstra 83, K.W. Wozniak 39, M. Wright 53, M. Wu 55, S.L. Wu 174, X. Wu 49, Y. Wu 88, E. Wulf 35, T.R. Wyatt 83, B.M. Wynne 46, S. Xella 36, M. Xiao 137, D. Xu 33a, L. Xu 33b,al, B. Yabsley 151, S. Yacoob 146b,am, R. Yakabe 66, M. Yamada 65, H. Yamaguchi 156, Y. Yamaguchi 117, A. Yamamoto 65, K. Yamamoto 63, S. Yamamoto 156, T. Yamamura 156, T. Yamanaka 156, K. Yamauchi 102, Y. Yamazaki 66, Z. Yan 22, H. Yang 33e, H. 30 CERN, Geneva, Switzerland Andronikashvili Institute of Physics, Iv. 30 CERN, Geneva, Switzerland Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 91 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, M 92 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus 92 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus 93 Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, United States Department of Physics, Massachusetts Institute of Technology, Cambridg 94 Group of Particle Physics, University of Montreal, Montreal, QC, Canad 94 Group of Particle Physics, University of Montreal, Montreal, QC, Cana 95 P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 96 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia f p y ( ) 97 National Research Nuclear University MEPhI, Moscow, Russia 97 National Research Nuclear University MEPhI, Moscow, Russia 97 National Research Nuclear University MEPhI, Moscow, Russia 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow 98 D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov M 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, G 100 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), Münc 101 Nagasaki Institute of Applied Science, Nagasaki, Japan 101 Nagasaki Institute of Applied Science, Nagasaki, Japan f y g y y g y J p 103 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy f y g y y g y J p 103 (a) INFN Sezione di Napoli; (b) Dipartimento di Fisica, Università di Napoli, Napoli, Italy 104 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, United States 106 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherla 106 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 107 Department of Physics, Northern Illinois University, DeKalb, IL, United States 107 Department of Physics, Northern Illinois University, DeKalb, IL, United States 108 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 108 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 109 Department of Physics, New York University, New York, NY, United States 109 Department of Physics, New York University, New York, NY, United States 110 Ohio State University, Columbus, OH, United States 111 Faculty of Science, Okayama University, Okayama, Japan y f y y y J p 112 Homer L. 30 CERN, Geneva, Switzerland 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States , y f g , g , , 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai, China 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of d 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 ( ) (b) ( ) 2 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Sa p , ﬁ , g ; p , 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Department, Shanghai Jiao Tong University, Shanghai, China 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 hysics, Nanjing University, Jiangsu; (d) School of Physics, Shandong Univ Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong; (e) Physics Departme y j g y J g f y g y g y p g J g y g 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 35 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Fe 35 34 Laboratoire de Physique Corpusculaire, Clermont Université and Uni 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende, Italy 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fisica, Università della Calabria, Rende 37 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati; (b) Dipartimento di Fis 7 (a) INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 215 nology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, 38 (a) AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow; (b) Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland Poland 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 40 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Po y f y y f 40 Physics Department, Southern Methodist University, Dallas, TX, United States 40 Physics Department, Southern Methodist University, Dallas, TX, United States y p y 41 Physics Department, University of Texas at Dallas, Richardson, TX, Un y p y Physics Department, University of Texas at Dallas, Richardson, TX, Unite 42 DESY, Hamburg and Zeuthen, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortm 44 Institut für Kern- und Teilchenphysik, Technische Universität Dresd 45 Department of Physics, Duke University, Durham, NC, United States 45 Department of Physics, Duke University, Durham, NC, United States 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinburgh, E 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United King 47 INFN Laboratori Nazionali di Frascati, Frascati, Italy 48 Fakultät für Mathematik und Physik, Albert-Ludwigs 48 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany 49 Section de Physique, Université de Genève, Geneva, Switzerland 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università 51 (a) E. 30 CERN, Geneva, Switzerland Javakhishvili Tbilisi State University, Tbilisi; (b) f y , J y, ; g gy y , y, , g 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53 ikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdo 54 II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France Laboratoire de Physique Subatomique et de Cosmologie, Université Gren y q q g 56 Department of Physics, Hampton University, Hampton, VA, United States 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, United States b 57 Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, MA, United States Laboratory for Particle Physics and Cosmology, Harvard University, Cam hysics and Cosmology, Harvard University, Cambridge, MA, United States Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für ht K l U i ität H id lb M h i G 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisch 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Hei 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c) ZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiros 60 Department of Physics, Indiana University, Bloomington, IN, United St 60 Department of Physics, Indiana University, Bloomington, IN, United States p f y y g 61 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, In 61 Institut für Astro- und Teilchenphysik, Leopold-Fr 61 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austr 62 University of Iowa, Iowa City, IA, United States 63 Department of Physics and Astronomy, Iowa State University, Ame 63 Department of Physics and Astronomy, Iowa State University, Ames, IA, United State 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 65 KEK, High Energy Accelerator Research Organization, Tsukuba, J 66 Graduate School of Science, Kobe University, Kobe, J 67 Faculty of Science, Kyoto University, Kyoto, Japan 68 Kyoto University of Education, Kyoto, Japan y y f y J p 69 Department of Physics, Kyushu University, Fukuoka, Japan 69 Department of Physics, Kyushu University, Fukuoka, Japan 70 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 70 Instituto de Física La Plata, Universidad Nacional de La Plata 70 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 71 71 Physics Department, Lancaster University, Lancaster, United Kingdom y p y g 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Ita 72 (a) INFN Sezione di Lecce; (b) Dipartimento di Matematica e Fisica, Un 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 74 Department of Physics, Jožef Stefan Institute and University of Ljublja 75 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdo 75 School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom f y y y y f g 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77 Department of Physics and Astronomy, University C 78 Louisiana Tech University, Ruston, LA, United States 79 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France 80 79 Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris- 80 Fysiska institutionen, Lunds universitet, Lund, Sweden y 81 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 82 81 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 81 Departamento de Fisica Teorica C-15, Universidad Aut 82 Institut für Physik, Universität Mainz, Mainz, German 83 School of Physics and Astronomy, University of Manchester, Manch 84 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 84 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 85 Department of Physics, University of Massachusetts, Amherst, MA, United Sta 85 Department of Physics, University of Massachusetts, Amherst, MA 86 Department of Physics, McGill University, Montreal, QC, Canada 87 School of Physics, University of Melbourne, Victoria, Australia 87 School of Physics, University of Melbourne, Victoria, Australia 88 Department of Physics, The University of Michigan, Ann Arbor, MI, United Stat 88 Department of Physics, The University of Michigan, Ann Arbor, 89 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United S 89 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United States p f y y g y g 90 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 90 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy p y 91 B.I. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 Fermi, Università di Pisa, Pisa, Italy p y 124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United St 124 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 125 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa; (b) Faculd p f y y, y f g , g , , 125 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa; (b) Faculdade de Ciências, Universidade de Lisboa, Lisboa; (c) Department of Physics, University of Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; (f) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 126 I tit t f Ph i A d f S i f th C h R bli P h C h R bli Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP Laboratorio de Instrumentacao e Fisica Experimental de Particulas LIP, Lisboa; Faculdade de Ciências, Universidade de Lisboa, Lisb Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minho, Braga; ( ) p Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de Fisica, Universidade do Minh ( ) bra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisb Coimbra, Coimbra; (d) Centro de Física Nuclear da Universidade de Lisboa, Lisboa; (e) Departamento de , ; , ; p , , g ; p y Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 126 p g p y Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep Fisica and CEFITEC of Faculdade d Cosmos and CAFPE, Universidad de Granada, Granada (Spain); (g) Dep 126 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 126 Institute of Physics, Academy of Sciences of the Czech Republic, 127 Czech Technical University in Prague, Praha, Czech Republic 128 Faculty of Mathematics and Physics, Charles University in Prague, P y f y y g 129 State Research Center Institute for High Energy Physics, Protvino, Russia 130 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 130 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 130 Particle Physics Department, Rutherford Appleton Laboratory, Di y p , f pp y, , 131 Physics Department, University of Regina, Regina, SK, Canada 131 Physics Department, University of Regina, Regina, SK, Canada 132 Ritsumeikan University, Kusatsu, Shiga, Japan 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 133 (a) INFN Sezione di Roma; (b) Dipartimento di F 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Univ 133 (a) INFN Sezione di Roma; (b) Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 134 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy 135 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy p y 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA – Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des sciences Université Mohammed V – Agdal Rabat Morocco 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre Na 136 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassa Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies Université Hassan II, Casablanca; Centre National de l Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA – Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Universi Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA – Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda sciences, Université Mohammed V – Agdal, Rabat, Morocco , g , , 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States g es sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France h i i i f lif i i d 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales d 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energie 138 137 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’En 138 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States anta Cruz Institute for Particle Physics, University of California Santa Cruz 139 Department of Physics, University of Washington, Seattle, WA, United States 139 Department of Physics, University of Washington, Seattle, WA, United States 140 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 141 140 Department of Physics and Astronomy, University of Sheﬃel 141 Department of Physics, Shinshu University, Nagano, Japan 142 Fachbereich Physik, Universität Siegen, Siegen, Germany 143 Department of Physics, Simon Fraser University, Burnaby, BC, Cana p f y y y 144 SLAC National Accelerator Laboratory, Stanford, CA, United States y f 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Exp Sciences, Kosice, Slovak Republic 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Sciences Kosice Slovak Republic 145 (a) Faculty of Mathematics, Physics & Informatics, Comenius Unive y f , y f , y, ; p f y , f p y f y f Sciences, Kosice, Slovak Republic 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand, Johannesburg, South Africa Sciences, Kosice, Slovak Republic Sciences, Kosice, Slovak Republic 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand Johannesburg South Africa Sciences, osice, Slo ak epublic 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) School of Physics, University of the Witwatersrand Johannesburg South Africa , , p 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, University of Johannesburg, Johannesburg; (c) Wi d J h b S h Af i 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Department of Physics, 146 (a) Department of Physics, University of Cape To 146 (a) Department of Physics, University of Cape Town, Cape Town; (b) Witwatersrand, Johannesburg, South Africa cs, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 147 (a) Department of Physics, Stockholm University; (b) The Oskar Klei 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 148 Physics Department, Royal Institute of Technology, Stockholm, Sweden 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, United States 149 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, Un 49 Departments of Physics & Astronomy and Chemistry, Stony Brook Uni 150 Department of Physics and Astronomy, University of Sussex, Brig 151 School of Physics, University of Sydney, Sydney, Australia 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 152 Institute of Physics, Academia Sinica, Taipei, Taiwan 153 Department of Physics, Technion: Israel Institute of Technology, Hai p f y f gy f 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 154 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel 54 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Av 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 55 Department of Physics, Aristotle University of Thessaloniki, Thessalon 155 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece p f y , y f , , 156 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan p f y y f 156 International Center for Elementary Particle Physics and Department of Physics, The Univers 56 International Center for Elementary Particle Physics and Department 157 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 1 8 157 Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 158 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 159 Department of Physics, University of Toronto, Toronto, ON, Canada p f y y f 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronomy, York University, Toronto, ON, Canada 160 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronom p f y y 161 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan p f y y 161 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 162 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 162 Department of Physics and Astronomy, Tufts University, Medford, MA, United States 163 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 64 Department of Physics and Astronomy, University of California Irvine, 164 Department of Physics and Astronomy, University of California Irvine, Irvine 164 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, United States 165 (a) (b) (c) à p f y y y f f 165 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento di Chimica, Fisica e Ambiente, Università 166 165 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP, Trieste; (c) Dipartimento d 65 (a) INFN Gruppo Collegato di Udine, Sezione di Trieste, Udine; (b) ICTP 166 Department of Physics, University of Illinois, Urbana, IL, United States 67 Department of Physics and Astronomy, University of Uppsala, Uppsala 167 Department of Physics and Astronomy, University of Uppsala, Uppsala, Swe 167 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrón 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamen 168 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecul de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 69 Department of Physics, University of British Columbia, Vancouver, BC, 169 Department of Physics, University of British Columbia, Vancouver, BC, Canad 170 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 171 170 Department of Physics and Astronomy, University of Victoria, Victoria, BC, Canada 171 Department of Physics, University of Warwick, Coventry, United Kingdom 172 172 Waseda University, Tokyo, Japan 30 CERN, Geneva, Switzerland Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States 113 112 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United Sta 112 Homer L. Dodge Department of Physics and Astronomy, Univ 113 Department of Physics, Oklahoma State University, Stillw 113 Department of Physics, Oklahoma State University, Stillwater, OK, United States 113 Department of Physics, Oklahoma State University, Stillwater, OK, United States p f y y 114 Palacký University, RCPTM, Olomouc, Czech Republic ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 216 115 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 115 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 116 LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France 117 Graduate School of Science, Osaka University, Osaka, Japan 118 Department of Physics, University of Oslo, Oslo, Norway 119 Department of Physics, Oxford University, Oxford, United Kingdom p f y f y f g 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 120 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università d 121 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 121 Department of Physics, University of Pennsylvania, Philadelphia, PA, United States 121 Department of Physics, University of Pennsylvania, Phi 122 Petersburg Nuclear Physics Institute, Gatchina, Russia g y 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy g y 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fe 123 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 / y w Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. w Also at Academia Sinica Grid Computing, Institute of Physics, A w Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. x Also at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, Fra x Also at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Did y Also at School of Physical Sciences, National Institute of Science Education and Research, Bhubaneswar, India. y Also at School of Physical Sciences, National Institute of Science Education and Resea y z Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. so at Dipartimento di Fisica, Sapienza Università di Roma, Roma, It z Also at Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. aa Also at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. aa Also at Moscow Institute of Physics and Technology State University, Dolgo ab Also at Section de Physique, Université de Genève, Geneva, Switzerland. ab Also at Section de Physique, Université de Genève, Geneva, Switzerland. ac Also at International School for Advanced Studies (SISSA), Trieste, Italy. c Also at International School for Advanced Studies (SISSA), Trieste ac Also at International School for Advanced Studies (SISSA), Trieste, Italy. d ad Also at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, United States of America. ad Also at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, Unite ae Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ae Also at School of Physics and Engineering, Sun Yat-sen University, Guangzhou, China. ae Also at School of Physics and Engineering, Sun Yat-sen Univers af Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow af Also at Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Physics, M.V. Lomonosov Moscow State University, Moscow, Russia af Also at Faculty of Physics, M.V. Lomonosov Moscow State Univ ag Also at National Research Nuclear University MEPhI, Moscow, Russia. ag Also at National Research Nuclear University MEPhI, Moscow, Russia. ah Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 f Also at Department of Physics, California State University, Fresno, CA, United States of America. f Also at Department of Physics, California State University, Fresno, CA, United States of America. g Also at Tomsk State University, Tomsk, Russia. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. h Also at CPPM, Aix-Marseille Université and CNRS/IN2P3 i Also at Università di Napoli Parthenope, Napoli, Italy. i Also at Università di Napoli Parthenope, Napoli, Italy. j Also at Institute of Particle Physics (IPP), Canada. k Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. k Also at Department of Physics, St. Petersburg State Polytechn s, St. Petersburg State Polytechnical University, St. Petersburg, Russ k Also at Department of Physics, St. Petersburg State Polytechnical University, St. Petersburg, Russia. l Also at Chinese University of Hong Kong, China. y g g m Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece. m Also at Department of Financial and Management Engineering, University of the Aegean, Chios, Gree m Also at Department of Financial and Manageme n Also at Louisiana Tech University, Ruston, LA, United States of America. n Also at Louisiana Tech University, Ruston, LA, United States of America. o Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spa o Also at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. p Also at Department of Physics, The University of Texas at Austin, Austin, TX, United States of America. p Also at Department of Physics, The University of Texas at Austin, Austin, TX, Uni q Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. q Also at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. q Also at Institute of Theoretical Physics, Ilia State University, Tb r Also at CERN, Geneva, Switzerland. s Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. s Also at Ochadai Academic Production, Ochanomizu University, Tokyo, Japan. t Also at Manhattan College, New York, NY, United States of Am u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. u Also at Institute of Physics, Academia Sinica, Taipei, Taiwan v Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France v Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. 172 Waseda University, Tokyo, Japan 173 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel 173 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel artment of Physics, University of Wisconsin, Madison, WI, United States Department of Physics, University of Wisconsin, Madison, WI, United Sta 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 175 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 176 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 176 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 177 Department of Physics, Yale University, New Haven, CT, United St 77 Department of Physics, Yale University, New Haven, CT, United States 178 Yerevan Physics Institute, Yerevan, Armenia 178 Yerevan Physics Institute, Yerevan, Armenia Yerevan Physics Institute, Yerevan, Armenia 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France y 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France 179 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, Franc a Also at Department of Physics, King’s College London, London, United Kingdom. a Also at Department of Physics, King’s College London, London, United Kingdom. b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. b Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. c Also at Novosibirsk State University, Novosibirsk, Russia. c Also at Novosibirsk State University, Novosibirsk, Russia. d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom. d Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom. e Also at TRIUMF, Vancouver, BC, Canada. 217 ATLAS Collaboration / Physics Letters B 740 (2015) 199–217 ah Also at Institute for Particle and Nuclear Physics, Wigner Resea ah Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, B ai Also at Department of Physics, Oxford University, Oxford, United Kingdom. ai Also at Department of Physics, Oxford University, Oxford, United Kingdom. j Also at Department of Physics, Nanjing University, Jiangsu, China ak Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. ak Also at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United States of America. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United am Also at Discipline of Physics, University of KwaZulu-Natal, Durban, South Africa p y , y , , an Also at University of Malaya Department of Physics Kuala Lumpur Malaysia ∗Deceased.
https://openalex.org/W3172021204	https://diposit.ub.edu/dspace/bitstream/2445/182942/1/pkab056.pdf	English	null	Association between Smoking and Molecular Subtypes of Colorectal Cancer	JNCI cancer spectrum	2,021	cc-by	10,958	Cancer Louis, MO, USA; 15Division of Gastroenterology, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA; 16Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 17Clinical and Translational Epidemiology Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA; 18Division of Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany; 19Genetic Tumour Epidemiology Group, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 20Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada; 21Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 22Cancer Epidemiology & Intelligence Division, Cancer Council Victoria, Melbourne, Australia; 23Melbourne School of Population and Global Health, The University of Melbourne, Melbourne, Australia; 24Nutrition and Metabolism Section, International Agency for Research on Cancer, World Health Organization, Lyon, France; 25Oncology Data Analytics Program, Catalan Institute of Oncology- IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain; 26Department of Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 27Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA; 28Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA; 29Department of Medicine and Epidemiology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 30Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT, USA; 31Department of Nutrition, Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA; 32Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; 33Departments of Cancer Biology and Genetics and Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; 34Department of Radiation Sciences, Oncology Unit, Umea˚ University, Umea˚, Sweden and 35Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland & Labrador, Canada Correspondence to: Xiaoliang Wang, PhD, MPH, Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, M4-B402, Seattle, WA 98109, USA (e-mail: xwang23@fredhutch.org). 1Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Department of Epidemiology, University of Washington, Seattle, WA, USA; 3Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 5Department of Preventive Oncology, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg, Germany; 6German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany; 7Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Parkville, Victoria, Australia; 8Department of Clinical Pathology, Colorectal Oncogenomics Group, The University of Melbourne, Parkville, Victoria, Australia; 9University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, Parkville, Victoria, Australia; 10Genomic Medicine and Family Cancer Clinic, Royal Melbourne Hospital, Melbourne, Victoria, Australia; 11Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA; 12Division of Public Health Sciences, Department of Surgery, Washington University School of Medicine, St Louis, MO, USA; 13Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO, USA; 14Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA; 15Division of Gastroenterology, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA; 16Channing Division of Network Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 17Clinical and Translational Epidemiology Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, MA, USA; 18Division of Cancer Epidemiology, German Cancer Research Center, Heidelberg, Germany; 19Genetic Tumour Epidemiology Group, University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 20Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada; 21Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; 22Cancer Epidemiology & Intelligence Division, Cancer Council Victoria, Melbourne, Australia; 23Melbourne School of Population and Global Health, The University of Melbourne, Melbourne, Australia; 24Nutrition and Metabolism Section, International Agency for Research on Cancer, World Health Organization, Lyon, France; 25Oncology Data Analytics Program, Catalan Institute of Oncology- IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain; 26Department of Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 27Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA; 28Division of Research, Kaiser Permanente Northern California, Oakland, CA, USA; 29Department of Medicine and Epidemiology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 30Department of Internal Medicine, University of Utah Health Sciences Center, Salt Lake City, UT, USA; 31Department of Nutrition, Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA; 32Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA; 33Departments of Cancer Biology and Genetics and Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA; 34Department of Radiation Sciences, Oncology Unit, Umea˚ University, Umea˚, Sweden and 35Discipline of Genetics, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland & Labrador, Canada JNCI Cancer Spectrum (2021) 5(4): pkab056 JNCI Cancer Spectrum (2021) 5(4): pkab056 doi: 10.1093/jncics/pkab056 First published online 14 June 2021 Article Cancer Xiaoliang Wang , PhD, MPH,1,2, Efrat Amitay , PhD, MPH,3 Tabitha A. Harrison , MPH,1 Barbara L. Banbury, PhD,1 Sonja I. Berndt, PharmD, PhD,4 Hermann Brenner , MD, MPH,3,5,6 Daniel D. Buchanan , PhD,7,8,9,10 Peter T. Campbell , PhD,11 Yin Cao , ScD, MPH,12,13,14 Andrew T. Chan, MD, MPH,15,16,17 Jenny Chang-Claude , DrscHum,18,19 Steven J. Gallinger, MSc, MD, Frcsc,20 Marios Giannakis, MD, PhD,21 Graham G. Giles, PhD,22,23 Marc J. Gunter , PhD,24 John L. Hopper, PhD,7 Mark A. Jenkins , PhD,7 Yi Lin, MPH,1 Victor Moreno , PhD,25 Reiko Nishihara, PhD,21 Polly A. Newcomb, PhD, MPH,1,2 Shuji Ogino , MD, PhD,26,27 Amanda I. Phipps, PhD, MPH,1,2 Lori C. Sakoda , PhD, MPH,1,28 Robert E. Schoen , MD, MPH,29 Martha L. Slattery, PhD, MPH,30 Mingyang Song , MD, ScD,15,17,31 Wei Sun , PhD,1 Steven N. Thibodeau, PhD,32 Amanda E. Toland , PhD,33 Bethany Van Guelpen, PhD,34 Michael O. Woods , PhD,35 Li Hsu, PhD,1 Michael Hoffmeister , PhD,3 Ulrike Peters, PhD, MPH1,2 1Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 2Department of Epidemiology, University of Washington, Seattle, WA, USA; 3Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 5Department of Preventive Oncology, German Cancer Research Center (DKFZ), National Center for Tumor Diseases (NCT), Heidelberg, Germany; 6German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany; 7Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, The University of Melbourne, Parkville, Victoria, Australia; 8Department of Clinical Pathology, Colorectal Oncogenomics Group, The University of Melbourne, Parkville, Victoria, Australia; 9University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, Parkville, Victoria, Australia; 10Genomic Medicine and Family Cancer Clinic, Royal Melbourne Hospital, Melbourne, Victoria, Australia; 11Epidemiology Research Program, American Cancer Society, Atlanta, GA, USA; 12Division of Public Health Sciences, Department of Surgery, Washington University School of Medicine, St Louis, MO, USA; 13Alvin J. Siteman Cancer Center at Barnes-Jewish Hospital and Washington University School of Medicine, St. Louis, MO, USA; 14Division of Gastroenterology, Department of Medicine, Washington University School of Medicine, St. Received: 31 December 2020; Revised: 25 March 2021; Accepted: 30 April 2021 © The Author(s) 2021. Published by Oxford University Press. ( ) y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ng Wang, PhD, MPH, Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, M4-B402, Seattle, WA 98109 dhutch.org). Assessment and Harmonization of Tumor Marker Data Cigarette smoking has been established as a risk factor for CRC (10,11). Meta-analysis showed that current smokers had a 17% higher risk of developing CRC and 40% higher risk of CRC mortality than never-smokers (11). Recent evidence suggests that the association between smoking, including current smok- ing status, cumulative pack-years, duration of smoking or ces- sation periods, and CRC risk may differ by molecular characteristics. Several studies have found that smoking status has stronger associations with higher risks of MSI-high, CIMP- positive, or BRAF-mutated colorectal tumors but is less pro- nounced among MSI-low or microsatellite stable, CIMP- negative, or BRAF–wild-type CRC (12–19). In addition, heavier smoking was found to be associated with an increased risk for KRAS–wild-type CRC but not KRAS-mutated tumors (20,21). However, 2 studies found no statistically significant difference in the association between smoking and CRC risk by KRAS mu- tation (15,22). Recent meta-analyses showed a statistically sig- nificant positive correlation between ever-smoking and BRAF mutation, MSI high, and CIMP positivity in CRC (23,24). Details on data collection and harmonization of tumor marker data were summarized and published previously (25–27). Briefly, testing for MSI, BRAF gene mutations, KRAS gene muta- tions, and CIMP status was conducted previously by each study and according to individual study protocols. To harmonize markers across all studies, we created 2 categories for each marker for downstream analyses. In instances where studies categorized as MSI high (MSI-H), MSI low (MSI-L), and microsat- ellite stable (MSS), we collapsed MSI-L and MSS into an MSI-L/ MSS category. In instances where studies categorized as CIMP high, CIMP low, and CIMP negative, we collapsed CIMP-low and CIMP-high into the CIMP-positive (CIMPþ) category. We in- cluded any mutation identified by a study for BRAF and KRAS genes. Additionally, we combined markers to create subtype classi- fications: subtypes 1-5 were created according to JASS classifica- tion (28), and type 6-16 were numbered consecutively by the status of MSI, CIMP, BRAF, and KRAS (summarized in Figure 1). Only cases with all markers assessed are included in combined molecular classifications and corresponding analysis. However, most studies assessed CRC molecular subtypes only by individual marker status. In this study, we aimed to comprehensively assess the association between smoking and CRC risk both by individual markers (MSI status, CIMP status, KRAS and BRAF mutations) and by combinations of all 4 markers, using pooled individual-level data from a large consortium. Smoking and Other Exposure Data Data collection and harmonization of epidemiologic data have been described elsewhere (29,30). Briefly, demographic and en- vironmental risk factors were self-reported at in-person inter- views or via structured self-administered questionnaires. Data were collected at study entry, blood draw, or 1 to 2 years prior to sample ascertainment. A multistep, iterative data harmoniza- tion procedure was applied, and multiple quality-control checks were performed, reconciling each study’s unique protocols. Variables were combined into a single dataset with common definitions, standardized coding, and standardized permissible values. Abstract The associations differed statistically signiﬁcantly between all molecular subtypes, which was the most statistically signiﬁcant for CIMP and BRAF. Compared with never-smokers, smokers in the fourth quartile of pack-years had a 90% higher risk of CIMP-positive CRC (odds ratio ¼ 1.90, 95% conﬁdence interval ¼ 1.60 to 2.26) but only 35% higher risk for CIMP-negative CRC (odds ratio ¼ 1.35, 95% conﬁdence inter- val ¼ 1.22 to 1.49; Pdifference ¼ 2.1 x 10-6). The association was also stronger in tumors that were CIMP positive, MSI high, or KRAS wild type when combined (Pdifference < .001). Conclusion: Smoking was associated with differential risk of CRC subtypes deﬁned by molecular characteristics. Heavier smokers had particularly higher risk of CRC subtypes that were CIMP positive and MSI high in combination, suggesting that smoking may be involved in the development of colorectal tumors via the serrated pathway. Colorectal cancer (CRC) is one of the most common and fatal cancers (1). In the United States, there were an estimated 145 600 new cases and 51 020 deaths in 2019 (2). In addition, CRC is a disease with considerable genetic and molecular heterogene- ity (3). Molecular classification of CRC using clinically informa- tive genetic and epigenetic features has potential prognostic (4) and treatment implications (5). Mutations in the KRAS gene have been shown to promote the growth of colorectal adeno- mas in 30%-40% of sporadic CRC (6). Microsatellite instability (MSI), characterized by frequent alterations in tandemly re- peated DNA sequences, has been reported to occur in 10%-15% of CRC and associated with a favorable prognosis (7,8). In addi- tion, many MSI-high CRC also present the CpG island methyla- tor phenotype (CIMP) or BRAF c.1799T>A (p.V600E) mutations (9). and the Colon Cancer Family Registry with available tumor marker and smoking data. Participating studies were previously described and summarized in Table 1 (25,26). All participants provided written informed consent, and each study was ap- proved by the relevant research ethics committee or institu- tional review board. CRC cases were confirmed by medical record, pathology re- port, or death certificate by study protocol. Controls were indi- viduals without history of CRC at the time of case selection and were selected per study-specific matching criteria. Participants of non-European ancestry were excluded from the analysis be- cause of small sample size. Abstract Background: Smoking is associated with colorectal cancer (CRC) risk. Previous studies suggested this association may be restricted to certain molecular subtypes of CRC, but large-scale comprehensive analysis is lacking. Methods: A total of 9789 CRC cases and 11 231 controls of European ancestry from 11 observational studies were included. We harmonized smoking variables across studies and derived sex study–speciﬁc quartiles of pack-years of smoking for analysis. Four somatic colorec- tal tumor markers were assessed individually and in combination, including BRAF mutation, KRAS mutation, CpG island methylator phenotype (CIMP), and microsatellite instability (MSI) status. A multinomial logistic regression analysis was used ( ) y y This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 of 10 2 of 10 \| JNCI Cancer Spectrum, 2021, Vol. 5, No. 4 to assess the association between smoking and risk of CRC subtypes by molecular characteristics, adjusting for age, sex, and study. All statistical tests were 2-sided and adjusted for Bonferroni correction. Results: Heavier smoking was associated with higher risk of CRC overall and stratiﬁed by individual markers (Ptrend < .001). The associations differed statistically signiﬁcantly between all molecular subtypes, which was the most statistically signiﬁcant for CIMP and BRAF. Compared with never-smokers, smokers in the fourth quartile of pack-years had a 90% higher risk of CIMP-positive CRC (odds ratio ¼ 1.90, 95% conﬁdence interval ¼ 1.60 to 2.26) but only 35% higher risk for CIMP-negative CRC (odds ratio ¼ 1.35, 95% conﬁdence inter- val ¼ 1.22 to 1.49; Pdifference ¼ 2.1 x 10-6). The association was also stronger in tumors that were CIMP positive, MSI high, or KRAS wild type when combined (Pdifference < .001). Conclusion: Smoking was associated with differential risk of CRC subtypes deﬁned by molecular characteristics. Heavier smokers had particularly higher risk of CRC subtypes that were CIMP positive and MSI high in combination, suggesting that smoking may be involved in the development of colorectal tumors via the serrated pathway. to assess the association between smoking and risk of CRC subtypes by molecular characteristics, adjusting for age, sex, and study. All statistical tests were 2-sided and adjusted for Bonferroni correction. Results: Heavier smoking was associated with higher risk of CRC overall and stratiﬁed by individual markers (Ptrend < .001). Study Participants This study consisted of 9789 patients diagnosed with CRC and 11 231 controls from 11 observational studies within the Genetics and Epidemiology of Colorectal Cancer Consortium X. Wang et al. \| 3 of 10 Table 1. Demographic characteristics of participating studiesa Study Country (enrollment year) Study design Cases Controls Mean (SD) age, y Female, No. (%) No. % never-smokers No. % never-smokers CCFR United States, Canada, Australia (1996-2015) Case-control 2636 45.5 2083 47.2 54.0 (11.7) 2477 (52.5) CPSII United States (1992- 1999) Cohort 858 40.2 969 45.6 74.3 (6.6) 912 (49.9) DACHS Germany (2003-2016) Case-control 2322 46.7 3428 50.0 68.7 (10.6) 2291 (39.8) DALS United States (1990- 1993) Case-control 1096 42.7 1163 49.4 65.4 (9.7) 1017 (45.0) EDRN United States (2012- 2013) Case-control 195 56.9 349 73.1 60.5 (11.4) 262 (48.2) EPIC Sweden (1992-1998) Case-control 115 49.6 318 54.1 67.2 (7.7) 206 (47.6) HPFS United States (1986- 2012) Cohort 584 37.5 433 46.2 71.0 (8.9) 0 (0) MCCS Australia (1990-1994) Cohort 490 51.2 674 53.0 68.9 (8.8) 553 (47.5) NFCCR Newfoundland (2000- 2004) Case-control 477 28.3 458 37.6 59.9 (9.1) 376 (40.2) NHS United States (1976- 2013) Cohort 783 40.0 1071 44.1 67.7 (8.3) 1854 (100) NSHDS Sweden (1995-2005) Case-control 233 38.2 285 45.6 62.6 (8.2) 250 (48.3) Total 9789 43.6 11 231 48.7 64.8 (11.9) 10 198 (48.5) aCCFR ¼ Colorectal Cancer Family Registry; CPSII ¼ Cancer Society Cancer Prevention Study II; DACHS ¼ Darmkrebs: Chancen der Verhu¨ tung durch Screening Study; DALS ¼ Diet, Activity and Lifestyle Study; HPFS ¼ Health Professionals Follow-up Study; Kentucky ¼ the Kentucky case-control study; MCCS ¼ Melbourne Case-Control Study (in Melbourne Collaborative Cohort); NFCCR ¼ NewFoundland Case-Control Study; NHS ¼ Nurses’ Health Study; NSHDS ¼ the Northern Sweden Health and Disease Study. Table 1. Demographic characteristics of participating studiesa aCCFR ¼ Colorectal Cancer Family Registry; CPSII ¼ Cancer Society Cancer Prevention Study II; DACHS ¼ Darmkrebs: Chancen der Verhu¨ tung durch Screening Study; DALS ¼ Diet, Activity and Lifestyle Study; HPFS ¼ Health Professionals Follow-up Study; Kentucky ¼ the Kentucky case-control study; MCCS ¼ Melbourne Case-Control Study (in Melbourne Collaborative Cohort); NFCCR ¼ NewFoundland Case-Control Study; NHS ¼ Nurses’ Health Study; NSHDS ¼ the Northern Sweden Health and Disease Study. Smoking status was categorized into never-, former, and current smokers at baseline in each study. In addition, sex study–specific quartiles were created for smoking pack-years among ever-smokers. Study Participants Never-smokers were used as a reference group in all analyses. years). Age at diagnosis, sex, and study were adjusted as covari- ates in the models. According to a priori knowledge about CRC risk factors that have been associated with smoking, we further simultaneously adjusted for BMI, use of NSAIDs, history of screening, alcohol intake, and physical activity as sensitivity analyses. Demographic variables included age and sex. Age was de- fined as age at diagnosis for cases and age at selection for con- trols. Other lifestyle covariates included body mass index (BMI; defined as weight[kg]/height[cm2]), regular use of non-steroidal anti-inflammatory drugs (NSAIDs), history of colorectal screen- ing, alcohol intake, and physical activity. In analysis of combined marker status, CRC subtypes with at least 50 cases were assessed in their association with smoking. Similarly, we used multinomial logistic regression models, adjusting for age, sex, and study. The subtype with MSI-L/MSS, CIMP-, BRAF-wt and KRAS-wt was used as a reference group in the case-only analysis (Bonferroni corrected Pdifference threshold: .05/10 ¼ 5.0 x 10-3). Statistical Analyses Exploratory analysis of smoking–CRC association stratified by sex, colonic locations, and study design was also conducted for both individual and combined markers. All analyses were performed using R version 3.5.1 . All statistical tests were 2-sided. The distributions of individual tumor markers were summarized among CRC cases, and Pearson correlation test was used to assess the correlation among markers. We used multinomial logistic regression mod- els to estimate odds ratios (OR) and 95% confidence intervals (CIs) for the association of smoking with the risk of CRC sub- types. To account for multiple testing in case-control analysis, we used a Bonferroni corrected P value threshold of .05/16 (4 markers x 2 status x 2 smoking comparisons) ¼ 3.1 x 10-3 for cat- egorical smoking status and .05/8 (4 markers x 2 status x 1 group linear comparison) ¼ .006 for smoking pack-years. We used lo- gistic regression models to assess the differences in the associa- tions between smoking and molecularly mutated subtypes (BRAF mutated [mut], KRAS-mut, MSI-H, or CIMPþ), as compared with wild-type subtypes (BRAF-wild type [wt], KRAS-wt, MSI-L/ MSS, or CIMP negative [CIMP-], respectively) among cases only (Bonferroni corrected Pdifference threshold: .05/8 ¼ 6.3 x 10-3 for categorical smoking status and .05/4 ¼ 0.013 for smoking pack- Results Overall Distributions of Tumor Markers Overall Distributions of Tumor Markers Among the 21 020 participants, there was a larger proportion of never-smokers among controls (48.7%) than among CRC cases (43.6%; Table 1). Among the 9789 CRC cases with tumor data available, 11% of the tumors were BRAF-mut, 34% KRAS-mut, 15% MSI-H, and 18% CIMPþ (Table 2). In addition, the MSI-H, CIMPþ, and BRAF-mut subtypes were highly positively corre- lated with each other (Pearson correlation > 0.4; frequency pre- sented in Supplementary Table 1, available online). KRAS-mut tumors were inversely correlated with other markers (Pearson correlation < 0.1). 4 of 10 \| JNCI Cancer Spectrum, 2021, Vol. 5, No. 4 Association Between Smoking and Individual Marker Subtypes mut, KRAS-wt, MSI-H, and CIMPþ CRC subtypes among current smokers only but not among former smokers, after Bonferroni Figure 1. Associations between former and current smokers and risk of CRC subtypes deﬁned by combined marker status. Two-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 11 231) and case-only analysis (Pdifference). A Bonferroni corrected P value threshold of 5.0 x 10-3 was used for both case-control and case-only analyses. Error bars represent the 95% conﬁdence intervals (CIs). CIMP ¼ CpG island methylation phenotype; CRC ¼ colorectal cancer; MSI ¼ microsatellite instability; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. Downloaded from https://academic.oup.com/jncics/article/5/4/pkab056/6298599 by guest on 03 February 2022 academic.oup.com/jncics/article/5/4/pkab056/6298599 by guest on 03 February 2022 Figure 1. Associations between former and current smokers and risk of CRC subtypes deﬁned by combined marker status. Two-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 11 231) and case-only analysis (Pdifference). A Bonferroni corrected P value threshold of 5.0 x 10-3 was used for both case-control and case-only analyses. Error bars represent the 95% conﬁdence intervals (CIs). CIMP ¼ CpG island methylation phenotype; CRC ¼ colorectal cancer; MSI ¼ microsatellite instability; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. Figure 1. Associations between former and current smokers and risk of CRC subtypes deﬁned by combined marker status. Two-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 11 231) and case-only analysis (Pdifference). A Bonferroni corrected P value threshold of 5.0 x 10-3 was used for both case-control and case-only analyses. Error bars represent the 95% conﬁdence intervals (CIs). Overall Distributions of Tumor Markers CIMP ¼ CpG island methylation phenotype; CRC ¼ colorectal cancer; MSI ¼ microsatellite instability; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. Association Between Smoking and Individual Marker Subtypes Association Between Smoking and Individual Marker Subtypes mut, KRAS-wt, MSI-H, and CIMPþ CRC subtypes among current smokers only but not among former smokers, after Bonferroni correction (Pdifference < 6.3 x 10-3; Table 2). In case-control analysis, individuals who smoked were associ- ated with a higher risk of CRC overall and stratified by individ- ual marker subtypes except for KRAS-mut tumors (P < 3.1 x 10-3; Table 2). Associations were stronger among current smokers. For instance, current smoking was associated with a 2-fold risk in MSI-H CRC (OR ¼ 2.01, 95% CI ¼ 1.68 to 2.40), whereas former smoking was associated with higher risk in MSI-H CRC (OR ¼ 1.27, 95% CI ¼ 1.11 to 1.46), compared with never-smokers. In case-only analyses, the association between smoking status and CRC risk was statistically significantly stronger for BRAF- We further assessed the dose-response relationship between smoking and CRC subtypes. Compared with non- smokers, higher pack-years of smoking were associated with higher risk of CRC among all subtypes in case-control analysis (P < 1.6 x 10-3; Ptrend < .001; Table 3). In case-only analysis, the association between pack-years and molecular subtypes was statistically significantly stronger for BRAF-mut, CIMPþ, and MSI-H subtypes compared with wild-type or negative CRC cases after Bonferroni correction (Pdifference < 6.3 x 10-3; Table 3). The largest difference in case-control risk estimates were seen for X. Wang et al. \| 5 of 10 Table 2. Association between former or current smoking and individual molecular subtypes of colorectal cancer, compared with never- smokers Table 2. Association between former or current smoking and individual molecular subtypes of colorectal cancer, compared with never- smokers Marker and status No. Association Between Smoking and Individual Marker Subtypes of cases Never-smokers Smokers Former smokers Current smokers OR (95% CI) OR (95% CI) Pa Pdifference b OR (95% CI) Pa Pdifference b BRAF mut 1008 1.00 (Referent) 1.43 (1.23 to 1.66) 3.9 x 10-6 1.89 (1.54 to 2.33) 1.5 x 10-9 wt 7695 1.00 (Referent) 1.17 (1.09 to 1.25) 7.5 x 10-6 .03 1.28 (1.16 to 1.41) 1.7 x 10-6 1.0 x 10-4 KRAS mut 2484 1.00 (Referent) 1.16 (1.05 to 1.28) 3.6 x 10-3 1.11 (0.95 to 1.29) .18 wt 4892 1.00 (Referent) 1.21 (1.12 to 1.31) 1.8 x 10-6 .36 1.40 (1.25 to 1.56) 5.0 x 10-9 3.3 x 10-3 CIMP þ 1382 1.00 (Referent) 1.37 (1.20 to 1.56) 2.1 x 10-6 1.82 (1.52 to 2.18) 8.6 x 10-11 6232 1.00 (Referent) 1.16 (1.08 to 1.25) 5.2 x 10-5 .03 1.22 (1.09 to 1.36) 3.7 x 10-4 5.6 x 10-7 MSI MSI-H 1334 1.00 (Referent) 1.27 (1.11 to 1.46) 5.4 x 10-4 2.01 (1.68 to 2.40) 1.1 x 10-14 MSI-L/MSS 7828 1.00 (Referent) 1.18 (1.10 to 1.26) 2.1 x 10-6 .43 1.27 (1.15 to 1.40) 3.8 x 10-6 1.2 x 10-6 aTwo-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 11 231). A Bonferroni corrected P value threshold of 6.3 x 10-3 was used for case-control analyses. CI ¼ conﬁdence interval; CIMP ¼ CpG island methylation phenotype; MSI ¼ microsatellite instability; MSI-H ¼ MSI-high; MSI-L ¼ MSI- low; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. bTwo-sided Wald test was used to calculate the P values from the case-only analysis. A Bonferroni corrected P value threshold of 3.1 x 10-3 was used for case-only analyses. aTwo-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 11 231). A Bonferroni corrected P value threshold of 6.3 x 10-3 was used for case-control analyses. CI ¼ conﬁdence interval; CIMP ¼ CpG island methylation phenotype; MSI ¼ microsatellite instability; MSI-H ¼ MSI-high; MSI-L ¼ MSI- low; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. bTwo-sided Wald test was used to calculate the P values from the case-only analysis. A Bonferroni corrected P value threshold of 3.1 x 10-3 was used for case-only analyses. Exploratory Stratified Analysis When stratified by colonic location, BRAF-mut, CIMPþ, and MSI- H status were more frequent in proximal colon cancer, com- pared with distal colon or rectal cancer (Supplementary Table 2, available online). For proximal colon, current smoking and higher pack-years were associated with higher risk of BRAF- mut, KRAS-wt, CIMPþ, and MSI-H tumors (Supplementary Tables 3 and 4, available online) and higher risk of type 14 (Supplementary Figures 2 and 3, available online). Although sample sizes were limited, a similar trend of smoking–CRC asso- ciation was observed in distal colon and rectal cancer. Current smoking was associated with higher risk of type 8 distal colon cancer (only CIMPþ), but this did not remain statistically signifi- cant after Bonferroni correction (Supplementary Figure 2, avail- able online). Interaction analysis between smoking and colonic location was not statistically significant. Association Between Smoking and Individual Marker Subtypes (only MSI-H; Pdifference ¼ 4.8 x 10-3) and type 14 CRC (Pdifference ¼ 7.3 x 10-8) compared with never-smokers after Bonferroni cor- rection (Figure 1, B). BRAF-mut and CIMPþ CRC. Participants in the highest quartile of smoking pack-years had nearly a 2-fold risk for CRC if they had BRAF-mut (OR ¼ 1.92, 95% CI ¼ 1.58 to 2.33) or CIMPþ tumors (OR ¼ 1.90, 95% CI ¼ 1.60 to 2.26), compared with non- smokers, respectively. In comparison, the risk of BRAF-wt or CIMP- CRC was increased by only 36.9% (Pdifference ¼ 2.7 x 10-6) and 34.8% (Pdifference ¼ 2.4 x 10-6) among heaviest smokers, re- spectively. There was no statistically significant difference after Bonferroni correction in the associations of pack-years of smok- ing with KRAS-mut or KRAS-wt CRC (Pdifference ¼ 9.6 x 10-3). Sensitivity analysis that additionally adjusted for BMI, family history of CRC, CRC screening history, NSAID use, or alcohol in- take did not meaningfully change our conclusions (data not shown). Higher smoking pack-years was also statistically signifi- cantly associated with higher risk of 4 CRC subtypes after Bonferroni correction (types 4, 3, 14, and 1; Figure 2). Similarly, the association between smoking pack-years and CRC risk was strongest in type 14 (OR per quartile ¼ 1.37, 95% CI ¼ 1.22 to 1.53). When compared with type 4 in case-only analyses, higher smoking pack-years was associated only with higher risk of type 14 (Pdifference ¼ 1.8 x 10-5) and type 1 CRC (MSI-H, CIMPþ, BRAF-mut, and KRAS-wt; Pdifference ¼ 2.5 x 10-4). Association Between Smoking and Combined Marker Subtypes Overall distribution of CRC cases by smoking status and com- bined marker subtypes are summarized in Supplementary Figure 1 (available online). Of 16 possible combined CRC sub- types, 10 had 50 or more cases and were included in the analy- sis. Among them, former smoking was statistically significantly associated with higher risk of 4 CRC subtypes after Bonferroni correction (types 4, 3, 14, 1; Figure 1, A) compared with never- smokers. Comparatively, current smoking was associated with higher risk of CRC for 6 subtypes, but only 3 remained statisti- cally significant after Bonferroni correction (types 5, 14, 1; Figure 1, B). The strongest association for both former and current smoking was observed in type 14 (MSI-H, CIMPþ, BRAF-wt, and KRAS-wt), where former and current smoking was associated with 87% and 264% higher risk of type 14 CRC compared with never-smokers, respectively. Using type 4 (all markers wild type/negative) as reference in case-only analyses, we observed no statistically significant differences between risks of CRC sub- types among former smokers. However, cases with current smoking were statistically significantly more likely to be type 5 When stratified by sex, similar trends of associations were observed, although the risk estimates varied slightly between sexes. For instance, current smoking was associated with a 90% and 66% increase in CIMPþ tumors among females and males, respectively (Supplementary Tables 5 and 6, available online). In addition, the dose–response association was stronger for BRAF- mut and CIMPþ CRC among females. In combined marker 6 of 10 \| JNCI Cancer Spectrum, 2021, Vol. 5, No. 4 Table 3. Association between quartiles of smoking pack-years and individual molecular subtypes of colorectal cancer compared with nonsmokers Marker and status No. Discussion In this large study, we found that smoking was associated with higher risk of all molecular CRC subtypes, and the association was statistically significantly stronger for BRAF-mut, MSI-H, or CIMPþ CRC cases. We also found that smoking had a statisti- cally significantly stronger association with CRC subtypes that display MSI-H and CIMPþ status. Smoking is a well-established carcinogen for CRC (35). Meta- analyses of epidemiological studies have consistently found a statistically significant association, and dose-response relation- ships, between smoking and CRC risk (10,11,36). However, knowledge on the underlying mechanisms of smoking in CRC molecular subtypes is limited. In general, tobacco smoke con- tains a variety of toxic chemicals (37), many of which can induce DNA damage (38). Tobacco exposure has also been associated with CIMP in other cancer types, including lung (39,40), bladder (41), and head and neck cancer (42). Therefore, it is biologically plausible that smoking promotes colorectal tumor growth and progression by epigenetic alterations. In addition, the detoxifi- cation of smoking-induced carcinogens are metabolized by phase I and phase II enzymes such as CYP (P-450) family genes, which lead to the production of abnormal DNA and mutations in genes such as KRAS and BRAF (43). Our results are consistent with previous evidence that smok- ing is associated with higher risk of CRC subgroups classified by individual marker status, including similar findings from 1 of the participating studies (19). Current smoking was associated with almost 2-fold higher risk of CRC with MSI-H, CIMPþ, or BRAF-mut compared with never smoking (12). A study in 2 pro- spective cohorts found that a longer cessation period was asso- ciated with MSI-H and CIMPþ CRC, but not with MSS or CIMP- CRC, compared with current smokers (13). In addition, longer duration of smoking was found to be associated with increased risk of MSI-H CRC (14). Several cohort and case-control studies also found that higher smoking pack-years were associated with higher risks of CRC with MSI-H, CIMPþ, or BRAF-mut, com- pared with wild-type or negative CRC subtypes (12–14,17). In a population-based, case-control study, current cigarette smoking and higher pack-years were found to be statistically signifi- cantly associated with higher risk of MSI-H than MSS colon tumors (16). Association Between Smoking and Combined Marker Subtypes bTwo-sided Wald test was used to calculate the P values from the case-only analysis. A Bonferroni corrected P value threshold of .013 for case-only analyses. analysis, current smoking was most strongly associated with type 14 CRC in both sexes (Supplementary Figures 4 and 5, avail- able online). Interaction analysis between smoking and sex was not statistically significant. Further stratification by sex in prox- imal colon tumors did not suggest statistically significant differ- ence (data not shown). In addition, similar trends were also observed when stratified by study design (Supplementary Tables 7 and 8 and Supplementary Figures 6 and 7, available online). statistically significant (6,20,21). In contrast, a case-cohort study (648 cases) in the Netherlands observed a non–statistically sig- nificant increase in KRAS-wt CRC risk among former smokers but not among current smokers (20). Individual markers were not independent from each other. CIMPþ CRC tumors tend to have a high frequency of MSI and BRAF mutation (9,31–34). However, few studies have assessed the combined subtypes of CRC. A prospective cohort study found that smoking 20 or more cigarettes per day was associ- ated with higher risks of MSI-L/MSS and CIMPþ CRC, regardless of BRAF mutation status (17). No statistically significant associa- tion was found in CIMP- tumors. Another analysis in 2 prospec- tive cohorts also found that smoking 40 or more pack-years of cigarettes was associated with higher risk of CIMPþ and MSI-H CRC compared with never-smokers (13). Consistent with previ- ous findings, we found that higher smoking pack-years were statistically significantly associated with higher risk of CIMPþ and MSI-H CRC, regardless of BRAF mutation status. Association Between Smoking and Combined Marker Subtypes of cases Smoking, pack-years Ptrend a Pdifference b Quartile 1 OR (95% CI) Quartile 2 OR (95% CI) Quartile 3 OR (95% CI) Quartile 4 OR (95% CI) BRAF mut 921 0.81 (0.62 to 1.06) 1.49 (1.20 to 1.84) 1.69 (1.37 to 2.08) 1.92 (1.58 to 2.33) 1.0 x 10-14 wt 7233 0.99 (0.89 to 1.11) 1.21 (1.09 to 1.34) 1.16 (1.05 to 1.29) 1.37 (1.24 to 1.51) 2.5 x 10-11 2.7 x 10-6 KRAS mut 2331 1.04 (0.89 to 1.21) 1.12 (0.96 to 1.29) 1.17 (1.01 to 1.35) 1.23 (1.07 to 1.41) 1.0 x 10-3 Wt 4547 0.93 (0.82 to 1.06) 1.28 (1.14 to 1.43) 1.22 (1.09 to 1.37) 1.48 (1.33 to 1.65) 2.1 x 10-14 9.6 x 10-3 CIMP þ 1247 1.05 (0.85 to 1.30) 1.33 (1.09 to 1.62) 1.62 (1.34 to 1.95) 1.90 (1.60 to 2.26) 8.9 x 10-16 5966 1.00 (0.89 to 1.12) 1.20 (1.08 to 1.33) 1.15 (1.03 to 1.28) 1.35 (1.22 to 1.49) 2.1 x 10-9 2.4 x 10-6 MSI MSI-H 1239 1.10 (0.90 to 1.35) 1.26 (1.04 to 1.53) 1.53 (1.28 to 1.84) 1.66 (1.39 to 1.99) 9.0 x 10-11 MSI-L/MSS 7255 0.98 (0.89 to 1.09) 1.24 (1.12 to 1.36) 1.16 (1.05 to 1.29) 1.38 (1.25 to 1.52) 7.0 x 10-12 3.9 x 10-3 aTwo-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 10 199). A Bonferroni corrected P value threshold of 6.3 x 10-3 was used for case-control analyses. CI ¼ conﬁdence interval; CIMP ¼ CpG island methylation phenotype; MSI ¼ microsatellite instability; MSI-H ¼ MSI-high; MSI-L ¼ MSI- low; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. bTwo-sided Wald test was used to calculate the P values from the case-only analysis. A Bonferroni corrected P value threshold of .013 for case-only analyses. Table 3. Association between quartiles of smoking pack-years and individual molecular subtypes of colorectal cancer compared with nonsmokers aTwo-sided Wald test was used to calculate the P values from the case-control analysis (N controls ¼ 10 199). A Bonferroni corrected P value threshold of 6.3 x 10-3 was used for case-control analyses. CI ¼ conﬁdence interval; CIMP ¼ CpG island methylation phenotype; MSI ¼ microsatellite instability; MSI-H ¼ MSI-high; MSI-L ¼ MSI- low; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. Discussion Similar to our results on KRAS mutation status, sev- eral observational studies found that smoking status and pack- years were associated only with higher risk of KRAS-wt but not KRAS-mut tumors, although the differences were not Furthermore, the CIMPþ and MSI-H tumors are more likely to arise from serrated polyp pathways, as compared with tradi- tional adenoma-carcinoma pathways. It is estimated that 10%- 20% of CRCs arise via serrated polyp-carcinoma pathway (44). DNA methylation is key to the development of this type of X. Wang et al. \| 7 of 10 Figure 2. Associations between smoking pack-years and risk of CRC subtypes deﬁned by combined marker status. Two-sided Wald test was used to calculate the P val- ues from the case-control analysis (N control ¼ 10 199) and case-only analysis (Pdifference). A Bonferroni corrected P value threshold of .005 was used for both case-con- trol and case-only analyses. Error bars represent the 95% conﬁdence intervals (CIs). CIMP ¼ CpG island methylation phenotype; CRC ¼ colorectal cancer; MSI ¼ microsatellite instability; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. Figure 2. Associations between smoking pack-years and risk of CRC subtypes deﬁned by combined marker status. Two-sided Wald test was used to calculate the P val- ues from the case-control analysis (N control ¼ 10 199) and case-only analysis (Pdifference). A Bonferroni corrected P value threshold of .005 was used for both case-con- trol and case-only analyses. Error bars represent the 95% conﬁdence intervals (CIs). CIMP ¼ CpG island methylation phenotype; CRC ¼ colorectal cancer; MSI ¼ microsatellite instability; MSS ¼ microsatellite stable; mut ¼ mutated; OR ¼ odds ratio; wt ¼ wild type. cancer (45). CIMPþ phenotype is frequently observed in precur- sor serrated lesions and colorectal polyps, ranging between 40% and 80% (46,47). MSI-H phenotype has also been observed in 20%-36% of serrated adenomas (48,49). Consistent with previous evidence, our exploratory analysis showed that BRAF-mut, CIMPþ, and MSI-H were preferentially located in the proximal colon. However, we observed similar associations with small variations, when stratified by location, suggesting that our find- ing cannot be explained by tumor location. It is also estimated that serrated adenocarcinoma has a less favorable survival than traditional adenocarcinoma (50), which could be partially be- cause of the interaction between smoking and the enrichment of BRAF mutations and CIMP expression levels. Discussion Therefore, better understanding of the risk factors of these molecular character- istics may help provide insights to the trajectory of serrated car- cinogenesis and preventive and therapeutic implications. because of recall bias. However, we observe similar trends of smoking-CRC associations when stratified by study design We also found almost identical estimates using random-effect meta-analysis across study-specific estimates of smoking–CRC associations (data not shown). In exploratory stratified analysis, we found potential variation in smoking–CRC association by sex or colonic locations. However, these exploratory results warrant further investigation in the future. Although we adjusted for several potential confounders in sensitivity analysis, we could not rule out the possibility of unmeasured confounding. Lastly, our study population was of European ancestry only. Therefore, our conclusions may not be generalizable to other race and eth- nicity groups. In conclusion, we found that heavier smoking was associ- ated with higher risk among all subtypes of CRC, particularly for those that may arise from serrated polyp pathways. These find- ings may help better understand the tumorigenesis of serrated adenomas and provide insights to targeted CRC prevention and treatment. Several features in our study provided the opportunity to systematically evaluate associations between smoking and mo- lecular subgroups of CRC. First, this is the largest study to inves- tigate these associations with sufficient statistical power for primary analysis. In addition, we combined CRC subtypes by all 4 tumor markers, providing a more comprehensive analysis for tumor characteristics. With sufficient sample size, we were the first to extend the combined subtype analysis beyond 5 previ- ously defined subtypes (28) and thus found a statistically signifi- cant association between smoking and new subtypes, suggesting a stronger impact of smoking on the serrated polyp- carcinoma pathway. Furthermore, smoking variables and other CRC risk factors were assessed and harmonized among all par- ticipating studies, which allowed us to further adjust for poten- tial confounders in sensitivity analysis. Funding NSHDS: Swedish Cancer Society; Cancer Research Foundation in Northern Sweden; Swedish Research Council; J C Kempe Memorial Fund; Faculty of Medicine, Umea˚ University, Umea˚, Sweden; and Cutting-Edge Research Grant from the County Council of V€asterbotten, Sweden. OFCCR: The Ontario Familial Colorectal Cancer Registry was supported in part by the NCI/ NIH under award U01 CA167551 and award U01/U24 CA074783 (to SG). Additional funding for the OFCCR and ARCTIC testing and genetic analysis was through and a Canadian Cancer Society CaRE (Cancer Risk Evaluation) pro- gram grant and Ontario Research Fund award GL201-043 (to BWZ), through the Canadian Institutes of Health Research award 112746 (to TJH), and through generous support from the Ontario Ministry of Research and Innovation. OSUMC: OCCPI funding was provided by Pelotonia and HNPCC fund- ing was provided by the NCI (CA16058 and CA67941). SCCFR: The Seattle Colon Cancer Family Registry was supported in part by the NCI/NIH under awards U01 CA167551, U01 CA074794 (to JDP), and awards U24 CA074794 and R01 CA076366 (to PAN). Funding NFCCR: This work was supported by an Interdisciplinary Health Research Team award from the Canadian Institutes of Health Research (CRT 43821); the National Institutes of Health, US Department of Health and Human Serivces (U01 CA74783); and National Cancer Institute of Canada grants (18223 and 18226). The authors wish to acknowledge the contribution of Alexandre Belisle and the genotyping team of the McGill University and Genome Quebec Innovation Centre, Montreal, Canada, for genotyping the Sequenom panel in the NFCCR samples. Funding was provided to Michael O. Woods by the Canadian Cancer Society Research Institute. NSHDS: Swedish Cancer Society; Cancer Research Foundation in Northern Sweden; Swedish Research Council; J C Kempe Memorial Fund; Faculty of Medicine, Umea˚ University, Umea˚, Sweden; and Cutting-Edge Research Grant from the County Council of V€asterbotten, Sweden. OFCCR: The Ontario Familial Colorectal Cancer Registry was supported in part by the NCI/ NIH under award U01 CA167551 and award U01/U24 CA074783 (to SG). Additional funding for the OFCCR and ARCTIC testing and genetic analysis was through and a Canadian Cancer Society CaRE (Cancer Risk Evaluation) pro- gram grant and Ontario Research Fund award GL201-043 (to BWZ), through the Canadian Institutes of Health Research award 112746 (to TJH), and through generous support from the Ontario Ministry of Research and Innovation. OSUMC: OCCPI funding was provided by Pelotonia and HNPCC fund- ing was provided by the NCI (CA16058 and CA67941). SCCFR: The Seattle Colon Cancer Family Registry was supported in part by the NCI/NIH under awards U01 CA167551, U01 CA074794 (to JDP) and awards U24 CA074794 and R01 NIH (award U01 CA167551). The CCFR Set-1 (Illumina 1 M/ 1M-Duo) and Set-2 (Illumina Omni1-Quad) scans were sup- ported by NIH awards U01 CA122839 and R01 CA143247 (to GC). The CCFR Set-3 (Affymetrix Axiom CORECT Set array) was supported by NIH award U19 CA148107 and R01 CA81488 (to SBG). The CCFR Set-4 (Illumina OncoArray 600 K SNP array) was supported by NIH award U19 CA148107 (to SBG) and by the CIDR, which is funded by the NIH to Johns Hopkins University, contract number HHSN268201200008I. The SCCFR Illumina HumanCytoSNP array was supported through NCI award R01 CA076366 (to PAN). Funding Additional fund- ing for the OFCCR/ARCTIC was through award GL201-043 from the Ontario Research Fund (to BWZ), award 112746 from the Canadian Institutes of Health Research (to TJH), through a Cancer Risk Evaluation (CaRE) Program grant from the Canadian Cancer Society (to SG) and through generous support from the Ontario Ministry of Research and Innovation. The content of this manuscript does not neces- sarily reflect the views or policies of the NCI, NIH, or any of the collaborating centers in the CCFR, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government, any cancer registry, or the CCFR. CPS-II: The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II (CPS-II) cohort. This study was con- ducted with institutional review board approval. DACHS: This work was supported by the German Research Council (BR 1704/6-1, BR 1704/6-3, BR 1704/6-4, CH 117/1-1, HO 5117/ 2-1, HE 5998/2-1, KL 2354/3-1, RO 2270/8-1 and BR 1704/17-1), the Interdisciplinary Research Program of the National Center for Tumor Diseases (NCT), Germany, and the German Federal Ministry of Education and Research (01KH0404, 01ER0814, 01ER0815, 01ER1505A and 01ER1505B). DALS: National Institutes of Health (R01 CA48998 to ML Slattery). EDRN: This work is funded and supported by the NCI, EDRN Grant (U01 CA 84968-06). EPIC: The coordination of EPIC is financially supported by the European Commission (DGSANCO) and the International Agency for Research on Cancer. Funding The national cohorts are supported by Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Generale de l’Education Nationale, Institut National de la Sante et de la Recherche Medicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), Federal Ministry of Education and Research (BMBF), Deutsche Krebshilfe, Deutsches Krebsforschungszentrum and Federal Ministry of Education and Research (Germany); the Hellenic Health Foundation (Greece); Associazione Italiana per la Ricerca sul Cancro-AIRCItaly and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (the Netherlands); ERC-2009-AdG 232997 and Nordforsk, Nordic Centre of Excellence programme on Food, Nutrition and Health (Norway); Health Research Fund (FIS), PI13/00061 to Granada, PI13/01162 to EPIC-Murcia, Regional Governments of Andalucıa, Asturias, Basque Country, Murcia and Navarra, ISCIII RETIC (RD06/0020) (Spain); Swedish Cancer Society, Swedish Research Council and County Councils of Ska˚ne and V€asterbotten (Sweden); Cancer Research UK (14136 to EPIC-Norfolk; C570/A16491 and C8221/A19170 to EPIC-Oxford), Medical Research Council (1000143 to EPIC-Norfolk, MR/M012190/1 to EPICOxford) (United Kingdom). Harvard cohorts (HPFS, NHS): HPFS is supported by the National Institutes of Health (P01 CA055075, UM1 CA167552, U01 CA167552, R01 CA137178, R01 CA151993, R35 CA197735, K07 CA190673, and P50 CA127003) and NHS by the National Institutes of Health (R01 CA137178, P01 CA087969, UM1 CA186107, R01 CA151993, R35 CA197735, K07CA190673, and P50 CA127003). MCCS cohort recruitment was funded by VicHealth and Cancer Council Victoria. The MCCS was further supported by Australian NHMRC grants 509348, 209057, 251553 and 504711 and by infrastructure provided by Cancer Council Victoria. Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index and the Australian Cancer Database. NFCCR: This work was supported by an Interdisciplinary Health Research Team award from the Canadian Institutes of Health Research (CRT 43821); the National Institutes of Health, US Department of Health and Human Serivces (U01 CA74783); and National Cancer Institute of Canada grants (18223 and 18226). The authors wish to acknowledge the contribution of Alexandre Belisle and the genotyping team of the McGill University and Genome Quebec Innovation Centre, Montreal, Canada, for genotyping the Sequenom panel in the NFCCR samples. Funding was provided to Michael O. Woods by the Canadian Cancer Society Research Institute. Funding Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO): National Cancer Institute (NCI), National Institutes of Health (NIH), US Department of Health and Human Services (U01 CA164930, U01 CA137088, R01 CA059045, U01 CA164930). Genotyping/Sequencing services were provided by the Center for Inherited Disease Research (CIDR) (X01- HG008596 and X-01-HG007585). CIDR is fully funded through a federal contract from the National Institutes of Health to Johns Hopkins University, contract number HHSN268201200008I. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA015704. The Colon Cancer Family Registry (CCFR, www. coloncfr.org) is supported in part by funding from the NCI, There are also limitations. We did not investigate all 16 pos- sible combinations of CRC subtypes, and the conclusions could not be inferred for the rarer subtypes. Both case-control and co- hort studies were included. There is a possibility of misclassifi- cation of smoking status, especially in case-control studies 8 of 10 \| JNCI Cancer Spectrum, 2021, Vol. 5, No. 4 8 of 10 NIH (award U01 CA167551). The CCFR Set-1 (Illumina 1 M/ 1M-Duo) and Set-2 (Illumina Omni1-Quad) scans were sup- ported by NIH awards U01 CA122839 and R01 CA143247 (to GC). The CCFR Set-3 (Affymetrix Axiom CORECT Set array) was supported by NIH award U19 CA148107 and R01 CA81488 (to SBG). The CCFR Set-4 (Illumina OncoArray 600 K SNP array) was supported by NIH award U19 CA148107 (to SBG) and by the CIDR, which is funded by the NIH to Johns Hopkins University, contract number HHSN268201200008I. The SCCFR Illumina HumanCytoSNP array was supported through NCI award R01 CA076366 (to PAN). Additional fund- ing for the OFCCR/ARCTIC was through award GL201-043 from the Ontario Research Fund (to BWZ), award 112746 from the Canadian Institutes of Health Research (to TJH), through a Cancer Risk Evaluation (CaRE) Program grant from the Canadian Cancer Society (to SG) and through generous support from the Ontario Ministry of Research and Innovation. The content of this manuscript does not neces- sarily reflect the views or policies of the NCI, NIH, or any of the collaborating centers in the CCFR, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government, any cancer registry, or the CCFR. CPS-II: The American Cancer Society funds the creation, maintenance, and updating of the Cancer Prevention Study-II (CPS-II) cohort. This study was con- ducted with institutional review board approval. Funding DACHS: This work was supported by the German Research Council (BR 1704/6-1, BR 1704/6-3, BR 1704/6-4, CH 117/1-1, HO 5117/ 2-1, HE 5998/2-1, KL 2354/3-1, RO 2270/8-1 and BR 1704/17-1), the Interdisciplinary Research Program of the National Center for Tumor Diseases (NCT), Germany, and the German Federal Ministry of Education and Research (01KH0404, 01ER0814, 01ER0815, 01ER1505A and 01ER1505B). DALS: National Institutes of Health (R01 CA48998 to ML Slattery). EDRN: This work is funded and supported by the NCI, EDRN Grant (U01 CA 84968-06). EPIC: The coordination of EPIC is financially supported by the European Commission (DGSANCO) and the International Agency for Research on Cancer. The national cohorts are supported by Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Generale de l’Education Nationale, Institut National de la Sante et de la Recherche Medicale (INSERM) (France); German Cancer Aid, German Cancer Research Center (DKFZ), Federal Ministry of Education and Research (BMBF), Deutsche Krebshilfe, Deutsches Krebsforschungszentrum and Federal Ministry of Education and Research (Germany); the Hellenic Health Foundation (Greece); Associazione Italiana per la Ricerca sul Cancro-AIRCItaly and National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (the Netherlands); ERC-2009-AdG 232997 and Nordforsk, Nordic Centre of Excellence programme on Food, Nutrition and Health (Norway); Health Research Fund (FIS), Cancer Research UK (14136 to EPIC-Norfolk; C570/A16491 and C8221/A19170 to EPIC-Oxford), Medical Research Council (1000143 to EPIC-Norfolk, MR/M012190/1 to EPICOxford) (United Kingdom). Harvard cohorts (HPFS, NHS): HPFS is supported by the National Institutes of Health (P01 CA055075, UM1 CA167552, U01 CA167552, R01 CA137178, R01 CA151993, R35 CA197735, K07 CA190673, and P50 CA127003) and NHS by the National Institutes of Health (R01 CA137178, P01 CA087969, UM1 CA186107, R01 CA151993, R35 CA197735, K07CA190673, and P50 CA127003). MCCS cohort recruitment was funded by VicHealth and Cancer Council Victoria. The MCCS was further supported by Australian NHMRC grants 509348, 209057, 251553 and 504711 and by infrastructure provided by Cancer Council Victoria. Cases and their vital status were ascertained through the Victorian Cancer Registry (VCR) and the Australian Institute of Health and Welfare (AIHW), including the National Death Index and the Australian Cancer Database. References of colorectal cancer and patient survival. Gastroenterology. 2015;148(1):7 p gy ( ) 5. Kocarnik JM, Shiovitz S, Phipps AI. Molecular phenotypes of colorectal cancer 5. Kocarnik JM, Shiovitz S, Phipps AI. Molecular phenotypes of colorectal cancer and potential clinical applications. Gastroenterol Rep (Oxf). 2015;3(4):269–276. Kocarnik JM, Shiovitz S, Phipps AI. Molecular phenotypes of colorecta J , , pp p yp and potential clinical applications. Gastroenterol Rep (Oxf). 2015;3(4):269–276. and potential clinical applications. Gastroenterol Rep (Oxf). 2015;3(4):26 6. Slattery ML, Anderson K, Curtin K, et al. Lifestyle factors and Ki-ras muta- tions in colon cancer tumors. Mutat Res. 2001;483(1-2):73–81. tions in colon cancer tumors. Mutat Res. 2001;483(1-2):73–81. 7. Jass JR. HNPCC and sporadic MSI-H colorectal cancer: a review of the morpho- logical similarities and differences. Fam Cancer. 2004;3(2):93–100. Downloaded from https://academic.oup.com/jncics/article/5/4/pkab056/6298599 by guest on 03 February 2022 8. Samowitz WS, Curtin K, Ma KN, et al. 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EDRN: We ac- knowledge all the following contributors to the development of the resource: University of Pittsburgh School of Medicine, Department of Gastroenterology, Hepatology and Nutrition and Biomedical Informatics. EPIC: Where authors are identified as personnel of the International Agency for Research on Cancer/ World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessar- ily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization. Harvard cohorts (HPFS, NHS): The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T.H. Chan School of Public Health, and those of participating registries as required. References Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study. Mutat Res. 2008;652(1):54–64. 21. 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https://openalex.org/W3112627153	http://vital.lib.tsu.ru/vital/access/services/Download/vtls:000788099/SOURCE1	Russian	null	COMPOSITION OF WEEVIL AMBER FAUNAS (CURCULIONOIDEA) IN THE EOCENE OF EUROPE	null	2,020	cc-by	981	МИНИСТЕРСТВО НАУКИ И ВЫСШЕГО ОБРАЗОВАНИЯ РОССИЙСКОЙ ФЕДЕРАЦИИ ТОМСКИЙ ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ КОНЦЕПТУАЛЬНЫЕ И ПРИКЛАДНЫЕ АСПЕКТЫ НАУЧНЫХ ИССЛЕДОВАНИЙ И ОБРАЗОВАНИЯ В ОБЛАСТИ ЗООЛОГИИ БЕСПОЗВОНОЧНЫХ Сборник статей V Международной конференции 26–28 октября 2020 г. г. Томск, Россия Томск Издательство Томского государственного университета 2020 МИНИСТЕРСТВО НАУКИ И ВЫСШЕГО ОБРАЗОВАНИЯ РОССИЙСКОЙ ФЕДЕРАЦИИ ТОМСКИЙ ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ МИНИСТЕРСТВО НАУКИ И ВЫСШЕГО ОБРАЗОВАНИЯ РОССИЙСКОЙ ФЕДЕРАЦИИ ТОМСКИЙ ГОСУДАРСТВЕННЫЙ УНИВЕРСИТЕТ Таким образом, строение радулы B. contortrix заметно отличается от трёх других видов, которые более сходны между собой. При этом радулы представителей рода Opisthorchophorus отличаются и от B. tentaculata, которая имеет промежуточную пластинку с симметричным и большим по числу зубчиков резцом. Автор выражает искреннюю благодарность к.б.н., ст. науч. сотр. ИЦИГ СО РАН А.В. Катохину за предоставленную возможность работы со сборами битиниид из Восточной Сибири. Литература 1. Беэр С.А. Биология возбудителя описторхоза. М.: Товарищество научных изданий КМК, 2005. 335 с. 2. Старобогатов Я.И., Прозорова Л.А., Богатов В.В. Саенко Е.М. Моллюски // Определитель пресноводных беспозвоночных России и сопредельных территорий / под общ. ред. С.Я. Цалолихина. Т. 6: Моллюски, полихеты, немертины. СПБ.: Наука, 2004. С. 9–491. 3. Старобогатов Я.И., Ситникова Т.Я. Новый простой метод приготовления препаратов радулы моллюсков // Тр. Зоол. Ин-та АН СССР. 1985. Т. 135. С. 20–21. 4. Белякова-Бутенко Ю.В. О морфологии балхашской битинии (Gastropoda, Bithyniidae) // Зоологический журнал. 1969. Т. 58. С. 811–815. 5. Русинек О.Т., Ситникова Т.Я., Кондратистов Ю.Л. Состояние Иркутского очага описторхоза и вопросы его дальнейшего изучения // Изв. Иркут. гос. ун-та. Сер. «Биология, экология». 2012. Т. 5, № 4. С. 125–134. 6. Kim J.J. Radula tooth structure of eight bithyniid snails observed by scanning electron microscopy // Korean Journ. Malacol. 2015. Vol. 31 (1). P. 49–59. УДК 565.768.1 DOI: 10.17223/978-5-94621-931-0-2020-22 СОСТАВ ЯНТАРНЫХ ФАУН ДОЛГОНОСИКООБРАЗНЫХ ЖУКОВ (CURCULIONOIDEA) В ЭОЦЕНЕ ЕВРОПЫ Легалов А.А. Легалов А.А. Институт систематики и экологии животных СО РАН, Новосибирск, Россия fossilweevils@gmail.com Институт систематики и экологии животных СО РАН, Новосибирск, Россия fossilweevils@gmail.com Аннотация. Проведен анализ структуры фауны долгоносикообразных жуков эоценовых янтарей Европы. Выявлено 143 вида из восьми семейств. Показаны отличия фаун ранне- и среднеэоценовых янтарей. ф у р р р Ключевые слова. Жесткокрылые, таксономическая структура, палеоген, энтомофауна. 94 COMPOSITION OF WEEVIL AMBER FAUNAS (CURCULIONOIDEA) IN THE EOCENE OF EUROPE Legalov A.A. Institute of Systematic and Ecology of Animals, SB RAS, Novosibirsk, Russia fossilweevils@gmail.com Institute of Systematic and Ecology of Animals, SB RAS, Novosibirsk, Russia fossilweevils@gmail.com Abstract. The analysis of the structure of the weevil fauna from the Eocene amber of Europe is given. 143 species from eight families are discovered. The differences between the faunas of the early and the middle Eocene amber are shown. Keywords: Coleoptera, taxonomic structure, Paleogene, insect fauna. Из эоценовых янтарей Европы описано 143 вида Curculionoidea (1 вид из семейства Nemonychidae; 16 видов из семейства Anthribidae, 6 видов из семейства Belidae, 10 видов из семейства Rhynchitidae, 13 видов из семейства Brentidae, 71 вид из семейства Curcuionidae, 2 вида из семейства Platypodidae и 24 вида из семейства Scolytidae) [1–3]. Семейство Nemonychidae отмечено только в балтийском янтаре, где представлено одним видом. Три современных подсемейства семейства Anthribidae найдены в эоценовом янтаре. Подсемейство Urodontinae зарегистрировано только для балтийского янтаря. Вид подсемейства Choraginae найден в ровенском янтаре. Представители Anthribinae встречаются во всех эоценовых янтарях. Семейство Belidae представлено несколькими видами подсемейства Oxycoryninae. Семейство Rhynchitidae обнаружено в эоценовом янтаре. Подсемейство Sayrevilleinae представлено трибой Sanyrevilleini с пятью видами рода Baltocar. Две трибы Auletini и Rhynchitini из подсемейства Rhynchitinae, встречаются в янтарях. Семейство Brentidae представлено в эоценовом янтаре подсемействами Apioninae и Nanophyinae. Семейство Curculionidae – крупнейшее в современной фауне, преобладает среди долгоносикообразных жуков в эоценовых янтарях. Зарегистрированы представители 8 подсемейств (Erirhininae, Molytinae, Lixinae, Dryophthorinae, Cossoninae, Conoderinae, Curculioninae и Entiminae). Семейство Platypodidae лишь недавно обнаружено в балтийском янтаре, где его представители очень редки, что принципиально отличает фауну эоценового янтаря от фауны миоценового доминиканского янтаря, где Platypodidae являются одной из доминантных групп. Семейство Scolytidae отмечено во всех эоценовых янтарях. Фауны Scolytidae ровенского и балтийского янтарей различны. ф б Установлено, что фауны долгоносикообразных жуков раннего и среднего эоцена Европы существенно отличались друг от друга. Раннеэоценовый янтарь Уаз сильно отличается от среднеэоценовых 95 янтарей. Ни один из известных в янтаре Уаз родов долгоносикообразных жуков не встречается в балтийском и ровенском янтаре. Вымершая триба Sciabregmini, триба Conoderini, ныне распространенная в Южной Африке, и триба Eudiagogini, известная в современной американской фауне, отмечены в янтаре Уаз, но не найдены в среднеэоценовом янтаре. Сравнение состава семейства долгоносиков на уровне триб дает основание считать ровенский янтарь как бы «обедненным балтийским янтарем». Все роды, найденные в ровенском янтаре, за исключением Stenommatomorphus, являются общими, но общих видов не обнаружено. Эти различия обусловлено не только временем захоронения, но, по-видимому, и тем, что янтарь Уаз формировался из смолы покрытосеменных, а среднеэоценовые – из смолы голосеменных. Institute of Systematic and Ecology of Animals, SB RAS, Novosibirsk, Russia fossilweevils@gmail.com Таким образом, эти смолы производились в существенно разных лесных сообществах. Ровенский и балтийский янтари можно рассматривать как одновозрастные. Фауны ровенского и балтийского янтарей самостоятельные, поскольку общие виды долгоносикообразных жуков в них отсутствуют. Исследования поддержаны грантами РФФИ № 18-04-00243-a и 19-04-00465-a, а также программой фундаментальных научных исследований на 2013–2020 гг., проект № VI.51.1.5 (AAAA-A16-116121410121-7). Исследования поддержаны грантами РФФИ № 18-04-00243-a и 19-04-00465-a, а также программой фундаментальных научных исследований на 2013–2020 гг., проект № VI.51.1.5 (AAAA-A16-116121410121-7). Литература 1. Legalov A.A. A review of the Curculionoidea (Coleoptera) from European Eocene ambers // Geosciences. 2020. Vol. 10, № 1 (16). P. 1–74. 2. Bukejs A., Alekseev V.I., Legalov A.A. A new Eocene genus of the subtribe Tylodina (Coleoptera: Curculionidae) and notes concerning local differences of Baltic amber in the Kaliningrad Region // Fossil Record. 2020. Vol. 23. P. 75–81. 3. Legalov A.A., Poinar G.O., Jr. Two new species and new findings of Curculionoidea (Insecta: Coleoptera) in Baltic amber // Ukrainian Journal of Ecology. 2020. Vol. 10, № 2. P. 357–364. УДК 59.002 УДК 59.002 DOI: 10.17223/978-5-94621-931-0-2020-23 Лябзина С.Н.1, 2, Зинников Д.Ф.2 1Петрозаводский государственный университет, , Петрозаводск, Россия 2 Всероссийский центр карантина растений, Петрозаводск, Россия slyabzina@petrsu.ru Аннотация. В полевой практике по зоологии беспозвоночных и другим предметам, помимо традиционных методов сбора насекомых, мы можем 96
https://openalex.org/W1976375976	https://europepmc.org/articles/pmc3233469?pdf=render	English	null	Active Protein Aggregates Produced in Escherichia coli	International journal of molecular sciences	2,011	cc-by	6,568	Špela Peternel 1,2,* and Radovan Komel 1,2 1 Laboratory for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; E-Mail: radovan.komel@mf.uni-lj.si 2 Medical Centre for Molecular Biology, Medical faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia * Author to whom correspondence should be addressed; E-Mail: spela.peternel@ki.si; Tel.: +386-1-4760-262; Fax: +386-1-4760-300. Received: 12 October 2011; in revised form: 11 November 2011 / Accepted: 11 November 2011 / Published: 22 November 2011 Abstract: Since recombinant proteins are widely used in industry and in research, the need for their low-cost production is increasing. Escherichia coli is one of the best known and most often used host organisms for economical protein production. However, upon over- expression, protein aggregates called inclusion bodies (IBs) are often formed. Until recently IBs formation represented a bottleneck in protein production as they were considered as deposits of inactive proteins. However, recent studies show that by choosing the appropriate host strain and designing an optimal production process, IBs composed from properly folded and biologically active recombinant proteins can be prepared. Such active protein particles can be further used for the isolation of pure proteins or as whole active protein particles in various biomedical and other applications. Therefore interest in understanding the mechanisms of their formation as well as their properties is increasing. Keywords: E. coli; active protein aggregates; inclusion bodies; IBs Int. J. Mol. Sci. 2011, 12, 8275-8287; doi:10.3390/ijms12118275 Int. J. Mol. Sci. 2011, 12, 8275-8287; doi:10.3390/ijms12118275 International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms OPEN ACCESS Review 1. Introduction The need for low-cost protein production is increasing as the use of proteins is expanding to various areas; from research to a range of commercial applications in pharmaceutical, chemical and food industry, cosmetics as well as biomedical applications (e.g., tissue cultures) and diagnostics. Bacterial host systems for recombinant protein production are still very attractive, as they are usually genetically Int. J. Mol. Sci. 2011, 12 Int. J. Mol. Sci. 2011, 12 8276 well-characterized having a large number of cloning vectors and mutant host strains available and they grow rapidly at high density on inexpensive substrates. It is difficult to decide which combination of host organism-promoter system would be the best for specific recombinant protein production; therefore this still has to be optimized for each product individually. Since Escherichia coli have all the above described advantages [1] and the laboratory strains are regarded as GRAS (Generally Recognized As Safe) organisms, it is still one of the most commonly used bacterial host system, not only on laboratory scale use, but also for production of therapeutic proteins [2–5]. bacterial host system, not only on laboratory scale use, but also for production of therapeutic proteins [2–5]. However, during recombinant protein production in E. coli, proteins often tend to aggregate into protein particles called inclusion bodies (IBs). Until recently, IBs were considered as deposits of misfolded and inactive proteins and represented bottleneck in recombinant protein production. Therefore many pharmaceutically interesting proteins have been disregarded for commercialization [6]. Nevertheless, the latest studies on protein aggregation have shown that protein aggregation into IBs does not necessarily imply protein inactivation [7–10] thus studies on protein aggregation has become an important subject in many fields, including biology, medicine and biotechnology [11]. This article gives an overview on the latest trends in recombinant protein production in E. coli, their aggregation and the possible applications of such protein particles (IBs). 3. Protein Aggregation and IBs Formation The aim of recombinant protein production is to yield high amounts of the desired proteins. As a result the host organism is often forced to produce proteins above the cells physiological capacity. Upon over-expression, a high amount of proteins are constantly formed that cannot be simultaneously processed by the protein synthesis machinery, thus the quality control system is activated [25]. Additionally, E. coli has simple protein folding machinery, which lacks post–translational modifications. Due to the reducing potential of the E. coli cytoplasmic redox state, the production of proteins possessing more disulfide bonds still remains a challenge. The combination of all these factors, together with the exceeded solubility of over-expressed recombinant protein leads to the aggregation of recombinant proteins, which often gives rise to IBs formation. IBs were for a long time understood as inert deposits of misfolded and inactive proteins in some way separated from the cellular repair mechanism. However, it is now known that protein aggregation into IBs is reversible [26] and dependent on the physiological state of the host organism. IBs are very dynamic structures that are continuously formed in the host cell and simultaneously the proteins are also released from them and refolded or degraded by the cell repair mechanisms [27]. Since protein aggregates represented an obstacle in recombinant protein production, various mechanisms that would enable production of soluble proteins in bacteria were studied. Yields of soluble protein expression can be increased by chaperone co-expression [28], fusion of target protein with suitable fusion tags [29], choosing appropriate host strain and promoter system or modifying the cultivation conditions (e.g., growth temperature, media composition) [17]. However, studies showed, that protein solubility does not imply protein activity, as large amount of the proteins found soluble in the cytoplasm can be biologically inactive [7,8]. The formation of protein aggregates is a self-assembly process in bacterial cells. As there is no compartmentalization, proteins are simultaneously synthesized on multiple locations in the bacterial cytoplasm and various transitional folding states of the target protein are formed (Figure 1). Some of the folding intermediates that fail to fold into a native conformation are immediately degraded by the cells’ repair mechanisms, while others aggregate into smaller proto-aggregates (“soluble aggregates”) [30]. During this nucleation, predominantly target recombinant protein is incorporated into the proto-aggregates by cross-molecular stereospecific interactions, while the other non-homologous cellular (and even recombinant) proteins are excluded from this seeding events [25]. Int. J. Mol. Sci. 2011, 12 Int. J. Mol. Sci. 2011, 12 Int. J. Mol. Sci. 2011, 12 8277 Yet strong production of recombinant proteins and thus unusually high transcription rates could result in a stressful situation for the host cell [23] that has a negative impact on productivity and protein quality [24]. Therefore a fragile balance between high culture density and high protein yield versus high protein quality should be maintained for optimal results. Yet strong production of recombinant proteins and thus unusually high transcription rates could result in a stressful situation for the host cell [23] that has a negative impact on productivity and protein quality [24]. Therefore a fragile balance between high culture density and high protein yield versus high protein quality should be maintained for optimal results. 2. E. coli and Recombinant Protein Production E. coli is one of the most widely used hosts for the production of recombinant proteins. Nevertheless, choosing an optimal expression system is vital for an efficient protein production process and is often dependant on recombinant protein itself. There are many different E. coli strains and vector systems, however B strains, such as BL21, combined with pET vectors, became popular due to their efficiency in recombinant protein production [2,12]. Because of well controlled promoters, this bacterial factory enables high recombinant protein production yields (up to 50% of total cell proteins). It proved to be a very efficient system also for the production of active protein particles called non-classical IBs (ncIBs) [7,13–15]. However, as recombinant protein production represents stress for the host cell and thus the whole cell machinery has to adapt to an over-expression of foreign protein, quality product can only be produced when the whole bioprocess is optimized [15]. Therefore in addition to the host organism, a production media should also be optimized and quality inoculum prepared for the reproducibility of the production process. Addition of some essential microelements to the basic LB media enables higher accumulation of recombinant proteins [16]. Furthermore, lowering the cultivation temperature was proven to effectively limit the in vivo aggregation of recombinant proteins [17], hence higher amounts of soluble proteins are formed [18] and the quality of the proteins is improved, including the ones trapped inside IBs [10,13,14,19,20]. Suboptimal growth temperatures slow down all the cell processes, including transcription and translation [17] thus proteins have more time to fold properly. The extended time period between synthesis and deposition, as well as rapidly exceeded solubility of target protein, results in aggregation of better folded proteins [20,21]. While satisfactory protein yields could be achieved in the shake flask culture, extremely high yields could only be obtained by high cell density fermentation, using finely tuned expression systems [22]. 3. Protein Aggregation and IBs Formation This leads to the rapid growth of proto-aggregates that are later fused together in an IB that continually grows further to form the final IB as depicted in Figure 2 [21,31]. Inside the IB, the network of partially folded proteins is formed and studies show that this network has an amyloid-like structure [32]. Properly folded protein precursors are trapped into this network (Figure 3) [21]. A similar self-assembly process of amyloid-like structures has also been observed in yeasts [33], fungi [34], plants [35] and can be observed in many Int. J. Mol. Sci. 2011, 12 8278 mammalian (human as well as animal) degenerative disorders formation is an ubiquitous process of many different organisms. mammalian (human as well as animal) degenerative disorders [36] thus it seems that amyloid formation is an ubiquitous process of many different organisms. thus it seems that amyloid Figure 1. Formation of inclusion bodies ( compartmentalized, proteins foreign genes, the cell repair mechanisms are overloaded thus various transitional folding states of the recombinant protein are simultaneously present in the cell. Due to various reasons (hydrophobicity, exceeded solubility, these protein precursors start to aggregate into small “proto together into (usually) a single IB. This IB grows in the cell as a sphere until it reaches the bacterial cell wall and it is th stay in one of the cells and grow further, while the other cell remains empty and protein production and IB formation begins translocation to the cell pole before cell division is inclusion bodies (IBs). Since bacterial c start to form on multiple locations. During over the cell repair mechanisms are overloaded thus various transitional folding states of the recombinant protein are simultaneously present in the cell. Due to various exceeded solubility, cross-molecular stereospecific interactions) these protein precursors start to aggregate into small “proto-aggregates” that are then glued together into (usually) a single IB. This IB grows in the cell as a sphere until it reaches the bacterial cell wall and it is then prolonged into a cylinder. When the cell divides stay in one of the cells and grow further, while the other cell remains empty and protein production and IB formation begins de novo. 3. Protein Aggregation and IBs Formation proteins inside IB. IBs are composed from the network of misfolded/partially folded proteins inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH s in strong contraction of the protein network, thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. IBs are composed from the network of inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. Figure 3. Network of proteins inside IB. misfolded/partially folded proteins The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH (around 4) results in strong contraction of the protein network and the properly folded proteins are trapped inside and their extraction is prevented. proteins inside IB. IBs are composed from the network of misfolded/partially folded proteins inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH s in strong contraction of the protein network, thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. IBs are composed from the network of inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. Figure 3. Network of proteins inside IB. misfolded/partially folded proteins The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH (around 4) results in strong contraction of the protein network and the properly folded proteins are trapped inside and their extraction is prevented. proteins inside IB. IBs are composed from the network of misfolded/partially folded proteins inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH s in strong contraction of the protein network, thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. 3. Protein Aggregation and IBs Formation Studies show that the process of IB translocation to the cell pole before cell division is an energy dependent process Since bacterial cytoplasm is not During over-expression of the cell repair mechanisms are overloaded thus various transitional folding states of the recombinant protein are simultaneously present in the cell. Due to various molecular stereospecific interactions) aggregates” that are then glued together into (usually) a single IB. This IB grows in the cell as a sphere until it reaches the n the cell divides, the IBs stay in one of the cells and grow further, while the other cell remains empty and protein . Studies show that the process of IB energy dependent process [37]. Int. J. Mol. Sci. 2011, 12 8279 Figure 2. Protein folding and aggregation in conformation during protein synthesis are present in the cell together wit machinery maintains kinetic equilibrium between soluble and aggregated forms of the protein. Soluble fraction is composed aggregates. Inside the soluble aggre aggregates are further aggregated into insoluble aggregates called inclusion bodies. The process in reversible and it is controlled by the cell quality control mechanism. Protein folding and aggregation in E. coli. Many proteins fail to fold to the native conformation during protein synthesis, therefore various transitional folding intermediates are present in the cell together with properly folded proteins. Cells’ quality control machinery maintains kinetic equilibrium between soluble and aggregated forms of the protein. Soluble fraction is composed of single protein molecules as well as soluble aggregates. Inside the soluble aggregates, properly folded proteins are also trapped. Soluble aggregates are further aggregated into insoluble aggregates called inclusion bodies. The process in reversible and it is controlled by the cell quality control mechanism. Many proteins fail to fold to the native therefore various transitional folding intermediates h properly folded proteins. Cells’ quality control machinery maintains kinetic equilibrium between soluble and aggregated forms of the single protein molecules as well as soluble properly folded proteins are also trapped. Soluble aggregates are further aggregated into insoluble aggregates called inclusion bodies. The process in reversible and it is controlled by the cell quality control mechanism. Figure 3. Network of proteins inside IB. misfolded/partially folded proteins The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH (around 4) results in strong contraction of the protein network and the properly folded proteins are trapped inside and their extraction is prevented. 3. Protein Aggregation and IBs Formation IBs are composed from the network of inside which the properly folded proteins are trapped. The protein network is loosely connected at neutral pH. The transfer of IBs into acidic pH thus the IBs are more compact and the properly folded proteins are trapped inside and their extraction is prevented. It seems that after the proto-aggregates are spaces between the proto-aggregates are filled with hydrophobic or stereospecific interactions with a mild detergent [7], these proto seems as if they were imbedded into a cotton matrix fills the spaces both among and inside the proto (Figure 4b). aggregates are fused into a single IB and this IB grows fur aggregates are filled with a mass of proteins incorporated to IB by stereospecific interactions. If the surface proteins are removed from the IBs’ surface these proto-aggregates can be observed under electron microscope and it imbedded into a cotton-like amorphous matrix (Figure 4a) matrix fills the spaces both among and inside the proto-aggregates which gives the IB a porous surface single IB and this IB grows further, the a mass of proteins incorporated to IB by . If the surface proteins are removed from the IBs’ surface an be observed under electron microscope and it like amorphous matrix (Figure 4a). This amorphous aggregates which gives the IB a porous surface Int. J. Mol. Sci. 2011, 12 8280 Figure 4. IB observed with a scanning electron microscope. The IBs washed with water (b) and additionally rapidly washed in mild detergent (a) [7]. It seems that IBs are composed of small proto-aggregates imbedded into a cotton-like amorphous matrix (a). However, the amorphous matrix fills the spaces both among and inside the proto-aggregates which gives the IB a porous surface (b). 3. Properties of Properly Folded Protein Aggregates The shape and size of IBs is very much dependant on the host bacterial strain. So the IBs wer found to be spherical [7,38], ellipsoidal [31,39], cylindrical [40,41] and even tear-shaped [42] rangin in size form 50–700 nm [7,42]. 3. Properties of Properly Folded Protein Aggregates 3. Properties of Properly Folded Protein Aggregates The shape and size of IBs is very much dependant on the host bacterial strain. So the IBs were found to be spherical [7,38], ellipsoidal [31,39], cylindrical [40,41] and even tear-shaped [42] ranging in size form 50–700 nm [7,42]. The shape and size of IBs is very much dependant on the host bacterial strain. Int. J. Mol. Sci. 2011, 12 Int. J. Mol. Sci. 2011, 12 8281 Figure 5. The diverse population of IBs after 24 h production. The protein production starts immediately after the inducer is added to the medium. Therefore some of the IBs in the population have been growing for 24 h and formed cylinders that occupy almost the entire cell space. In contrast, other IBs started growing de novo after each bacterial cell division. Therefore the whole range from small spherical IBs, that have been formed in the final hour of cultivation, to large spheres that have been growing for several hours all the way to the cylinders are simultaneously present in the one sample. The activity of proteins inside IBs varies significantly, depending on the target protein as well as the production conditions. The active protein particles, ncIBs, are composed from significant amounts of properly folded and biologically active proteins, trapped into the network of misfolded proteins. Thus they possess some interesting properties that can be exploited for various biotechnological, as well as biomedical, applications. Such ncIBs are extremely fragile and soluble in contrast to classical IBs. They are soluble in mild detergents and even in buffers usually used to wash and store classical IBs [7,13]. Therefore optimization of isolation and washing process is necessary in order not to impair the structure of IBs or the protein trapped inside [40,43]. It was shown that sonication, often used for bacterial cell disruption, can damage the structure of ncIBs. Furthermore, the structure of properly folded proteins trapped inside ncIBs can also be destroyed and significant proportion of biologically active protein can thus be lost [40]. Another interesting property of IBs (classical as well as non classical) is their irreversible The activity of proteins inside IBs varies significantly, depending on the target protein as well as the production conditions. The active protein particles, ncIBs, are composed from significant amounts of properly folded and biologically active proteins, trapped into the network of misfolded proteins. Thus they possess some interesting properties that can be exploited for various biotechnological, as well as biomedical, applications. Such ncIBs are extremely fragile and soluble in contrast to classical IBs. They are soluble in mild detergents and even in buffers usually used to wash and store classical IBs [7,13]. Therefore optimization of isolation and washing process is necessary in order not to impair the structure of IBs or the protein trapped inside [40,43]. 3. Protein Aggregation and IBs Formation So the IBs were found to be spherical [7,38], ellipsoidal [31,39], cylindrical [40,41] and even tear-shaped [42] ranging in size form 50–700 nm [7,42]. There is usually one and only rarely two IBs present in the bacterial cell. So after the bacterial cell division, when two cells emerge, IB is present in only one cell while the other cell is empty. Recent study showed that IB is actively translocated to the cell pole before cell division and in this manner aggregated proteins can be removed from bacterial population in nature [37]. In bacterial culture where protein production is artificially induced, production of recombinant proteins in the vacant cells begins de novo and a new IB is formed (Figure 1), while the IBs that remain in the cells after cell division grow further. Therefore there are various populations of bacterial cells present simultaneously in the bacterial culture. Thus the shape and size of IBs is dependant not only on the host strain, but also on the time of cultivation. Based on our observation in the case of spherical IBs, they form spheres at the beginning of cultivation. However, when the IB grows and reaches the bacterial cell wall and it can no longer grow in one direction it starts to form the cylinder. Therefore, the population of IBs after long-term cultivation (24 h) is very diverse as some of the IBs in the population have been growing for 24 h and have formed large cylinders that nearly occupy the whole bacterial cell, ranging from smaller cylinders and large spheres all the way to small spheres that are just beginning to form (Figure 5). We Learn from Properly Folded Protein Aggregates and How Can We Use Them? Understanding protein folding and mechanisms of protein aggregates (IBs) formation in bacteria can serve as a model for protein aggregation in higher organisms and help us understand how and why human conformational diseases progress [32]. Besides this, understanding the properties of IBs could also give us an insight into the properties of protein aggregates formed in mammalian cells. This knowledge could serve as a background for identification of novel target sites for the development of novel and more efficient treatments for human conformational diseases. Furthermore, IBs, formed after over-expression of recombinant proteins in bacteria, are a highly pure protein deposit of target recombinant protein as it can represent more than 95% of all protein present inside the IBs [44]. Historically, IBs were considered as the main obstacle in protein production while isolating active proteins from IBs represented a great challenge. However, ncIBs composed from significant amount of properly folded and biologically active proteins, easily soluble in mild detergents, are ideal for protein isolation. Since ncIBs are extremely fragile and soluble, bacterial cell disruption process should be optimized. After bacterial cell disruption ncIBs are washed and collected with centrifugation, and the majority of the impurities are removed already in this step. The buffers used during cell disruption and for ncIBs washing should also be carefully chosen, not to dissolve the IBs [7,13,40,43]. While ncIBs are soluble in mild detergents, recombinant protein can be extracted from them in mild non-denaturing conditions in biologically active form, therefore no renaturation step is needed. The protein isolation process is thus simplified, less protein specific, more cost effective and environment-friendlier [7,13–15,45,46]. Since the need for recombinant proteins is constantly increasing, this process could be well exploited in the future. In the past few years with the development of nanobiotechnology the field of possible applications of protein particles (IBs) is broadening and protein micro- and nano-particles are becoming increasingly interesting. The IBs are therefore studied for various biomedical applications [47]. As IBs are large protein particles that can easily trigger the immune response, the idea of using the whole IBs as a vaccine was tested by several different research groups simultaneously. Successful protective immunity was reported for vaccines against several animal diseases (e.g., liver fluke, classical swine fever, salmonid rikettsial septicaemia …) [48–50] as well as against human oral infections (gingivitis and periodontitis) [51]. Int. J. Mol. Sci. 2011, 12 It was shown that sonication, often used for bacterial cell disruption, can damage the structure of ncIBs. Furthermore, the structure of properly folded proteins trapped inside ncIBs can also be destroyed and significant proportion of biologically active protein can thus be lost [40]. Another interesting property of IBs (classical as well as non-classical) is their irreversible contraction at low pH. It seems that the high proton concentration in acidic buffers induces a change in the network of unfolded/partially folded proteins and this leads to a strong contraction of the protein network and formation of more compact IBs [21]. Consequently the solubility of contracted ncIBs is greatly reduced. While at neutral pH the protein network is loosely bound, the extraction of native-like precursor molecules as well as some still soluble proteins having biologically inactive conformations is easy (Figure 3a) [8,13]. However, the contraction of protein network in acidic pH traps the properly folded precursors into the IB and their extraction prevented (Figure 3b). Thus when soluble ncIBs are needed for further applications, the buffers used for washing and storing the IBs should be carefully chosen to prevent ncIBs contraction. Int. J. Mol. Sci. 2011, 12 8282 5. Conclusion and Perspectives Protein aggregation into IBs has long been considered as an obstacle in protein production in bacteria. However, the knowledge gained during studies of protein aggregation in bacteria could help us understand the human progressive conformational diseases that represent a growing problem with increased prevalence in an aging society. Furthermore, with carefully designed bioprocess production of active and even soluble protein aggregates being possible, such aggregates can be used in biotechnology for the isolation of pure recombinant proteins and as micro-/nanoparticles in various biomedical and pharmacological applications as well as in other fields. As this is still an emerging and fast evolving discipline, the protocols for production and preparation of protein particles have to be redesigned and carefully optimized. A more thorough and systematic study on production and properties of such protein particles is needed in order to enable the design of particles with the desired properties (e.g., solubility, biological activity, size, shape…) [62]. Int. J. Mol. Sci. 2011, 12 Int. J. Mol. Sci. 2011, 12 8283 The field of nanobiotechnology is new and fast evolving, thus predicting various possibilities of IBs applications is difficult. However, it could be anticipated that such nanoparticles could be used in the development of new treatments and diagnostic techniques in medicine, biomedicine and pharmacology as well as biocatalysts in various industries (cosmetics, biotechnology, food and chemical industry) [47,61,62]. Acknowledgments The study was financially supported by a Young Researcher’s grant, by the research project “Micro and Nano Particles in Biotechnology” as well as by the program “Functional genomics and biotechnology for health” from the Slovenian Research Agency. We Learn from Properly Folded Protein Aggregates and How Can We Use Them? Furthermore, IBs composed from a suitable protein (pseudan) can be used as a coating in medical tubes to reduce or even inhibit biofilm formation [52]. On the other hand, recent studies showed, that IBs can be utilized as a scaffold material in tissue engineering, as they affect the mammalian cell attachment and proliferation [39,42,53]. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). © 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
https://openalex.org/W2898539421	https://www.scielo.br/j/cagro/a/BmVxvYZpLqZ4f4yCvGh4DjM/?lang=en&format=pdf	English	null	Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda	Ciência e Agrotecnologia	2,018	cc-by	7,911	RESUMO A lagarta do cartucho (LCM) (Spodoptera frugiperda) é um inseto polífago que causa danos em várias culturas. Inseticidas químicos sintéticos e plantas geneticamente modificadas são os métodos mais comumente empregados para o seu controle. Entretanto, existem muitos relatos da seleção de populações resistentes, o que justifica a busca por novas moléculas para o controle de S. frugiperda. O objetivo desse trabalho foi avaliar a toxicidade do óleo essencial de capim limão (Cymbopogon flexuosus) (OECL) e seu componente majoritário, citral, para LCM. Adicionalmente, a atividade anticolinestarase do OECL e do citral foram avaliadas usando a acetilcolinestarase (AChE) de Electrophorus electricus. OECL foi tóxico para LCM, quando incorporado em dieta artificial (CL50 = 1,35 mg mL-1), nas mais altas concentrações testadas, o tempo letal mediano (TL50) foi de 18,85 h. Os componentes majoritários do OECL foram identificados por cromatografia gasosa acoplada a espectometria de massas. Citral, o composto mais abundante, foi empregado em bioensaios com LCM. A atividade inseticida do citral foi estatisticamente similar àquela do OECL, demonstrando que o citral é responsável pela atividade inseticida do OECL. A inibição da AChE foi mensurada, sendo os valores de concentração inibitória media (CI50) encontrados para o OECL e citral, 650 e 405 vezes maiores, respectivamente, do que o detectado para o controle positivo, o inseticida metomil, sugerindo seletividade para organismo não-alvo. Os resultados encontrados tornam o citral e C. flexuosus promissores para serem empregados no desenvolvimento de novos produtos para o controle de S. frugiperda. Termos para indexação: Lemongrass; (2Z)-3,7-dimethylocta-2,6-dienal; monoterpenos; inseticida botânico; lagarta do cartucho. Ellison Rosario de Oliveira1, Dejane Santos Alves2, Geraldo Andrade Carvalho3, Bárbara Maria Ribeiro Guimarães de Oliveira1, Smail Aazza1, Suzan Kelly Vilela Bertolucci1 Ellison Rosario de Oliveira1, Dejane Santos Alves2, Geraldo Andrade Carvalho3, Bárbara Maria Ribeiro Guimarães de Oliveira1, Smail Aazza1, Suzan Kelly Vilela Bertolucc Ellison Rosario de Oliveira1, Dejane Santos Alves2, Geraldo Andrade Carvalho3, Bárbara Maria Ribeiro Guimarães de Oliveira1, Smail Aazza1, Suzan Kelly Vilela Bertolucci1 1Universidade Federal de Lavras/UFLA, Departamento de Agricultura/DAG, Lavras, MG, Brasil 2Universidade Tecnológica Federal do Paraná /UTFPR, Santa Helena, PR, Brasil 3Universidade Federal de Lavras/UFLA, Departamento de Entomologia/DEN, Lavras, MG, Brasil Corresponding author: agronomellison.oliveira@yahoo.com.br Received in June 20, 2018 and approved in August 6, 2018 1Universidade Federal de Lavras/UFLA, Departamento de Agricultura/DAG, Lavras, MG, Brasil 2Universidade Tecnológica Federal do Paraná /UTFPR, Santa Helena, PR, Brasil 3Universidade Federal de Lavras/UFLA, Departamento de Entomologia/DEN, Lavras, MG, Brasil Corresponding author: agronomellison.oliveira@yahoo.com.br Received in June 20, 2018 and approved in August 6, 2018 2018 \| Lavras \| Editora UFLA \| www.editora.ufla.br \| www.scielo.br/cagro All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License attribuition-type BY. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 http://dx.doi.org/10.1590/1413-70542018424013918 Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 http://dx.doi.org/10.1590/1413-70542018424013918 Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda ABSTRACT Fall armyworm (FAW) (Spodoptera frugiperda) is a polyphagous insect responsible for damage to several crops. Synthetic chemical insecticides and genetically modified plants are the most commonly used methods for FAW control. However, the selection of resistant populations has been reported in several studies, justifying the search for new molecules to be used in the control of S. frugiperda. The aim of the present study was to evaluate the toxicity of lemongrass (Cymbopogon flexuosus) essential oil (LEO) and its major component (citral) in relation to FAW. Additionally, the anticholinesterase activity of LEO and citral was evaluated using acetylcholinesterase (AChE) from Electrophorus electricus. The LEO was toxic to FAW when added to an artificial diet (LC50 = 1.35 mg mL-1) at the highest concentrations tested, and the median lethal time (LT50) was 18.85 h. Major components of LEO were identified by gas chromatography-mass spectrometry, and citral, the most abundant component, was used in FAW bioassays. The insecticidal activity of citral was statistically similar to that of LEO, demonstrating that citral was responsible for the insecticidal activity of LEO. Inhibition of AChE was measured, and the mean inhibitory concentration (IC50) values for LEO and citral were 650- and 405-fold higher, respectively, than that verified for the positive control (methomyl insecticide), suggesting selectivity for non-target organisms. Based on these results, citral and C. flexuosus have the potential to be applied in the development of new products for the control of S. frugiperda. Index terms: Lemongrass; (2Z)-3,7-dimethylocta-2,6-dienal; monoterpenes; botanical insecticide; fall armyworm. Insects Spodoptera frugiperda were reared from caterpillars and pupae obtained from maize in an experimental field of the Federal University of Lavras, Minas Gerais, Brazil. To perform the bioassays, S. frugiperda caterpillars were used at 48 h of age from the second reproduction of laboratory specimens fed artificial diet (Parra, 2001). Adults were fed 10% honey aqueous solution. All insects were kept in an acclimatized room at 25 ± 2 ºC, 70 ± 10% RH and with a 12 h photoperiod. In this context, secondary plant metabolites are identified as a promising tool in the control of insect pests. Substances from plants are often less toxic to non- target organisms, such as natural enemies, pollinators and humans (Isman, 2017; Pavela; Benelli, 2016). Among secondary plant metabolites, essential oils have received increasing attention for their insecticidal activity and sublethal effects (Krinski; Foerster, 2016; Regnault-Roger, 2013; Silva et al., 2017b; Walia et al., 2017). Furthermore, essential oils are compounds that may reduce the potential for resistance to pests because of their complex chemical composition that may direct their performance at different sites and at different stages of development of the target insect (Akhtar et al., 2012). Essential oil The tillers of C. flexuosus were collected from the Horto of Medicinal Plants at Federal Lavras University. Plants of C. flexuosus were cultivated in soil with organic fertilizer (cow dung) at a dose of 3.0 kg m-2 irrigated periodically with plant spacing of 45 cm (21°14’43” S, 44°59’59” W). The plants were identified on the basis of morphological features and deposited in the herbarium of the Department of Biology at Federal Lavras University (UFLA). The fresh plant leaves were collected at 20 cm from the soil at seven months of age. The plant material (1000 g) was steam-distilled for 90 min in a Marconi MA480 essential oil distiller (Piracicaba, São Paulo, Brazil). The LEO was separated by decantation for 20 min and stored in a freezer at -10 °C until chemical analyses and biological tests. Species from the genus Cymbopogon of the Poaceae family are widely known for producing lemongrass essential oil (LEO) and for their insecticidal properties. Among the species from this genus, Cymbopogon flexuosus (Steud.) Wats. (Poaceae) is one of the most widely grown plants producing essential oil in tropical and subtropical regions of India, Indonesia, Madagascar, and countries in Africa and South America (Ganjewala; Luthra, 2010). Toxicity of this plant is observed against stored grain pests (Caballero-Gallardo; Olivero-Verbel; Stashenko, 2012), insect-borne diseases (Tawatsin et al., 2001; Tennyson et al., 2013; Vera et al., 2014), and oilseed pests (Hernández-Lambraño, Caballero-Gallardo; Olivero-Verbel, 2014). INTRODUCTION phenological stages of several crops, both in productive and surrounding areas, because of intensive cultivation (Casmuz et al., 2010; Murúa et al., 2015). The fall armyworm (FAW) Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is considered one of the primary pests occurring in the most important agricultural commodities in the Americas. The FAW is a polyphagous insect that reproduces throughout the The high population density and economic damages caused by S. frugiperda lead to great dependence on the use of synthetic chemical insecticides and genetically modified plants for control. However, the Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda 409 MATERIAL AND METHODS indiscriminate and intensive use of these technologies, associated with the high reproductive rate and dispersion of this insect, has led to the selection of resistant populations (Farias et al., 2016; Omoto et al., 2016; Santos-Amaya et al., 2017; Yu; McCord, 2007), justifying the constant search for molecules that may be used to control this insect. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Toxicity to citral for FAW The reference citral (neral and geranial mixture with 95% purity; Sigma-Aldrich®) was added to the artificial diet of FAW at concentrations of (0.521, 1.042 and 1.58 mg mL-1 diet), as previously described. Mass spectra from the database of NIST/EPA/NHI (National Institute of Standards and Technology - NIST, 2008) were compared to identify the chemical constituents of samples. Additionally, the retention indices from the Adams (2007) literature were compared with retention indices calculated based on the equation of Dool and Kratz (1963) relative to coinjection of a standard solution of n-alkanes (C8-C20; Sigma-Aldrich®, St. Louis, USA) and coinjection with citral (neral and geranial mixture with 95% purity; Sigma-Aldrich®). The bioassay was performed in a completely randomized design consisting of eight treatments: the estimated values for the LC20 (0.675 mg mL-1), LC50 (1.35 mg mL-1) and LC90 (2.053 mg mL -1) of the LEO; the estimated values for the LC20 (1.58 mg mL-1), LC50 (1.042 mg mL-1) and LC90 (0.521 mg mL-1) of citral, and two controls, diet with water and food coloring agent added and diet with Tween® 80 aqueous solution and dye added. Citral concentrations were calculated using the values obtained in the quantitative chromatographic analysis of the essential oil, using the formula LC = [(c x p)/100], where LC = expected lethal concentration of citral, c = lethal concentration of the essential oil and p = percentage of citral in the constitution of the LEO. Inhibition of enzymatic activity of the AChE The inhibitory activity of AChE was determined according to the methodology described by Aazza, Lyoussi and Miguel (2011) with modifications. In this respect, 4.25 μL of Tris-HCl buffer (0.1 M, pH = 8) and 25 μL of the test constituent (LEO or citral) were dissolved in ethanol at different concentrations from 0.01 to 2.24 mg mL-1. Subsequently, 25 μL of AChE enzyme (0.22 U/mL) (Type-VI-S, EC 3.1.1.7, Sigma-Aldrich®) was added and homogenized. After incubation at 37 °C for 15 min, 75 μL of 15 mM acetylcholine iodide substrate (Sigma-Aldrich®) and 475 μL of 3 mM 5,5’-dithiobis-(2- nitrobenzoic acid) (Sigma -Aldrich®) were added, and the resulting solution was incubated at room temperature for 30 min. The same procedure was performed for the positive control, a commercial product based on methomyl (Lannate®, DuPont™), and for the negative control (ethanol). The solutions were transferred to microplates, and the absorbance was measured at 405 nm in a TECAN Infinite® M200 PRO microplate reader. GC-MS analysis of C. flexuosus essential oil The quantitative analysis of C. flexuosus essential oil was performed using an Agilent® 7890A chromatograph operated with the HP GC ChemStation data processing system ver. A.01.14 and equipped with a CombiPAL Autosampler System (CTC Analytic AG, Switzerland) and flame ionization detector (GC-FID). Samples were prepared by diluting the essential oil with ethyl acetate (10 mL L-1). Volume injection was 1.0 μL in split mode at a 50:1 injection ratio. An HP-5ms fused silica capillary column (30 m length x 250 μm internal diameter x 0.25 μm film thickness) (California, USA) was used. Helium gas was used as the carrier gas with flow of 1.0 mL min-1. The injector and detector temperatures were maintained at 240 °C. The initial oven temperature was 60 °C maintained for 1 min, followed by a temperature ramp of 3 °C min-1 up to 240 °C, followed by a ramp of 10 °C min-1 up to 250 °C, with the isothermal condition maintained for 1 min. The concentrations of the constituents were expressed by the Note that LEO is considered as a minimum risk insecticide according to the United States Environmental Protection Agency (EPA, 2015); however, to the best of our knowledge, LEO insecticidal activity against S. frugiperda has not been reported to date. Thus, the aim of the present study was to chemically characterize the C. flexuosus essential oil and evaluate the toxicity of LEO and that of the major constituent to S. frugiperda. Additionally, the potential inhibitory effects of the essential oil and citral were evaluated in vitro on the acetylcholinesterase (AChE) enzyme from electric eel (Electrophorus electricus), which can provide information on the selectivity of these metabolites to non-target organisms. 410 OLIVEIRA, E. R. de et al. average relative percentage of area of the chromatographic peaks ± the standard deviation of three analyzed samples. replicate; the experimental plot consisted of one individual caterpillar. Insect development was evaluated daily until the pupal stage, with records of the larval survival every 24 h for 264 h, the larval phase duration and the pupal weight. Qualitative analyzes were performed on an Agilent® 7890A chromatograph coupled to an Agilent® MSD 5975C mass selective detector (Agilent Technologies, California, USA) operated by electronic impact ionization at 70 eV in scan mode at a speed of 1.0 scan s-1, with a mass acquisition interval of 40-400 m/z. The operating conditions were the same as those used in GC-DIC analyses. Time-concentration-mortality responses for FAW fed diet containing LEO LEO at the range of concentrations from 0.5 to 4.0 mg mL-1 diet was leached in aqueous 0.01 g mL-1 Tween® 80 (Polysorbate 80; Sigma-Aldrich®) (20 mL) and incorporated into artificial diet (200 mL) at 40 °C. To ensure that the aqueous solution of Tween® 80 was homogenized with the essential oil, 10 drops of food coloring agent (Arcolor®) were added using a Pasteur pipette. Dietary pieces (1 cm diameter x 1.5 cm height, weight = 9 ± 0.35 g) were transferred to glass tubes (8 cm x 2.5 cm) in which a caterpillar at 48 h of age was introduced, previously fed artificial diet without the addition of essential oil. A completely randomized design was used composed of LEO leached at different concentrations and the two controls: diet with water and food coloring agent added and diet with Tween® 80 aqueous solution and dye added, totaling 14 treatments. The experimental plot consisted of one caterpillar maintained individually, and 60 replicates were used per treatment. Insect survival was evaluated every 24 h for 11 days. Survival data after 72 h were used to calculate the median lethal concentration (LC50). Each treatment consisted of 60 replicates, each represented by a 48-h-old caterpillar maintained in a glass tube (8 cm x 2.5 cm) containing an artificial diet of the same size (1 cm diameter x 1.5 cm height). Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Sublethal effects of LEO for FAW The LEO [0.675 mg mL-1 diet (LC20) and 1.35 mg mL-1 diet (LC50)] was incorporated into the artificial diet as described in the subitem above. The control treatments were diet with water and food coloring agent added and diet with aqueous solution Tween® 80 and dye added. Each treatment consisted of 120 replicates with one caterpillar per Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda 411 Statistical analyses The survival data of insects over time were subjected to survival analysis by applying the Weibull model through the Survival package (Therneau, 2015) of the R software® (R Core Team, 2018). After selecting the most appropriate mathematical model through residue analysis, the contrast analysis was performed to verify the similarity among the used treatments to form congeners, with the median lethal time (LT50) calculated for each formed group. To verify the data fitting to the model, the Kolmogorov-Smirnov test was applied. RT = retention time; RI = retention index calculated in relation to the n-alkane series (C8-C20) on HP-5 MS column in the elution order; Ni = non-identified constituent. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 RT = retention time; RI = retention index calculated in relation to the n-alkane series (C8-C20) on HP-5 M order; Ni = non-identified constituent. GC-MS analysis of C. flexuosus essential oil The LEO was composed of 21 constituents, corresponding to 90.57% of the chemical composition. The major constituents of LEO were neral/Z-citral (32.59%) and geranial/E-citral (44.65%). These isomeric monoterpenes totaled 77.25% of this essential oil, whereas the other constituents summed to 13.32% of total oil content (Table 1). The major constituents generally found in C. flexuosus essential oil are the geranial and neral aldehydes, which together form citral and can vary in content according to numerous factors. The recorded content of citral (77.25%) in the present study was relatively low in relation to the content described in other studies (Adukwu et al., 2012; Ahmad; Viljoen, 2015; Silva et al., 2015). By contrast, the citral content reported in this study was higher than that in the studies of Anaruma et al. (2010) and Vera et al. (2014). To determine the concentration-mortality response, the data were subjected to Logit analysis using the drc package (Ritz; Streibig, 2016) of the R software® (R Core Team, 2018). Data on the duration of the larval period and weight of pupae were subjected to the Shapiro-Wilk test, using the Mvnormteste package (Jarek, 2012) to verify the normality. Subsequently, data were subjected to ANOVA and to the Scott-Knott test of the Laercio package (Silva, 2010). Constituent RT RI Area % 1 5-Hepten-2-one, 6-methyl- 7.820 985 1.58 2 Myrcene 7.971 990 0.45 3 Trans-linalool oxyde 11.711 1088 0.14 4 Linalool 12.171 1100 2.79 5 Exo-isocitral 14.127 1144 0.42 6 Photocitral A 14.345 1149 0.38 7 Citronellal 14.493 1152 0.29 8 Z-isocitral 15.003 1164 0.98 9 E-isocitral 15.815 1182 1.28 10 Estragole 16.513 1198 0.35 11 n-decanal 16.843 1205 0.42 12 Neral 18.523 1242 32.59 13 Geraniol 19.061 1254 0.93 14 Geranial 19.897 1273 44.65 15 Nerolic acid 22.168 1323 0.54 16 Ni m/z = 168 22.850 1339 2.31 17 Geranic acid 23.939 1363 0.18 18 Ni m/z = 169 24.479 1375 3.41 19 Geranyl acetate 24.882 1384 1.92 20 Ar-curcumene 29.165 1483 0.46 21 Sandacopimara-8(14), 15-diene 47.356 1966 0.22 RT = retention time; *RI = retention index calculated in relation to the n-alkane series (C8-C20) on HP-5 MS column in the elution order; Ni = non identified constituent Table 1: Chemical composition of the essential oil of Cymbopogon flexuosus leaves. Table 1: Chemical composition of the essential oil of Cymbopogon flexuosus leaves. 412 OLIVEIRA, E. R. de et al. The differences in the quantitative chemical composition of C. GC-MS analysis of C. flexuosus essential oil flexuosus observed in the present study can be explained by the variation in genotype, geographic origin, environmental factors, plant development stage, harvest season, fertilization type and the method of obtaining the essential oil (Gobbo-Neto; Lopes, 2007; Bakkali et al., 2008). Note that plants from the same species but from different origins can express different patterns of metabolites. group, which presented an LT50 greater than 264 h and a cumulative survival rate of 68%. The two controls, diet with water and food coloring added and diet with aqueous 0.01 g mL-1 Tween® 80 and dye added, formed the fourth group, with an LT50 greater than 264 h and a cumulative survival of 90% at the end of the test (Figure 1). After 72 h of feeding by the caterpillars on the artificial diet containing the LEO, the estimated concentration in causing the death of 50% of the population (LC50) was 1.33 ± 0.05 mg mL-1 diet. The LC90 was estimated at 2.81 ± 0.26 mg mL-1 diet, and the estimated LC20 was 0.84 ± 0.06 mg mL-1 diet (χ2 = 395.68; df = 351; p = 0.1088). Time-concentration-mortality responses for FAW fed diets containing LEO The C. flexuosus essential oil presented insecticidal activity for S. frugiperda caterpillars. The survival analysis of S. frugiperda caterpillars fed a diet containing LEO produced four congener groups (χ2 = 321; df = 13; p ≤ 0.01) for which the data were fitted to the Weibull distribution (D = 0.054409, p-value = 0.4092). The caterpillars fed diets containing the essential oil at the highest concentrations (2.25, 2.5 and 4.0 mg mL-1 diet) formed the first group with the LT50 of only 18.85 h, and 100% of the insects were dead after 240 h. The second group consisted of treatments at intermediate concentrations (1.5, 1.75 and 2.0 mg mL-1 diet), with the LT50 of 106.5 h and accumulated survival at the end of the trial evaluation period of only 28%. The lowest concentrations (0.5 to 1.4 mg mL-1) formed the third The LEO was toxic to FAW, corroborating results in other studies in which the insecticidal activity of essential oil for S. frugiperda from other plant species of the genus Cymbopogon was verified, such as Cymbopogon winterianus (Silva el al., 2016; Silva et al., 2017a) and Cymbopogon citratus (Knaak et al., 2013). Specifically, for C. flexuosus, this report is the first of insecticidal activity against S. frugiperda, although this species is known for insecticidal activity on other lepidopteran species (Hernández-Lambraño; Caballero- Gallardo; Olivero-Verbel, 2014). In the present study, fast insecticidal activity was also verified for the highest concentrations of LEO (LT50 of only 18.85 h). This result was reinforced by analyzing Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Figure 1: Survival curves for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. With: Group 1 = diet with C. flexuosus essential oil added (2.25, 2.5 and 4.0 mg mL-1 diet); Group 2 = diet with C. flexuosus essential oil added (1.5, 1.75 and 2.0 mg mL-1 diet); Group 3 = diet with C. flexuosus essential oil added (0.5, 1.0, 1.1, 1.2, 1.3 and 1.4 mg mL-1 diet); Group 4 = diet with water and dye added and diet with aqueous 0.01 g mL-1 Tween® 80 added. Figure 1: Survival curves for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Sublethal effects of LEO for FAW In this context, the toxicity of LEO is consistent with other studies that evaluated the insecticidal potential of Cymbopogon species on Lepidoptera. Labinas and Crocomo (2002) verified insecticidal activity of the essential oil of C. winterianus at the concentration of 5.0 mg mL-1 for S. frugiperda neonate caterpillars, conferring 85% mortality. Kolani et al. (2016) observed a strong antifeedant effect on third instar caterpillars of the essential oil of Cymbopogon schcoenanthus (LC50 52.39 mg mL-1) and inhibition of adult emergence of Plutella xylostella based on a feeding method. Murcia-Meseguer et al. (2018) applied 1 µL of C. winterianus at the concentration of 500 mg mL-1 to third instar caterpillars of Spodoptera exigua and verified a mortality of 42%, in addition to developmental interruptions and reduction in pupal size. The treatment consisting of artificial diet with added LEO (1.35 mg mL-1 diet) caused 65.83% mortality after 264 h of evaluation with a median lethal time (LT50) of 102.6 h. For the concentration of 0.675 mg mL-1 essential oil, the accumulated mortality was 29.17%, and the LT50 was greater than 264 h. The control diets with water and food coloring agent added and with aqueous 0.01 g mL-1 Tween® 80 and dye added did not differ significantly from one another, with accumulated survival of 94% (χ2 = 141.66; df = 3; p ≤ 0.05). The data were fitted to the Weibull distribution (D = 0.031315, p-value = 0.9729) (Figure 2). In addition to causing mortality, LEO increased the duration of the larval stage of S. frugiperda. For caterpillars that were fed an artificial diet containing essential oil at concentrations equivalent to the LC50 and LC20, an average increase in larval stage duration of up to 23% was observed The values of LC50 found in the present study demonstrate the potential of C. flexuosus for the control of S. frugiperda, because the LC50 value (1.33 ± 0.05 mg Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Figure 2: Survival curve for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of the Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. With: Group 1 = diet with C. flexuosus essential oil added at the concentration equivalent to the LC50 (1.35 mg mL-1 diet); Group 2 = diet with C. Time-concentration-mortality responses for FAW fed diets containing LEO With: Group 1 = diet with C. flexuosus essential oil added (2.25, 2.5 and 4.0 mg mL-1 diet); Group 2 = diet with C. flexuosus essential oil added (1.5, 1.75 and 2.0 mg mL-1 diet); Group 3 = diet with C. flexuosus essential oil added (0.5, 1.0, 1.1, 1.2, 1.3 and 1.4 mg mL-1 diet); Group 4 = diet with water and dye added and diet with aqueous 0.01 g mL-1 Tween® 80 added. Figure 1: Survival curves for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. With: Group 1 = diet with C. flexuosus essential oil added (2.25, 2.5 and 4.0 mg mL-1 diet); Group 2 = diet with C. flexuosus essential oil added (1.5, 1.75 and 2.0 mg mL-1 diet); Group 3 = diet with C. flexuosus essential oil added (0.5, 1.0, 1.1, 1.2, 1.3 and 1.4 mg mL-1 diet); Group 4 = diet with water and dye added and diet with aqueous 0.01 g mL-1 Tween® 80 added. Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda 413 the risk function of the survival curve in which the (alpha) scale parameter was less than 1 (0.67) and therefore decreasing, which was evidence of significant mortality of caterpillars in the first hours of evaluation, suggesting the interaction of the constituents with sites of action in the nervous system of the caterpillars. mL-1 diet) is comparable to that of other natural products that are considered promising for use in the control of FAW, such as secondary metabolites from stem bark of Duguetia lanceolata A.St.-Hil. (Annonaceae) (LC50 946.5 μg mL-1 diet) (Alves et al., 2016). Sublethal effects of LEO for FAW flexuosus essential oil added at the concentration equivalent to the LC20 (0.675 mg mL-1 diet); Group 3 = control diets with water and food coloring agent added and with aqueous 0.01 g mL-1 Tween® 80 and dye added. Figure 2: Survival curve for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of the Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. With: Group 1 = diet with C. flexuosus essential oil added at the concentration equivalent to the LC50 (1.35 mg mL-1 diet); Group 2 = diet with C. flexuosus essential oil added at the concentration equivalent to the LC20 (0.675 mg mL-1 diet); Group 3 = control diets with water and food coloring agent added and with aqueous 0.01 g mL-1 Tween® 80 and dye added. Figure 2: Survival curve for Spodoptera frugiperda caterpillars fed artificial diet containing different concentrations of the Cymbopogon flexuosus essential oil. Where: S (t) = exp(-(time/δ)α); δ = skewness parameter; α = scale parameter. With: Group 1 = diet with C. flexuosus essential oil added at the concentration equivalent to the LC50 (1.35 mg mL-1 diet); Group 2 = diet with C. flexuosus essential oil added at the concentration equivalent to the LC20 (0.675 mg mL-1 diet); Group 3 = control diets with water and food coloring agent added and with aqueous 0.01 g mL-1 Tween® 80 and dye added. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 414 OLIVEIRA, E. R. de et al. in relation to that of the controls. However, for pupae weights, no significant difference was detected among treatments (Table 2). mortality of 71%. The third group consisted of treatments with concentrations equivalent to the LC20 values of the LEO (0.675 mg mL-1) and citral (0.521 mg mL-1), with an LT50 greater than 264 h and accumulated survival of 69.9%. The two controls, diet with water and food coloring agent added and diet with aqueous solution Tween® 80 added, formed the fourth group with a cumulative survival of 98.33% (Figure 3). This effect was also observed in other studies in which the action of botanical insecticides was evaluated on lepidopteran pests (Ansante et al., 2015; Cruz et al., 2016; Hummelbrunner; Isman, 2001; Kaleeswaran et al., 2018). Sublethal effects of LEO for FAW With regard to integrated pest management, the increase in the larval period observed with sublethal doses of insecticidal plants may be an important strategy when associated with biological control, for example. Essential oils generally show greater insecticidal activity than that of any of the components in their chemical constitution. However, this activity may be linked to synergistic or antagonistic interactions among the structural components of an essential oil in which more than one active constituent is responsible for bioactivity (Akhtar et al., 2012; Heshmati Afshar et al., 2017; Jiang et al., 2009). The increase in FAW larval phase duration did not lead to a reduction in pupal weight. This finding, combined with the rapid mortality, suggested a neurotoxic effect of the essential oil. The requirement for caterpillars to feed for a longer period may be associated with the higher energy expenditure required by an insect to metabolize the toxic compounds in the essential oil. However, in the present study, the insecticidal activity of the LEO was statistically equal to that of its major constituent (citral). Based on this result, the toxicity of this essential oil was due to the action of citral. This result of the current study is similar to that found by Tak, Jovel and Isman (2016) when evaluating the insecticidal activity of Cymbopogon citratus Stapf. (Poaceae) essential oil and its major constituents citral and limonene for Trichoplusia ni Hübner (Lepidoptera: Noctuidae). These authors verified that the citral toxicity was similar to that of the essential oil and that the insecticidal activity was attributed to this constituent. Similar results were also observed when the toxicity of Lippia alba (Mill) N. E. Brown essential oil was evaluated for S. frugiperda caterpillars, and the insecticidal property of this plant was also attributed to citral (Niculau et al., 2013). Averages followed by the same letter in the column do not differ by the Scott-Knott test (р ≤ 0.05%). nsValues were not significantly different. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Toxicity to citral for FAW flexuosus essential oil and LC20 (0.521 mg mL-1) of citral. Group 4: Controls, diet with water and food coloring agent added and diet with aqueous 0.01 g mL-1 Tween® 80 and dye added. Figure 3: Survival of Spodoptera frugiperda caterpillars after exposure to artificial diet containing Cymbopogon flexuosus essential oil and its pure major constituent (citral) at different concentrations. Where: S (t) = exp(- (time/δ)α); δ = skewness parameter; α = scale parameter. Group 1: LC90 (2.053 mg mL-1) of C. flexuosus essential oil and LC90 (1.58 mg mL-1) of citral. Group 2: LC50 (1.35 mg mL-1) of C. flexuosus essential oil and LC50 (1.042 mg mL-1) of citral. Group 3: LC20 (0.675 mg mL-1) of C. flexuosus essential oil and LC20 (0.521 mg mL-1) of citral. Group 4: Controls, diet with water and food coloring agent added and diet with aqueous 0.01 g mL-1 Tween® 80 and dye added. Although some research has been conducted, the mode of action of citral in insects is not well elucidated. Citral can reversibly inhibit purified AChE from the brain of Galleria mellonella L. (Lepidoptera: Pyralidae) (Keane; Ryan, 1999) and also promotes a similar neurophysiological effect on the neuromodulator, octopamine, in cockroaches (Price; Berry, 2006). However, to date, no assays have been performed that evaluate the inhibition of AChE purified from the brain of S. frugiperda by citral. Although the amino acid sequences in the AChEs of insects are well conserved, evolutionarily intraspecific differences in the amino acid residues are verified (Pang et al., 2009). Thus, because of the rapid mortality observed in the present study, only that citral acts on the nervous system of S. frugiperda can be suggested. Ahmed et al., 2012), citral and LEO caused 50% inhibition of enzyme activity at concentrations 405 and 654-fold higher, respectively, than those required to cause the same inhibition value as the positive control, methomyl commercial insecticide (Table 3). These values suggest low toxicity for non-target organisms.i This finding did not exclude the possibility that the mode of action of citral in S. frugiperda occurred through the inhibition of AChE, as verified in a study in which purified AChE was used from the brain of another lepidopteran species (Keane; Ryan, 1999). This AChE was employed because mammalian AChE differs from that found in insects by an amino acid residue, known as the insect-specific cysteine residue (Jankowska et al., 2018). Toxicity to citral for FAW Citral, the major constituent of the C. flexuosus essential oil, when evaluated at concentrations equivalent to those estimated in the quantitative chromatographic analysis of the essential oil, showed insecticidal activity for S. frugiperda caterpillars that was statistically equal to that of the C. flexuosus essential oil. Based on survival analysis, four congener groups were formed. The first group was formed by treatments corresponding to the LC90 of the LEO (2.053 mg ml-1) and the LC90 of citral (1.58 mg mL-1) with an LT50 of 44.5 h and a cumulative mortality of 93%. Similarly, the treatments with the LC50 of the LEO (1.35 mg mL-1) and the LC50 of citral (1.042 mg mL-1) formed the second group, with an LT50 of 120.5 h and accumulated Table 2: Duration of the larval stage (average ± standard error) of Spodoptera frugiperda when 48-h-old larvae were fed an artificial diet containing Cymbopogon flexuosus essential oil. Table 2: Duration of the larval stage (average ± standard error) of Spodoptera frugiperda when 48-h-old larvae were fed an artificial diet containing Cymbopogon flexuosus essential oil. Treatment Larval period (days) Pupa weight (mg)ns Water + Dye 16.90±0.16 a 243.83±2.25 Water + Dye + 1% Tween® 80 17.32±0.14 a 243.12±2.75 C. flexuosus – 0.675 mg mL-1 diet 18.36±0.37 b 242.17±3.32 C. flexuosus – 1.35 mg mL-1 diet 20.85±0.72 c 241.81±4.25 р ≤ 0.05 0.0000 0.968 F 24.472 0.0855 df 3 3 were fed an artificial diet containing Cymbopogon flexuosus essential oil. Averages followed by the same letter in the column do not differ by the Scott-Knott test (р ≤ 0.05%). nsValues were not significantly different. Averages followed by the same letter in the column do not differ by the Scott-Knott test (р ≤ 0.05%). nsValues were not significantly different. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda 415 Figure 3: Survival of Spodoptera frugiperda caterpillars after exposure to artificial diet containing Cymbopogon flexuosus essential oil and its pure major constituent (citral) at different concentrations. Where: S (t) = exp(- (time/δ)α); δ = skewness parameter; α = scale parameter. Group 1: LC90 (2.053 mg mL-1) of C. flexuosus essential oil and LC90 (1.58 mg mL-1) of citral. Group 2: LC50 (1.35 mg mL-1) of C. flexuosus essential oil and LC50 (1.042 mg mL-1) of citral. Group 3: LC20 (0.675 mg mL-1) of C. Toxicity to citral for FAW Few studies compare the inhibition of AChE in vertebrates with that in insects; however, differences are detected (Picollo et al., 2008; Jankowska et al., 2018).i Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 Inhibition of enzymatic activity of the AChE Field and semi-field studies must be conducted to assess whether the same pattern of results obtained in laboratory studies is maintained in the field, both for S. frugiperda and for other insects and food crops. This report is the first showing the use of LEO to control S. frugiperda. When AChE was used from electric eel (E. electricus), which is commonly used as a standard for seeking substances that inhibit AChE in the nervous system of humans, particularly for the treatment of neurogenerative diseases (Bozkurt et al., 2017; Mesquita et al., 2018) and pesticide monitoring (Assis et al., 2012; Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 416 OLIVEIRA, E. R. de et al. Concentration % Inhibition ± SD % Inhibition ± SD % Inhibition ± SD (mg.mL-1) C. flexuosus Citral Control (methomyl) 0.01 3.88 ± 2.14 3.70 ± 0.51 73.64 ± 0.23 0.02 5.81 ± 0.91 4.11 ± 0.99 79.86 ± 0.12 0.04 6.41 ± 1.11 7.05 ± 0.28 90.09 ± 0.25 0.07 10.48 ± 1.57 8.55 ± 0.24 98.35 ± 0.18 0.14 18.71 ± 0.05 16.08 ± 0.86 99.89 ± 0.37 0.28 25.59 ± 0.67 31.06 ± 0.52 99.99 ± 0.09 0.56 40.22 ± 0.15 51.57 ± 0.77 99.99 ± 0.07 1.12 61.30 ± 0.21 64.40 ± 0.36 99.99 ± 0.05 2.24 74.36 ± 0.37 75.57 ± 0.56 99.99 ± 0.05 Table 3: Percent inhibition of the acetylcholinesterase enzyme for C. flexuosus essential oil and citral. SD: Standard deviation. bition of the acetylcholinesterase enzyme for C. flexuosus essential oil and citral. CONCLUSIONS ADUKWU, E. C.; ALLEN, S. C. H.; PHILLIPS, C. A. The anti- biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus. Journal of Applied Microbiology, 113(5):1217-1227, 2012. The C. flexuosus essential oil caused high mortality for S. frugiperda caterpillars. To the best of our knowledge, this study is the first to investigate the insecticidal activity of C. flexuosus essential oil for S. frugiperda. When the major constituent (citral) was evaluated for toxicity to S. frugiperda, citral was affirmed as the monoterpenoid responsible for the insecticidal activity. The high IC50 values required to cause inhibition of AChE indicated selectivity for non-target organisms. Thus, because of the rapid mortality observed in the present study, citral possibly acted on the nervous system of S. frugiperda. Based on the results of this study, citral and C. flexuosus have promise for use in the development of new products for the control of S. frugiperda. AHMAD, A.; VILJOEN, A. The in vitro antimicrobial activity of Cymbopogon essential oil (lemon grass) and its interaction with silver ions. Phytomedicine, 22(6):657-665, 2015. AHMED, M. et al. Toxicological effect of herbicides (diuron and bentazon) on snake venom and electric eel acetylcholinesterase. Bulletin of Environmental Contamination and Toxicology, 89(2):229-233, 2012. AKHTAR, Y. L. et al. Effect of chemical complexity of essential oils on feeding deterrence in larvae of the cabbage looper. Physiological Entomology, 37(1):81-91, 2012. ACKNOWLEDGMENTS ALVES, D. S. et al. Selection of annonaceae species for the control of Spodoptera frugiperda (Lepidoptera: Noctuidae) and metabolic profiling of Duguetia lanceolata using nuclear magnetic resonance spectroscopy. Journal of Economic Entomology, 109(2):649-659, 2016. We thank the Minas Gerais Foundation for Research Support (FAPEMIG) for the financial support for the scholarship to the first author and the National Council of Scientific and Technological Development (CNPq) for the productivity research grant for the fourth and fifth authors. ANARUMA, N. D. et al. Control of Colletotrichum gloeosporioides (Penz.) Sacc. in yellow passion fruit using Cymbopogon citratus essential oil. Brazilian Journal of Microbiology, 41(1):66-73, 2010. REFERENCES AAZZA, S.; LYOUSSI, B.; MIGUEL, M. G. Antioxidant and antiacetyl cholinesterase activities of some commercial essential oils and their major compounds. Molecules, 16(9):7672-7690, 2011. ANSANTE, T. F. et al. Secondary metabolites from Neotropical Annonaceae: Screening, bioguided fractionation, and toxicity to Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Industrial Crops and Products, 74:969- 976, 2015. ADAMS, R. P. Identification of essential oil components by gas chromatography/mass spectrometry. 4th ed. Illinois: Allured, 2007. 804p. Ciência e Agrotecnologia, 42(4):408-419, Jul/Aug. 2018 417 Toxicity of Cymbopogon flexuosus essential oil and citral for Spodoptera frugiperda constituents against the cabbage looper, Trichoplusia ni (Lepidoptera). Industrial Crops and Products, 98:46-52, 2017. ASSIS, C. R. D. et al. 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W1579844804.txt	https://www.intechopen.com/citation-pdf-url/36332	en		Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques	InTech eBooks	2,012	cc-by	10,735	We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 6,900 186,000 200M Open access books available International authors and editors Downloads Our authors are among the 154 TOP 1% 12.2% Countries delivered to most cited scientists Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com 5 Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques L. Ciontea1, M.S. Gabor1, T. Petrisor Jr.1, T. Ristoiu1, C. Tiusan1,2 and T. Petrisor1 1Technical 2Institut University Cluj-Napoca, Material Science Laboratory, Napoca Jean-Lamour, UMR7198 CNRS-Nancy Université, Vandoeuvre les Nancy 1Romania 2France 1. Introduction Within this chapter we would like to address two main applications of Atomic Force Microscopy techniques. The first one illustrates the use of the Atomic Force Microscopy for the optimization of the morphological properties in multilayer stacks dedicated to spintronic devices, in which the electric current flows perpendicular to the layers (currentperpendicular-to-plane CPP geometry). The second part of our chapter presents the use of the Magnetic Force Microscopy as a tool for the micro-magnetic characterization of magnetic thin film dedicated to interface systems with high temperature superconductors. The performances of both spintronic and superconductor/ferromagnet interface devices are directly related to the optimal structural and magnetic properties of the constituent thin films. During the process of the complex sample growth, the structural, morphological and magnetic characterization represents one of the most important steps. Various ex-situ techniques are involved for characterization at microscopic and macroscopic scale. For the multilayer stacks, which require flatness and continuity of the constituent layers, the near field microscopy techniques represent one of the most commonly used tools. The standard atomic force microscopy (AFM) allows to extract precise information about the thin film surface topology in terms of roughness and morphology. This information is furthermore correlated with the crystallographic properties of the layers determined by diffraction techniques (X-Ray, electrons…). The magnetic force microscopy (MFM) operating mode provides a complete characterization of the micromagnetic properties of a magnetic film. This analysis at a microscopic scale is often correlated to the macroscopic magnetic properties measured using the magnetometry techniques. 2. The use of AFM technique for the characterization of multilayer stacks for spintronic devices After the discovery in 1989 of the giant magnetorezistive effect in magnetic multilayers by A. Fert and P. Grunberg (Baibich et al 1988, Binash et al 1989), the spintronics became one of www.intechopen.com 104 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale the most attractive research fields from both fundamental and applicative point of view. The spintronic devices, composed by alternating magnetic and nonmagnetic multilayer structures, represent nowadays one of the major issues of the sensors and data storage industries (Wolf et al 2001). The main functional property of a spintronic device is based on the skilful manipulation of the spin of the electrons carrying the charge current. In the current-perpendicular-to-plane (CPP) spintronic devices, the interfacial roughness has to be minimized in order to get flat and abrupt interfaces between the different layers. This avoids the intermixing and provides a perfect continuity with minimum thickness fluctuations of spacers sandwiched between adjacent layers. We chose to show here the characterization steps by AFM in a complex magnetic tunnel junction multilayer stack using as magnetic electrodes the full Heusler Co2FeAl film and the single crystal MgO as insulating barrier. Therefore, the magnetic tunnel junction is constituted of the MgO insulating barrier sandwiched between two magnetic metallic films (Tiusan et al 2007) (transition metal and Heusler alloy). The electric current crosses the insulator by quantum tunneling whose thickness is typically less than 12 monolayers. Therefore, one can easily understand that the extreme control of the interfacial roughness is a key parameter for proper operating the device. Analysis of our samples by AFM provides the surface topography of the layers. This has been correlated with their crystalline structure analyzed by X ray diffraction techniques. Our analysis allowed a deep understanding of the growth mechanisms by the sputtering of the layers constituent of the complex multilayer stack. The Co2FeAl (CFA) Heusler alloy has a cubic structure with a lattice parameter close to 0.573 nm. For this reason, in order to facilitate the epitaxial growth of the Heusler alloy, one can use single crystalline MgO(001) substrates. The MgO has a crystalline structure belonging to the Fm-3m space group, essentially formed by the interpenetration of two face centered cubic sub-lattices containing Mg and O atoms. Taking into account the CFA and MgO lattice parameters, the CFA should normally grow on MgO with the epitaxial relation: CFA(001)[110]\|\|MgO(001)[100], which implies a 45° in-plane rotation of the CFA lattice with respect to the MgO one. In this configuration the lattice mismatch between CFA and MgO is ( 2 / 2 aCFA  aMgO ) / aMgO  3.8% , which is adequate to promote epitaxy. However, in order to better adapt the lattice mismatch between the epitaxial film and the single crystalline substrate one can employ a buffer layer. We explored this possibility by using an epitaxial Cr film as buffer. Cr has a body centered cubic crystalline structure belonging to the Im-3m space group. The lattice parameter is 0.2884 nm and if one assumes a Cr(001)[110]\|\|MgO(001)[100] epitaxial relation of Cr on MgO, the lattice mismatch is 3.15%. Moreover, considering a cube-on-cube epitaxy of CFA on Cr, i.e. CFA(001)[100]\|\|Cr(001)[100] (see figure 1), the lattice mismatch between CFA and Cr is only -0.7%, and as a consequence, Cr is well suited to be used as a buffer layer for the epitaxial growth of CFA films on MgO(001) substrates. 2.1 Chromium buffer layer Although CFA Heusler alloy thin films were also grown on un-buffered MgO (001) single crystal substrates, we will first discuss the epitaxial growth of Cr (001) buffer layer. The process of epitaxial growth of CFA on MgO is essentially similar to that of Cr on MgO, even www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 105 Fig. 1. Schematic representation of the (a) CFA and (b) Cr/CFA bilayer epitaxy on MgO(001) single crystalline substrate. though it is more complicated due to the complex chemical ordering within the Heusler alloy. Therefore, we will first treat, in extensive details, the growth of Cr on MgO and use the drawn conclusions when presenting the epitaxial growth of CFA on MgO. The Cr films were deposited using standard DC magnetron sputtering on polished MgO (001) substrates in a system having a base pressure better than 4x10-9 torr. The Ar working gas pressure was maintained at 1.0 mtorr during sputtering and the deposition rate was around 0.1nm/s. The substrate temperature during growth was varied between room temperature (RT) and 600 °C. High angle specular X-Ray diffraction scans were performed to test the orientations present in the different Cr films. figure 2 shows a typical XRD symmetric scan for the Cr film grown at a substrate temperature of 400°C showing only the (002) film reflection. Regardless of the growth temperature, no evidence of other orientation, except for (002) was found. The inset in figure 2 shows the evolution of the Cr (002) peak with the growth temperature. A large decrease of the Cr (002) peak intensity and position shift can be observed for the sample grown at RT relative to the high temperature deposited ones. This suggests the occurrence of an important change in the structure at atomic level for this sample. Figure 3 shows the evolution of the in-plane and out-of-plane lattice parameters as a function of the growth temperatures extracted from symmetric and asymmetric X-Ray diffraction experiments. A 3.2% expansion of the out-of-plane lattice parameter from the bulk value is observed for the sample grown at RT. This growth with the out of-plane lattice expansion is not accompanied by clear in-plane lattice contraction, the small variation of the in-plane lattice parameter from the bulk value being within the measurement error limits. Nevertheless, a small increase of the in-plane lattice parameter can be argued. This behavior is consistent with the in-plane constant expansion due to the tensile strain in the growth plane produced by the lattice mismatch between MgO and Cr (-3.15%). The out-of-plane lattice distortion is rather difficult to explain. A similar growth temperature dependence of the lattice parameter was observed in triode sputtered V (001) epitaxial films on MgO (001) substrates (Huttel et al., 2005). A complex experimental analysis accompanied with ab-initio simulations (Huttel et al., 2007) showed that this low temperature out-of-plane lattice expansion is a metastable state and that it is most likely due to the inhomogeneous disorder created by the bombardment of the growing film by neutral atoms reflected off the target. Another possible mechanism for the lattice expansion is the residual stress of the thin films produced by sputtering at low pressures (Thornton & www.intechopen.com 106 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale Fig. 2. High angle symmetric scan for the MgO (001)//Cr (16.5nm) film grown at 400°C, showing the presence of Cr (002) reflection, in addition to the MgO (002) one. The peaks marked with S are substrate refections corresponding to the Cu Kα and WLα wavelengths. The inset shows the Cr (002) peak for films deposited at different temperatures. The vertical dashed line marks the position of the Cr bulk 002 reflections. The scans are shifted vertically for better visibility. Hoffman, 1989). At low sputtering pressures, because of the poor thermalization, there is an increase in high energy atoms arriving at the substrate and, due to the momentum transfer, Cr atoms are forced to into spaces too small to accommodate them under existing thermal equilibrium conditions and, as a result, an out-of-plane lattice expansion takes place (Hsieh et al., 2003). Following these assumptions, one should expect that a substrate temperature increase drives the system to its most stable state relaxing the strains and disorder (Huttel et al., 2007). This is indeed the case and for the sample grown at 400 °C a much smaller strain is observed, the out-of-plane lattice parameter being contracted by 0.12% while the in-plane lattice parameter expanded by 0.18% and, consistent with the in-plane tensile stress to the Cr to MgO lattice mismatch. Moreover, for the sample grown at 600 °C the strain is totally relaxed, most likely by formation of misfit dislocations, and the in-plane and out-of-plane lattice parameters are being consistent with the bulk values. As expected, the epitaxial quality of the Cr(001) films improves with increasing the growth temperature. The full width at half maximum (FWHM) of the rocking curves around Cr (002) and (011) reflections as a function of temperature are depicted in figure 3b. The figure indicates a FWHM of 0.74° for the (002) reflection and of 0.91° for the (011) reflection, for the sample deposited at 600 °C. These values are in agreement with the previously reported ones (Harp. & Parkin, 1994; Harp. & Parkin, 1996; Fullerton, 1993) and demonstrate a high degree of epitaxy. The main conclusion from the structural point of view is that it is possible to obtain epitaxial Cr(001) thin films on MgO(001) substrates by DC sputtering starting from room temperature and with improved crystalline quality at higher growth temperatures. Conventionally, it is considered that the epitaxial growth of Cr on insulating substrates by sputtering can be achieved only when the deposition takes place at elevated temperatures (Harp. & Parkin, www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 107 (a) (b) Fig. 3. (a) Evolution of the in-plane and out-of-plane lattice parameters of the Cr films with the growth temperature and of the (b) full width at half maximum of the rocking curves around Cr symmetric (002) and asymmetric (011) reflections. 1994; Harp. & Parkin, 1996; Fullerton et al., 1993a, 1993b), while our findings show a lower growth temperature required for epitaxy. This behavior is most likely related to our specific growth conditions which imply a relative low Ar pressure during deposition. In contrast to evaporation methods, sputtering deposition methods are characterized by an impulse transfer from accelerated energetic particles to the surface atoms of the target. The energy distribution of the sputtered species has a maximum around 5-10eV, presenting a high energy tail with a low percentage of high energy sputtered particles as well, that can increase by decreasing the Ar pressure (Depla & Mahieu, 2008). It has been shown that these energetic particles can modify the growth kinetics trough the formation of a high density of nucleation centers resulted from their energetic impact on the sample surface (Kalff et al., 1997). The maximum of the sputtered particles energy distribution can be shifted to lower energies by the thermalization process (Meyer et al., 1981), i.e. collisions with the working gas atoms between the target and the substrate. The www.intechopen.com 108 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale thermalization process is controlled by the total gas pressure in the sputtering chamber. Thus, by decreasing the Ar pressure, the thermalization is reduced, so the energy of the atoms arriving at the substrate increases, and eventually, they will have enough energy to undergo diffusion on the surface to a high-binding energy site at a lower surface temperature. The higher mobility of the ad-atoms, together with the increase in the density of nucleation centers, would explain why lower growth temperatures are required for epitaxy to occur under our specific growth conditions. Atomic force microscopy (AFM) was used to investigate the effect of the deposition temperatures on the films surface morphology. Figure 4 shows the AFM images for Cr films grown on MgO (001) at different temperatures. The deposition of Cr at room temperature yields a very flat featureless surface with a root mean square roughness (RMS) of 0.14 nm. As the temperature is increased to 400 °C and 600 °C, temperature dependent morphological features start to form. The surface of the 400 °C grown sample exhibits 3D rounded islands, while the surface of the 600 °C grown sample display larger, well defined and regulated ones, with the sides parallel to the substrate [110] and to the [1-10] directions. At the same time, the RMS increases from 0.45 nm for the film deposited at 400 °C to 1.25 nm for the film grown at 600 °C. 2 Fig. 4. AFM images (1x1 μm scans) showing the morphology of the MgO(001)//Cr(001) films grown at different temperatures. www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 109 It is a common feature for the mid to late transition metals that in conditions of thermodynamic equilibrium not to wet an oxide surface (Campbell, 1997). However, our growth conditions are far from thermodynamic equilibrium and in this case the film growth process is governed by kinetic effects. In a simplified picture layer by layer growth occurs when additional islands start to form on the surface after the previous layer has coalesced. Therefore, in order to prevent nucleation before coalescence, the ad-atoms have to be mobile enough to reach the island edges and to have enough energy to overcome the EhrlichSchwoebel energetic barrier (Ehrlich & Hudda, 1966; Schwoebel, 1969) encountered by the ad-atom upon descending a step. In the special case of non-wetting metals on oxides, the energetics has two essential features: the strong attraction to the edge of an island due to the lateral metal-metal bonding (Ei) and the energy difference (ΔE) between the absorption energy of a metal atom a oxide site and on a metal site (Campbell, 1997; Ernst et al., 1993). If the energy difference (ΔE) exceeds the lateral metal-metal bonding energy (Ei), then there will be no stable sites at the edges of the 2D islands and 3D clusters will form starting from the lowest coverage. On the other hand, if the energy difference (ΔE) is smaller than the lateral metal-metal bonding energy (Ei), the ad-atoms will stick to the edges of the 2D islands, provided that thermal energy is high enough for the ad-atom migration across the surface and for the down-stepping to occur. Moreover, the up-stepping activation barriers, which is approximately equal to the lateral metal-metal bonding energy (Ei) have to be large as compared to the thermal energy, so that the thermal thickening of the islands to be kinetically disallowed. This pseudo layer-bylayer growth mode usually leads to the formation of 2D islands below a critical coverage, after which additional layers grow in a layer-by-layer mode on top of these islands. The critical coverage is directly related to the density of 2D islands which is, in turn, linked to the density of nucleation centers. In sputtering deposition, the low percentage of high energy sputtered particles that can reach up to hundreds of eV of energy leads to the formation of a high density of nucleation centers (Kalff, 1997). This will eventually increase the critical coverage giving rise to the formation of at continuous layers at relative low growth temperatures. The arguments above, of kinetically governed growth, can explain the observed morphology of the Cr film deposited at room temperature (see figure 4). As the growth temperature increases, the ad-atoms have enough energy to surface diffuse overcoming the up-stepping energetic barrier, allowing the system to move in the actual thermodynamic equilibrium configuration of 3D islands on the MgO surface (see figure 4). The sample grown at 600 °C exhibits square shaped 3D crystalline clusters with the sides parallel to the substrate [110] and [1-10] directions proving the 45° in-plane rotation epitaxy (see figure1) of Cr on MgO. Another interesting feature of the 3D islands is the narrow size distribution. This aspect is connected with strain and strain relief mechanism. In lattice mismatched epitaxy, after an island is formed, the misfit strain relaxation in the island causes a strain concentration at the edges which increases monotonically with increasing island size. Since the ad-atoms tend to diffuse from the high strain sites to lower strain sites, the strain concentration at the edges will translate into an additional kinetic barrier for the ad-atoms to diffuse to the islands, thus the island growth rate is slowed down as the island size increases, leading to the formation of homogenously sized islands (Chen & Washburn, 1996). www.intechopen.com 110 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale The main result from the X-Ray Diffraction and Atomic Force Microscopy studies is that crystallinity improvements of Cr films deposited on MgO comes with the cost of surface morphology quality (see figures 5, 6 and 7). Since epitaxy is favored at high temperatures, while at surfaces al low temperatures it is difficult to select deposition temperatures that allow for both high degree of epitaxy and flat surfaces. However, the sample deposited at room temperature exhibits very good morphologic properties, while preserving the epitaxy. Therefore, to overcome the challenge of growing Cr films with both flat surface and high degree, one can choose to deposit the films at room temperature and to subsequently perform high temperature annealing stages. Figure 5 shows AFM images for two samples one deposited at room temperature, and the other one deposited at room temperature and post-annealed in vacuum at 600 °C for 20 minutes. As indicated by the images, the postannealing process preserves and even improves the surface morphology, the root mean square roughness being reduced from 0.17 nm down to 0.12 nm after annealing. As indicated by the X-Ray diffraction measurements (see figure 3), the as deposited film shows a rather large tetragonal lattice distortion that is associated with the growth process. After annealing the film at 600 °C the distortion is relaxed and the in-plane and out-of-plane lattice parameters regain the bulk value. The epitaxial quality of the film increases with annealing, as reflected by the decrease of the FWHM of the rocking-curve around the (002) reflection from 2.54° to 1.51°. The crystallinity also increases after annealing, the mean crystallite size reaching a value comparable with the thickness of the film (16.5 nm). Figure 6 shows a High Resolution Transmission Electron Microscopy (HRTEM) image of a Cr film deposited on MgO at room temperature and subsequently annealed at 600 °C. The image confirms the high quality single crystalline nature of the Cr film. Fig. 5. AFM images (1x1μm2 scans) showing the morphology of the MgO(001)//Cr(001) films grown at room temperature and post annealed at 600 °C. In conclusion, we showed that in order to obtain smooth epitaxial Cr films on MgO(001), by sputtering deposition, a two step process must be employed. First, the deposition must take place at low temperatures, to obtain textured films with a flat surface morphology. Secondly, in order to improve the structural properties of the films, a high temperature annealing stage must be performed. Following this procedure, the flat surface morphology is preserved, while the structural properties of the films are improved. www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 111 Fig. 6. High Resolution Transmission Electron Microscopy image of an MgO//Cr film deposited at room temperature and annealed at 600°C, confirming the single crystalline growth of Cr on MgO (by courtesy of E. Snoeck CEMES, TOULOUSE). 2.2 Co2FeAl (CFA) epitaxial films In a first stage we deposited CFA Heusler alloy thin films on un-buffered MgO(001) single crystal substrates by RF and DC sputtering. As in the case of Cr films, before deposition, the substrates were degassed in-situ at 600°C for 20 minutes. After cooling down to room temperature, a 5 nm thick MgO coating layer was deposited on the substrate. The CFA films with a thickness of 50 nm were sputtered at room temperature from a stoichiometric target (Co50%Fe25%Al25%) at 30 W under an Ar pressure of 1 mtorr. As seen in the case of the Cr buffer layer, depositing the films at high temperatures will increase the crystalline properties but also will degrade the surface morphology through the formation of large 3D clusters. In order to test if this is also valid for CFA we have grown two types of films: one consisting of films deposited at high temperatures and the other of films grown at room temperature and subsequently high temperature vacuum annealed. The surface morphology of the layers was studied by Atomic Force Microscopy and the results are depicted in figure 7. As seen from the figure 7 the films deposited at high temperatures have a granular structure with increasing grain size for higher deposition temperatures. In the case of the films grown at room temperature and ex-situ post annealed the surface is very flat and featureless, this characteristic being maintained even after annealing at 600 °C. Figure 8 shows the evolution of the Root Mean Square (RMS) surface roughness parameter and of the Maximum PeakValley (MPV) distance for CFA films deposited at various substrate temperatures and for CFA films grown at room temperature and ex-situ vacuum annealed. In the case of the annealed samples the RMS and the MPV remain at low values regardless of the annealing temperature. Still, a minimum of RMS of 0.13 nm and MPV of 1.48 nm is obtained for the sample annealed at 400 °C, with a small increase of RMS to 0.18 nm and of the MPV to 2.8 nm for the layer annealed at 600°C. In case of the samples deposited at various substrate temperatures, the RMS and the MPV distance show a monotonous increase from RMS=0.13 nm and MPV =1.48 nm, for the RT deposited sample, to RMS=8.3 nm and MPV =64 nm, for www.intechopen.com 112 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale the films grown at 600°C. For the latter, the MPV distance is even larger than the expected film thickness (50 nm). As seen form figure 7 the film consists of very large 3D clusters and most likely that the layer is not even continuous. This is expected since in conditions of thermodynamical equilibrium the metallic films have the tendency not to wet an oxide surface (see discussion in the previous paragraph). Fig. 7. AFM images showing the surface morphology of CFA films deposited at high temperatures (a), (b) and (c) and deposited at room temperature (d) and ex-situ vacuum annealed (e) and (f). www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 113 Fig. 8. Evolution of the Root Mean Square (RMS) surface roughness parameter and of the Maximum Peak-Valley (MPV) distance as a function of temperature for (a) CFA films deposited at various substrate temperatures and (b) for CFA films grown at room temperature and ex-situ vacuum annealed. Our analysis indicated that, due to the granular morphology, the CFA films deposited at high temperatures are not compatible with the manufacturing of magnetic tunnel junctions. Therefore, our efforts have been focused on the room temperature deposited and subsequently annealed samples. The 2θ/ω specular X-Ray diffraction measurements (see figure 9 (a) indicated that the CFA films are epitaxial even when deposited at room temperature and that the crystalline quality of the films increases with annealing (see figure 9 b), the best crystalline properties being obtained for the film annealed at 600 °C. However, the morphological properties of the film annealed at 600 °C are somewhat degraded relative to the one annealed at 400 °C, making the choice of the optimal annealing temperature relative difficult, since both the crystalline and morphological properties of the CFA films are of extreme importance for the further development of the magnetic tunnel junction multilayer stack. In addition to the films deposited directly on MgO we have grown epitaxial CFA films using a Cr buffer layer. The buffer layer, with a thickness of 20 nm, was deposited as described in the previous section. To ensure a high crystalline quality of the buffer layer, after growth the Cr film was annealed in-situ at 600 °C. After cooling down to room temperature the CFA films, with a thickness of 50 nm, were deposited by RF sputtering under 1.0 mtorr of Ar working pressure. As in the case of the films grown directly on MgO, the 2θ/ω specular X-Ray diffraction measurements (see figure 10 a) indicated that the CFA films are epitaxial even when deposited at room temperature and that the crystalline quality of the films increases with annealing (see figure 10 b), the best crystalline properties being obtained for the film annealed at the highest temperature. An interesting feature is that the values of the FWHM for the samples deposited on the Cr buffer layer are smaller as compared to the ones recorded for the films deposited directly on MgO (figure 9). This is a direct consequence of the smaller lattice misfit between CFA and www.intechopen.com 114 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale Fig. 9. (a) Specular 2θ/ω X-Ray diffraction patterns for the CFA films as a function of the annealing temperature; (b) FWHM of the rocking curve around the (004) CFA reflection and of the X-Ray diffraction coherence length along [004] direction versus the annealing temperature. Cr (-0.7%), the one than between CFA and MgO (-3.8%). If the lattice misfit is higher, the interfacial strain energy also increases. This energy is normally relaxed by the formation of dislocations in the film, which, of course, degrade the crystallinity of the epilayer. This is also sustained by the evolution of the mean size of the X-Ray coherently diffracting domains as a function of annealing temperature. The mean size has the same behavior, as in the case of MgO deposited CFA films (figure 9), correlating perfectly with the decrease of the FWHM, but the values are larger for the Cr buffered films. The increased size of the coherently diffracting domains indicates that the epitaxial perfection of the CFA lattice is spread over larger distances, the density of lattice dislocations being lower than in the case of the films grown on MgO. The surface morphology of the CFA films deposited by RF sputtering on Cr buffered MgO substrates was investigated by Atomic Force Microscopy. Figure 11 shows AFM images for the CFA films as-deposited at room temperature and post-annealed at 600 °C for 20 minutes. One can see that the surface of both films are very flat and featureless. For the as deposited film, the RMS roughness parameter is around 0.1 nm and slightly smaller for the annealed one. The MPV distance decreases from 1.7 nm, for the as-deposited film, to just less than 1 nm for the www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 115 (a) (b) Fig. 10. (a) Specular 2θ/ω X-Ray diffraction patterns for the Cr/CFA films as a function of the annealing temperature; (b) FWHM of the rocking curve around the (002) CFA reflection and of the X-Ray diffraction coherence length along [002] direction versus the annealing temperature, indicating the improvement of the crystallinity of the films with annealing. annealed one. These results indicate that, contrary to the case of the CFA films deposited directly on the MgO substrate, both the crystalline properties and the surface morphology of the Cr buffered CFA film improve with the increase of the annealing temperature. The growth of a high quality tunnel barrier in a magnetic tunnel junction is in general a very difficult task since the tunnel barrier must be very thin, typically 1-2 nm, and pinhole free in order to avoid metallic shorts in the junction. This implies that the lower electrode of the junction to be extremely flat, which is indeed the case of the Cr buffered CFA film annealed at 600°C. Using this optimized lower electrode we have grown the subsequent MgO tunnel barrier and the upper CoFe electrode. The AFM measurements have indicated a very flat morphology for the tunnel barrier (see figure 12 a), with a RMS roughness around 0.2 nm, which ensure the continuity of the barrier, as also suggested by the HRTEM images in figure 12 b. Moreover, the HRTEM images indicate that the whole magnetic tunnel junction stack is epitaxial. www.intechopen.com 116 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale Fig. 11. AFM images of Cr buffered CFA films as-deposited at room temperature and postannealed at 600°C for 20 minutes. Fig. 12. (a) AFM image on the MgO//Cr(15 nm)/CFA(20 nm)/MgO(2.2 nm) stack indicating the morphology in the tunnel barrier; b) High Resolution Transmission Electron Microscopy image of the magnetic tunnel junction indicating the crystallinity and continuity of the barrier. Based on this analysis, we succeeded to grow and pattern by optical lithography magnetic tunnel junctions which give significant tunnel magnetorezistive effects at room temperature. 3. The use of AFM/MFM techniques for the study of hybrid interface systems between high temperature superconductors and magnetic structures Concerning the second part of the chapter, we present the magnetic force microscopy as a powerful tool to get deep insight in the micro-magnetic properties of a magnetic thin film interfaced with a high temperature superconductor layer. More recently, a new hybrid research domain has emerged: superconducting spintronics, dealing with systems constituted by alternating superconducting and magnetic layers. The aim of these novel www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 117 systems is to drive new functionality of the constituent layers by taking profit on the mutual interfacial interaction between the superconductor and the magnetic film. For instance, vortices and the anisotropy of the electronic transport in the superconductor can be controlled by the stray-fields emerging from a modulated magnetic structure (Carneiro 2007, Hoffman et al 2008, Karapetrof et al 2009). The engineering of the micromagnetic properties in continuous or patterned magnetic thin films represent therefore a powerful tool to control the electronic transport in the superconductor. We illustrate the results concerning the modulation of the magnetic structure of Permalloy thin films via the thickness of the film. We follow the evolution of the magnetization from in-plane to out-ofplane by increasing the thickness and we present a direct correlation between the micromagnetic properties, measured by MFM, and the magnetic properties at macroscopic scale (magnetization hysteresis curves) measured by standard magnetometry. Furthermore, we exemplify the possibility to engineer the magnetic/micromagnetic properties in mezoscopic size magnetic objects patterned by optical lithography. Single objects and arrays of circular shape of mezoscopic dots with lateral size in the micron range, are considered. Alternatively, nanoscopic magnetic objects are elaborated using polystyrene balls shadowing/lift-off technique. In the past decade numerous research groups have addressed the problem of magnetic pinning in superconductors (Aladyshkin et al., 2009). The relevance of this subject lies in the possibility of attaining large pinning forces of vortices in type II superconductors, that are temperature independent, through the interaction between the vortex flux and the magnetization of a magnetic structure, i.e. ferromagnetic films, ferromagnetic micro-, or nanostructures. Large scale applications of superconductivity, such as energy transport, superconducting magnets, engines, generators, etc., require superconducting materials with a critical current density, Jc, greater than 1 MA/cm2 and high irreversibility lines. In order to fulfill these requirements, the only way is to avoid the vortex motion, which is responsible for the energy dissipation in a type II superconductor. Up to date, the method used to block the vortex movement is their pinning on normal impurities present in a superconductor, the socalled normal core pinning. This type of pinning relies on the tendency of the vortex normal core to attach itself to regions in the superconductor where superconductivity is suppressed, in an attempt to minimize the overall system energy. For this reason, normal zones within the superconductor are artificially created in different ways. These zones coexist with the natural occurring normal regions arising from inherent growth defects, such as grain boundaries, dislocations and the presence of secondary phases. Several ways of externally inducing normal regions in superconductors have been demonstrated. They include: irradiation by swift heavy ions, artificially introduced regular arrays of holes, artificially introduced nanoparticles that create columnar defects in superconducting films, or surface decoration of the substrate upon which the superconducting film is grown (Civale et al., 1991; Augieri et al., 2010; Mele et al.,2006; Sparing et al., 2007). However, classic nonmagnetic pinning becomes ineffective at high temperatures making it difficult to imagine practical applications of HTS materials. In this context, magnetic pinning, originating from the Zeeman interaction between a magnetization and the magnetic flux of the SC vortices, may be a valid alternative for www.intechopen.com 118 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale effective vortex pinning applications, as the pinning potential created by a magnetic structure, in this case displaying a stripe domain structure varying along the x axis, is expressed as U mp  Φ 0 M ( x )ds (3) ds being the superconducting layer thickness and M(x) the magnetization value, which, neglecting the domain wall contributions, takes the values of ±M0 (Bulaevskii et al., 2000). Even though the above expression of the pinning potential is calculated for a particular case of magnetic configuration, the point it stands for may be generalized to any magnetic distribution, and it is that magnetic pinning potential is independent of temperature. Of course, the Curie temperature of the ferromagnet has to be high enough so that there is no significant variation of the magnetization with temperature, in the superconducting regime of the SC film. This fact makes this type of pinning as the ideal case of superconducting vortex pinning, suitable for high temperature superconductors. Equating the magnetic pinning force, calculated from the pinning potential (3), as Ump/l, with l as the domain width, to the Lorentz force acting on the vortex line, stemming from the transport current in the SC layer, J0ds/c, gives a rough estimate of the critical current Jc ~ cM0 / l. (4) It can be seen that within the model proposed, the critical current density, Jc, increases as the perpendicular component of the magnetization, M0 increases, justifying the use in magnetic pinning experiments FM structures that exhibit a perpendicular anisotropy. In the following paragraphs we give an account of the results obtained on some magnetic systems exhibiting out-of-plane anisotropy investigated for possible magnetic pinning applications of superconducting vortices. They include permalloy continuous thin films, permalloy micronic disks and cobalt nanostructures. A particular emphasis is placed on the magnetic characterization of the above structures by means of magnetic force microscopy (MFM) and the information this analysis may give regarding the magnetic pinning characteristics of the respective investigated systems. 3.1 Permalloy thin films In the case of Permalloy, Py, (NixFe(1-x), x=19-21 at.%) thin films, perpendicular anisotropy originates from a negative magnetostriction constant correlated with an in- plane tensile strain of the films. In this case, the anisotropy constant is given by the expression (Saito et al., 1964): KU  3 L 2 (5) where  is the magnetostriction constant and  represents the strain of the film. The orientation of the magnetization within Py thin films is determined by the competition between the perpendicular anisotropy energy term and the magnetostatic energy. Since the uniaxial anisotropy is not strong enough to determine the perpendicular orientation of the magnetization with respect to the film surface, the resulting configuration corresponds to an out-of-plane deviation of the magnetization. The domain pattern stabilized by this www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 119 orientation is comprised of stripes in which the magnetization points alternatively, outwards and inwards with respect to the film surface, Figure 13. Because the deviation angle, , of the magnetization is less than 90o, this domain configuration is referred to as a weak stripe domain pattern. A consequence of the presence of weak stripe domains is the so called rotatable anisotropy. This phenomenon consists in the orientation of the domain stripes along the direction of a relatively weak, in-plane applied magnetic field, and the persistence of this orientation after the field is removed. The field magnitude is typically of few hundreds of Oe. The two properties of thin Py films recommend them for fundamental studies of magnetic pinning in superconductors, as they allow for the preparation of two different states with respect to the effects of vortex pinning, as illustrated in Figure 14. These two states consist in the parallel and perpendicular orientation of a current density, J, which is injected into a superconducting YBa2Cu3O7 (YBCO) strip, with respect to the orientation of the magnetic stripe domains of a Py film deposited on top. As it can be seen, the Lorentztype force acting on the superconducting vortices is directed perpendicularly to the current density. For the perpendicular configuration, Figure 14a, the vortices will not be acted upon by any pinning force as the vortex motion takes place along the stripe domains where the magnetization is constant. This configuration is referred to as a vortex guide configuration. On the other hand, in the parallel configuration, Figure 14b, the force due to the current density acts on the vortices so as to move them across the magnetic domains. In this case the vortex motion will be impeded by the presence of the periodic modulation of the magnetization. Thus, the vortices will by pinned by a periodic magnetic pinning potential. In order to be able to fabricate and study such systems, a careful analysis of the magnetism of the Py thin films is required. For this purpose we have fabricated several polycrystalline thin Py films by means of dc magnetron sputtering, deposited on Si (111) single crystal substrates. The thickness of the films was varied between 270 and 720 nm. The as deposited films were analyzed by means of vibrating sample magnetometry (VSM) and magnetic force microscopy (MFM). Fig. 13. Schematic representation of the spin distribution within the weak stripe domain configuration, as proposed by Chikazumi (Chikazumi, 1997). It is to be noted that the out-ofplane component of the magnetization has a sine variation along the y direction. The out-ofplane deflection angle of the spins is denoted by  The magnetization dependence on the externally applied field, Figure 15, has the characteristic shape of the films that exhibit weak stripe domains. The magnetic field was applied parallel to the film surface plane. The linear decrease of the magnetization from saturation is observed with a small hysteresis. The decrease is due to the out-of-plane rotation of the film magnetization, corresponding to the weak stripe formation. The www.intechopen.com 120 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale (a) (b) Fig. 14. Schematic representation of the vortex guide (a) and the periodic pinning potential (b) configurations of a Py/YBCO heterostructure. Fig. 15. Hysteresis loops of the (a) 270 nm, (b) 646 nm and (c) 730 nm thick Py thin films. The shape of the loop is typical for films having weak stripe domain configuration; (d) Variation of the normalized remanent magnetization, Mr/Ms, and of the in-plane saturation field, H\|\|sat, as a function of Py film thickness. hysteresis on the other hand, may be explained by the existence of a slight difference in the stripe domain pattern in the two branches. In order to test the expected rotatable magnetic anisotropy, the cycles were performed applying the magnetic field in two different perpendicular directions. No modification of www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 121 the loops were observed, proving thus the existence of rotatable anisotropy. Figure 15d presents the thickness dependence of two characteristic magnetic quantities, the magnetization at remanence, Mr, and the in-plane saturation field, H\|\|sat. As the film thickness increases, the magnetostatic energy of surface charges decreases, allowing for the perpendicular anisotropy to manifest by an increase of the deviation angle of the spins, , with respect to the film surface plane. This increased deviation leads to a decrease of the inplane magnetization remanence. Consequently, the larger magnetization perpendicular to the film plane leads to a higher in-plane saturation field. With respect to our goal to fabricate and study hybrid interface SC/FM systems for vortex pinning, higher  values are expected to have a more pronounced influence on the superconducting properties of the bottom SC layer. Concomitantly, as analytical calculations have shown (Saito et al., 1964, Chikazumi, 1997, Murayama, 1966), an increase of film thickness also leads to an increase of stripe periodicity, i.e. domain width. In the following we present the results of MFM measurements on the film domain structure and how it is affected by the film thickness. The domain structure of the films having thicknesses of 270 nm, 460 nm and 720 nm are shown in Figure 16. As it can be seen, the expected stripe domain configuration is present. Extracting a profile from the above images allows for an evaluation of the stripe wavelength, , and accordingly, of the domain width, d=, Figure 17. The domain width variation as a function of thickness has also been calculated according to the three models (Saito et al., 1964, Chikazumi, 1997, Murayama, 1966). For the calculation, the physical parameters entering the domain width expression were taken from (Ben Yousseff et al., 2004), so that Ms=826 emu/cm3, the out-of-plane anisotropy constant, KU=5×104 erg/cm2, while the exchange stiffness constant was taken to be A=1×10-6 erg/cm. Comparing the measured results with the three models, it is noted that the initial model of stripe domains, proposed by Saito et al. (Saito et al., 1964), in which the spin direction varies perpendicularly to the stripes in a zig-zag as a function of position, is the worst approximation to the domain width value. A sine variation (Chikazumi, 1997) of the spin direction yields a better approximation of the domain width. Yet, the best results are found using the model proposed (Murayama, 1966). Because of the fact that their model allows for the highest freedom in spin ordering, allowing for a variation of the spins with respect to all the coordinate axes, they are able to take into account the presence of closure domains at film surface. As confirmed by numerical simulations run on low out-ofplane anisotropy Py thin films (Vlasko-Vlasov et al., 2008, Ben Yousseff et al., 2004), the model proposed by Murayama is the closest in describing the actual physical picture in these systems, and thus yields the most satisfactory result in predicting the domain width dependence on the film thickness. Fig. 16. 5 m×5 m MFM images of the (a) 270 nm, (b) 460 nm and (c) 720 nm thick Py films. www.intechopen.com 122 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale Fig. 17. Domain width of the 270 nm, 460 nm and 720 nm thick Permalloy films. Domain width variation as a function of film thickess, calculated according to the three different models presented in the text. As far as the rotatable magnetic anisotropy is concerned, Figure 18 shows the orientation of the domain stripes in a remanent state after the application of an in-plane magnetic field along different directions. The value of the applied field was set at 650 Oe in order to ensure saturation of all the studied films. As it can be observed, the stripe domains align themselves along the direction of the applied magnetic field and do not change their orientation after the field is removed. The rotatable anisotropy feature of low out-of-plane anisotropy magnetic thin films is essential for the study of the influence of a periodic magnetic field on the superconducting properties of SC thin films, as it allows for a systematic modulation of the stray magnetic field produced by the FM layer (Belkin et al., 2008). Fig. 18. Rotatable anisotropy. Different magnetic stripe orientations correspond to the direction of the applied magnetic field for a 460 nm thick Py film. 3.2 Magnetic micro,- and nanostructures Periodic stripes of alternating "up" and "down" orientation of magnetization with respect to the film surface plane are expected to provide a strong pinning of the SC vortices when www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 123 these alternations are oriented perpendicularly to the direction of the vortex motion. Magnetic micro,- and nanostructures, such as regular dot arrays, provide an additional degree of freedom to the modulation, as they are also subject to a topographic periodicity, given by the specific pattern of the arrays defined by lithographic techniques. In these sense, even the sole presence of such structures in the vicinity of SC films may give rise to a pinning potential of the vortices, as demonstrated by Hoffmann et al. (Hoffmann et al., 2000) in their study of a Nb film deposited on top of a regular array of non-magnetic Ag dots. The flexibility in defining different configurations of dot lattices coupled with their respective magnetic configurations produce commensurability effects. These effects consist in resonant changes of the magnetoresistance of the SC layer, (Hext), with the appearance of equidistant minima having a period in H determined by lattice constant of the magnetic dot arrays. Also, these minima are visible in the field dependence critical current density, Jc(Hext), or in the magnetization curves, M(Hext). These effects are explained considering a strong magnetic pinning potential exerted by magnetic dots on the SC vortices, coupled with a structural match between the dot and vortex lattices. Thus, when the condition that an integer number of vortices per magnetic unit cell is fulfilled, a maximum number of vortices are pinned, producing resistivity minima or Jc maxima of the SC film. The field dependence of these effects is explained by the fact that the vortex lattice parameter is field-dependent. Because of the fact that the vortex lattice is triangular strong commensurability effects are produced by triangular dot lattice. Lateral structuring of the magnetic films into micro or nanostrucutres also influences their magnetic configuration, so that the magnetic state of the film will be altered when it is patterned into dot sutructures. Jubert and Allenspach (Jubert and Allenspach, 2004) constructed a phase diagram of the magnetic states of circular nanometric dots as a function of their thickness and diameter, based on micromagnetic simulations. By varying the lateral size and thickness of the dots one is able to stabilize different magnetic states: in-plane single domain (IPSD), vortex (V) and out-of-plane single domain (OPSD). Carneiro (Carneiro, 2004) calculated the interaction energy between a SC vortex and an inplane and out-of-plane magnetized dipole showing that different attraction interaction profiles exist between the dipole and the vortices in the two cases. While in the case of out-of-plane magnetic dipoles the largest pinning potential arises at the center of the dipole, in the case of an in-plane dipole configuration pinning is present at the edges of the object where the variation of the magnetization is highest. Larger, micronic, dots that may be able to accommodate a multi-domain magnetic configuration are also interesting in the study of FM/SC structures as their net magnetic moment may be tuned by magnetizing the sample in a field lower than the saturation field (Aladyshkin et al., 2009). Also larger dots may be able to stabilize "giant" vortices that are able to carry more than one flux quanta (Aladyshkin et al., 2009). In view of the above arguments, it can be seen that within the context of FM/SC heterostructures, magnetic micro,- and nanostructures present a special importance as they can produce effective magnetic pinning that can be modulated both by the dot lattice, as well as their magnetic state. In this sense we have fabricated micronic Py disks having diameters of 10, 5 and 1m. Also, Co nanostructures were grown using an alternative lithographic process that involves the use of self-assembled polystyrene nano-spheres as a shadow mask. www.intechopen.com 124 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale 3.2.1 Permalloy microstructures Figure 19 presents the magnetic images of circular, 350 nm thick Py dots having a diameter of (a)10, (b)5 and (c)1 m. For the 10 m dots, the diameter is well above the stripe domain width, estimated to be around 300 nm, according to the model proposed by Murayama (Murayama, 1966). As a consequence, the disks exhibit a well defined striped domain pattern. The stripes that are situated at the center of the disk have the same orientation and are very much similar to the case of a continuous film. As the stripes are situated closer to the edge of the microstrucutres, they tend to curve along the side of the dots, so as to prevent any magnetic charge build-up at the edges, and so to reduce the magnetostatic energy of the system. These closure domain - type structures have a limited spatial extension with respect to the overall area of the dots. As the diameter of the dots is reduced to 5 m, even though this dimension is still considerably higher than the domain width, the stripe pattern is almost entirely pertaining to the closure domains. As a consequence, the MFM images of the 5 m dots is composed of alternating bright and dark concentric circles. Again, locally the parallel stripe order is maintained. However, this is not the only domain configuration that can be observed. Another domain pattern that is observed, resembles the one present in the 10 m structures. It consists of straight magnetic stripes in the center and elongated circles at the edges as closure domains. It can be noted that the surface "covered" by the closure domains is much larger than in the 10 m case, reaching 100% for the circular stripe pattern. In view of the evolution that was observed so far, the domain pattern for 1 m dots is not surprising. As the diameter of the microstructures is reduced towards the limiting value of the stripe width, the competition between the anisotropy and magnetostatic energies results in the formation of stripe domains in the form of concentric rings. For the 1m disks, the single bright/dark alternation in the MFM images indicates the existence of solely two magnetic domains. Fig. 19. MFM images of (a)10 m (40 m×40 m), (b) 5 m (40 m×40 m), (c) 1 m (20 m×20 m), Py dots. 3.2.2 Cobalt nanostructures Co nanostructures were obtained using the polystyrene nanosphere lithography, a method described in detail in (Canpean et al., 2009). 170 nm and 40 nm thick films were deposited on a nanoshpere patterned Si substrate. After lift-off, triangular Co structures remain on the substrate. Even though a large difference in the deposited film thickness was prepared, the height of the nano-structures in the case of the two films did not differ significantly. In the www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 125 case of the 40 nm thick film, the mean height of the magnetic structures was of 12 nm, while for the thicker 170 nm film, the resulting dots were only 25 nm high. The large difference in the expected value of the height of the dots and the actual one is probably due to the shadowing effect produced by a divergent beam of sputtered atoms and also by the large diameter of the spheres (450 nm), which accentuate these effects. The MFM analyses performed on the two films are shown in Figure 20. For the 25 nm high structures, the dots are in a mono-domain state. The MFM contrsast consists of a dark/bright formation corresponding to an in-plane magnetic dipole structure. If the initial film thickness is decreased, the magnetic configuration of the nanostructures changes, as can be seen in Figure 20b, into a vortex-like spin configuration. The MFM contrast is typical for nanostructures in this configuration, having a bright center and the rest of the dot exhibiting a darker shade. According to the diameter-thickness phase diagram (Jubert and Allenspach, 2004) both dots should have been in the magnetic vortex state, and moreover the behavior predicted by the phase diagram is that for thicker dots the vortex state becomes more stable for a larger range of dot diameters. Our findings point out a different behavior, in the sense that thicker structures are found to be in the single domain state. Although no concrete explanation was found for this trend, we suspect that the particular shape of the dots, in our case triangular, may play an important role in modifying the magnetic phase diagram. Fig. 20. MFM images of Co nanostructures having different thicknesses: (a) 20 nm (magnetic dipoles) and (b) 12 nm (magnetic vortices). 4. Conclusions Within this chapter we illustrated the use of the Atomic Force Microscopy techniques as a powerful tool in characterization of complex thin film systems. Usually, when elaborating multilayer thin film stacks, the constituent layers have to be continuous and to present small roughness. Therefore, the AFM characterization will provide important information about the film surface topology in studies related to roughness reduction. Furthermore, high resolution analysis of the film topology can be used in studies of thin film growth mechanisms when the growth parameters (substrate temperature, deposition rate, etc…) are varied. Within the field of the Spintronics, for the elaboration of devices such as the magnetic tunnel junction, the extreme control of roughness of the constituent layers and the www.intechopen.com 126 Scanning Probe Microscopy – Physical Property Characterization at Nanoscale continuity of the insulating tunnel barrier is required. The interfacial roughness induces here fluctuations in electronic transport properties (perpendicular to the stack) with detrimental effects on the functional properties of the MTJ device. Moreover, the Magnetic Force Microscopy operating mode represents a versatile tool for the characterization of the micromagnetic properties of continuous magnetic thin films or mezoscopic size patterned magnetic objects. We have chosen here to illustrate the use of the MFM for characterizing the micromagnetic features of continuous magnetic films and patterned magnetic objects which started recently to be used for magnetic pinning of superconducting vortices in high temperature superconductors. 5. Acknowledgments This work has been partially supported by CNCSIS-UEFISCSU, project number PNII IDEI No. 4/2010, code ID-106 and by POS CCE ID. 574, code SMIS-CSNR 12467. 6. References Aladyshkin, A. Yu; Silhanek, A. V.; Gillijns, W. & Moshchalkov, V. V. (2009). 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Ultrathin metal films and particles on oxide surfaces: structural, electronic and chemisorptive properties, Surface Science Reports, Vol.27, Issues 1-3, (May 1998), pp. 1-111, ISSN 0167-5729 www.intechopen.com Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques 127 Chen, Y. & Washburn, J. (1996). Structural Transition in Large-Lattice-Mismatch Heteroepitaxy, Physics Review Letters, Vol.77, Issue 19, (November 1996), pp. 40464049, ISSN 1079-7114 (online) Chikazumi, S. (1997). Physics of Ferromagnetism, Oxford Science Publications, ISBN 0-19851776-9, New York, United States Civale, L.; Marwick, A. D.; Worthington, T. K.; Kirk, M. A.; Thompson, J. R.; Krusin-Elbaum, L.; Sun, Y.; Clem, J. R.; Holtzberg, F. (1991). Vortex confinement by columnar defects in YBa2Cu3O7 crystals: Enhanced pinning at high fields and temperatures, Phys. Rev. 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Guiding superconducting vortices with magnetic domain walls, Physical Review B, Vol.77, Issue 13, (April 2008), pp. 134518-134524, ISSN 1550-235x (online) www.intechopen.com Scanning Probe Microscopy-Physical Property Characterization at Nanoscale Edited by Dr. Vijay Nalladega ISBN 978-953-51-0576-3 Hard cover, 242 pages Publisher InTech Published online 27, April, 2012 Published in print edition April, 2012 Scanning probe microscopy (SPM) is one of the key enabling tools for the advancement for nanotechnology with applications in many interdisciplinary research areas. This book presents selected original research works on the application of scanning probe microscopy techniques for the characterization of physical properties of different materials at the nanoscale. The topics in the book range from surface morphology analysis of thin film structures, oxide thin layers and superconducting structures, novel scanning probe microscopy techniques for characterization of mechanical and electrical properties, evaluation of mechanical and tribological properties of hybrid coatings and thin films. The variety of topics chosen for the book underlines the strong interdisciplinary nature of the research work in the field of scanning probe microscopy. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: L. Ciontea, M.S. Gabor, T. Petrisor Jr., T. Ristoiu, C. Tiusan and T. Petrisor (2012). Characterization of Complex Spintronic and Superconducting Structures by Atomic Force Microscopy Techniques, Scanning Probe Microscopy-Physical Property Characterization at Nanoscale, Dr. Vijay Nalladega (Ed.), ISBN: 978-95351-0576-3, InTech, Available from: http://www.intechopen.com/books/scanning-probe-microscopy-physicalproperty-characterization-at-nanoscale/characterization-of-complex-spintronic-and-superconducting-structures InTech Europe InTech China University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 © 2012 The Author(s). Licensee IntechOpen. 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https://openalex.org/W2058493200	https://www.beilstein-journals.org/bjnano/content/pdf/2190-4286-5-174.pdf	English	null	Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages	Beilstein journal of nanotechnology	2,014	cc-by	9,088	Abstract Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocaliza- tion showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endo- cytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages Dagmar A. Kuhn1, Dimitri Vanhecke1, Benjamin Michen1, Fabian Blank2, Peter Gehr3, Alke Petri-Fink1 and Barbara Rothen-Rutishauser1,2 Full Research Paper Open Access Address: 1Adolphe Merkle Institute, University of Fribourg, Chemin des Verdiers 4, 1700 Fribourg, Switzerland, Phone +41 26 300 95 02, 2Respiratory Medicine, University Hospital of Bern, Murtenstrasse 50, 3008 Bern, Switzerland and 3Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000 Bern 9, Switzerland Email: Barbara Rothen-Rutishauser - barbara.rothen@unifr.ch * Corresponding author Keywords: cell lines; endocytosis; inhibition; nanoparticles; uptake proteins Beilstein J. Nanotechnol. 2014, 5, 1625–1636. doi:10.3762/bjnano.5.174 Received: 04 February 2014 Accepted: 28 August 2014 Published: 24 September 2014 This article is part of the Thematic Series "Biological responses to NPs". Guest Editor: R. Zellner © 2014 Kuhn et al; licensee Beilstein-Institut. License and terms: see end of document. Introduction It is important to highlight that the use of inhibitors must be optimized for each cell and NP type, since an inhibitor might show a high speci- ficity in one experiment but cause side effects in another [32]. The use of positive controls to show that an inhibitor only affects one endocytotic pathway without interfering with other uptake mechanism(s) is mandatory [33]. There are many different inhibitors described, and we will focus only on the most commonly used drugs to study NP uptake. Cytochalasin D can depolymerize actin filaments [34,35] and can therefore be used to study actin-dependent uptake mecha- nisms, that is, phagocytosis and macropinocytosis. Larger parti- cles, such as polystyrene particles of 1 µm in diameter, can be used to run the experiment under controlled conditions. Phagocytosis and macropinocytosis are both dependent on actin [15,23]. Phagocytosis is carried out by professional phagocytes (i.e., monocytes/macrophages, neutrophils and dendritic cells), which in turn form intracellular phagosomes. Macromolecule and particle uptake is triggered via the interaction of the responsible receptors on the cell surface and the ligands. Macropinocytosis, which is also actin-driven, forms protru- sions at the outer cell membrane which then again fuse with the cell membrane by taking up larger fragments or debris [14]. Chlorpromazine hydrochloride which inhibits clathrin-medi- ated endocytosis, induces a loss of clathrin and adaptor protein complex 2 from the surface of the cell [31,36]. It is thus classi- fied as an inhibitor for clathrin-mediated endocytosis [37,38]. Monodansylcadaverine (MDC), a competitive inhibitor, blocks the enzyme transglutaminase 2, which is necessary for receptor crosslinking in the region of clathrin-coated pits [31,39,40]. Furthermore, chlorpromazine and MDC are specific in inhibiting the uptake of the serum protein transferrin [41]. Consequently, fluorescently labelled transferrin can be used to investigate clathrin-mediated endocytosis [32,41,42]. Clathrin-mediated endocytosis is very well studied and is, like most pinocytotic pathways, a form of receptor-mediated endo- cytosis. This abundant pathway is essential for the uptake of many molecules such as low-density lipoprotein and transferrin [24,25]. When clathrin-mediated endocytosis is initiated, the so-called “coated pits” come into play consisting of transmem- brane receptors and cytosolic proteins, such as clathrin and the AP2 adaptor complex [20]. Caveolae and lipid raft internalizations are known to be inhib- ited by nystatin, filipin and methyl-β-cyclodextrin (mβcd) through depletion of the cholesterol from the cell membrane by forming inclusion complexes with cholesterol [31,43]. Introduction pharmaceutical applications, specifically for targeted drug delivery [1-6], biosensing [7] and bio-medical imaging [8]. In order to develop optimal NPs for biomedical use, much atten- In recent years, the use of engineered nanoparticles (NPs) (defined as <100 nm in three dimensions according to ISO TS 27687:2008) has witnessed a strong rise in biomedical and 1625 1625 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. tion is given to the understanding of the basic mechanism of NP interactions with cellular systems at the single cellular level [9-11]. It has already been shown that different NP properties, such as size, shape, material and surface coating, as well as the cell type, age, interaction with other cells and the cellular envi- ronment, influence NP uptake and the cellular behavior as well as the down-stream response of the cells [11-16]. (subdomains), which consist of glycosphingolipids and high amounts of cholesterol [27]. The protein which gives shape and structure in caveolin-mediated endocytosis is caveolin-1, a dimeric protein which binds cholesterol onto the cellular surface for uptake and intracellular trafficking (lipid homeostasis) [28]. Also located at the site of lipid rafts is flotillin-1, an integral membrane protein which forms a hetero-oligomer with flotillin-2 [29]. In addition to the aforementioned uptake mecha- nisms, clathrin- and caveolin-independent endocytosis as well as passive diffusion of NPs across the cell plasma membrane are also addressed [17,30]. The term endocytosis describes two different cellular uptake mechanisms: pinocytosis, which involves the uptake of fluids and molecules within small vesicles and phagocytosis, which is responsible for engulfing large particles (e.g., microorganisms and cell debris). Pinocytosis covers macropinocytosis, clathrin- mediated endocytosis, caveolin-mediated endocytosis and clathrin- and caveolin-independent endocytosis [8,17-20]. However, not all cell types are equipped with the required machinery to perform the entire spectrum of endocytotic path- ways. Therefore, these pathways are specific to types of cells and subsequently determine the trafficking and intracellular fate of particles [21]. Red blood cells are a common example, as they do not have any phagocytic receptors on their surface and no actin–myosin system, therefore they serve as a model for non-phagocytic cells to study how NPs penetrate through cell membranes [22]. To elaborate on the most important cellular endocytotic uptake mechanism of NPs, specific pharmacological substances which inhibit specific pathways can be used [31]. Introduction It was also shown that mβcd inhibits clathrin-mediated endocytosis, since clathrin uptake requires cholesterol as well [11,44]. All of these mentioned inhibitors form aggregates which accumulate cholesterol and separate it from the membrane structures. Finally, the cholera toxin subunit b (ctx-b) [45] has been shown On the other hand, caveolin-mediated endocytosis is respon- sible for the homeostasis of cholesterol [20]. The static struc- tures of caveolae form flask-shaped invaginations in the cell membrane. Many cell types such as the capillary endothelium, type I epithelial cells, muscle cells as well as fibroblasts, ex- hibit caveolin-mediated endocytosis, which occurs at the site of the lipid rafts [20,26]. These rafts are plasma membrane regions 1626 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. Figure 1: Characterization of polystyrene particles. (A) Characteristics of the particles as measured by dynamic light scattering, zeta potential and transmission electron microscopy in water and unsupplemented cell culture medium. Transmission electron microscopy images of (B) 1 µm particles, (C) NPs, and (D) a mixture of 1 µm particles and NPs. Figure 1: Characterization of polystyrene particles. (A) Characteristics of the particles as measured by dynamic light scattering, zeta potential and transmission electron microscopy in water and unsupplemented cell culture medium. Transmission electron microscopy images of (B) 1 µm particles, (C) NPs, and (D) a mixture of 1 µm particles and NPs. to enter the cells by caveolin-mediated endocytosis, therefore this protein can be used to control the inhibitors in any experi- mental setting. microscopy (TEM) revealed a core radius of 520 nm for the microparticles and 30.9 nm for the NPs. The latter, however, exhibited a larger polydispersity (Figure 1). Both methods confirm the experimentally obtained values. Measurements were carried out in water and unsupplemented RPMI medium and showed that all analyzed particles remained stable and monodisperse in biological medium. The zeta potential indi- cated a negative charge for both particle types which was slightly reduced, but still negative, when the particles were suspended in unsupplemented RPMI medium (Figure 1). To show that the endocytotic uptake route of choice is depen- dent upon the particle size, we deployed two fluorescently labelled polystyrene particles of significantly different sizes, that is, 40 nm and 1 µm in diameter. These particles were chosen as they are easy to detect by fluorescence methods and available in different sizes [46]. Introduction Moreover, they have a narrow size distribution and are considered to be suitable for biomed- ical applications [47,48] since they are considered non-toxic at applied physiological concentrations [49]. Finally, two of the most relevant cell types in regard to uptake and interaction of (nano) particles at any barrier system (i.e., macrophages and epithelial) [50,51] were included to demonstrate that not only the applied particle dimensions and uptake pathways are deter- minant, but the actual cell types as well. Expression of endocytotic uptake proteins in both cell types In order to define particle uptake routes, it is crucial in the first step to determine the presence of the endocytotic proteins which are involved in endocytosis in both cell types (Figure 2). To achieve this, laser scanning microscopy (LSM) was applied as the primary tool for this investigations. Flotillin-1 and clathrin heavy chain could be visualized in J774A.1 cells, but caveolin-1 was not detected. Clathrin heavy chain was detected within the cells, both at the cell membrane and in the cytosol. Flotillin-1 was, however, only observed in the cytosol. In A549 cells, clathrin heavy chain and caveolin-1 were located in the cells both at the cell membrane as well as in the cytoplasm, albeit to a lower extent than in the macrophages. Flotillin-1 could not be detected in A549 cells. Particle characterization In the first step, both particles were thoroughly investigated prior to cellular experiments. The particle size measured with dynamic light scattering (DLS) revealed an average hydrody- namic radius of approximately 581 nm for the microparticles and 28 nm for the nanoparticles. Transmission electron 1627 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. Table 1: Analysis of the different endocytotic inhibitors regarding specificity and efficiency. J774A.1 cells Inhibitors c (µM) Exposure (t) Transferrin Ctx-b 1 µm PS Cell morph.a Chlorpromazine (clathrin) 100 30 min No inhibition No inhibition No inhibition – MDC (clathrin) 250 1 h 30 min Inhibition No inhibition No inhibition + MβCD (clathrin, caveolin) 10·103 30 min No inhibition No inhibition No inhibition + CytoD (phag., macrop) 4 1 h 30 min No inhibition No inhibition Inhibition + A549 cells Inhibitors c (µM) Exposure (t) Transferrin Ctx-b 1 µm PS Cell morph.a Chlorpromazine (clathrin) 100 30 min Inhibition No inhibition No inhibition + MDC (clathrin) 250 1 h 30 min No inhibition No inhibition No inhibition – MβCD (clathrin, caveolin) 10·103 30 min Inhibition Inhibition No inhibition + CytoD (phag., macrop.) 4 1 h 30 min No inhibition No inhibition No inhibition – aCell morphology: no cellular impairment (+) and cellular damage (-). Table 1: Analysis of the different endocytotic inhibitors regarding specificity and efficiency. aCell morphology: no cellular impairment (+) and cellular damage (-). optimal concentration, exposure time and cell impairment in both cell types (Table 1 and Figure 3). The cell morphology was assessed by LSM (Figure 3) and the cytotoxicity by lactate dehydrogenase (LDH) assay (see Figure S1, Supporting Infor- mation File 1). Trypan blue staining marked the integrity of the cell membrane for cells which were impaired by the inhibitor (red insets, Figure 3) and revealed the following percentage of dead cells for J774A.1 cells (n = 3): negative control 20% (SD ± 9.5%), triton 100% (SD ± 0%), chlorpromazine 46.1% (SD ± 2.7%). For A549 cells, the following percentages of dead cells were revealed: negative control 0% (SD ± 0%), triton 100% (SD ± 0%), MDC 78.3% (SD ± 15.5%), cytochalasin D 10.3% (SD ± 9.6%). Fluorescently labelled transferrin was used together with specific inhibitors as a control to investigate clathrin-mediated endocytosis and fluorescent ctx-b with inhibi- tors as a control to analyze caveolin-mediated endocytosis. Polystyrene particles of 1 µm diameter were used to demon- strate the inhibition of phagocytosis. Particle characterization The uptake of fluores- cently labelled transferrin was blocked by applying 250 µM MDC in J774A.1 cells, whereas MDC could not inhibit clathrin-mediated endocytosis in A549 cells. In addition, treat- ment of A549 cells with MDC resulted in cell impairment as shown by LSM (Figure 3). Chlorpromazine did not inhibit transferrin uptake and also severely impaired the morphology of the J774A.1 cells. However, 100 μM chlorpromazine inhibited the clathrin-mediated endocytosis of A549 cells. Mβcd could neither inhibit transferrin (clathrin-mediated) nor ctx-b (cave- olin-mediated) uptake by J774A.1 macrophages. On the contrary, 10 mM mβcd inhibited clathrin- as well as caveolin- Figure 2: LSM images demonstrate the presence of the different endocytotic uptake proteins within J774A.1 macrophages and A549 epithelial cells. The LSM images show the presence of endocytotic proteins clathrin heavy chain, caveolin-1 and flotillin-1 in J774A.1 cells and A549 cells. The white arrows in the upper panel of each image (XY orthogonal plane) represent the position of the XZ slice shown in the lower picture. The apical side of the cells corresponds to the bottom line of the images. Endocytotic uptake proteins are shown in green and the actin cytoskeleton in red. Scale bar: 8 µm. Figure 2: LSM images demonstrate the presence of the different endocytotic uptake proteins within J774A.1 macrophages and A549 epithelial cells. The LSM images show the presence of endocytotic proteins clathrin heavy chain, caveolin-1 and flotillin-1 in J774A.1 cells and A549 cells. The white arrows in the upper panel of each image (XY orthogonal plane) represent the position of the XZ slice shown in the lower picture. The apical side of the cells corresponds to the bottom line of the images. Endocytotic uptake proteins are shown in green and the actin cytoskeleton in red. Scale bar: 8 µm. Particle uptake by the two cell types in the presence of endocytotic uptake inhibitors In order to resolve the uptake routes for the given particle size, both particles were tested together with the optimal inhibitor concentration for both cell types. Conditions were chosen such that the uptake of the relevant control substance was completely inhibited and no impaired cell morphology was observed. Both cell lines were exposed to either 1 µm PS particles or 40 nm PS NPs at a concentration of 20 µg/mL for 1 hour either after preincubation with endocytotic inhibitors (preinhibition experi- ment) or in coexposure (continuous experiment) with endocy- totic inhibitors (chlorpromazine, MDC, mβcd, cytochalasin D or none). Intracellular particles were visualized by LSM (Figure 5). Figure 3: Investigation of cell morphology after inhibitor treatment. Healthy cells (green inset) retained their cellular structure after inhibitor treatment. Impaired cells (red inset) showed membrane damage, loss of integrity and loss of viability. (A) chlorpromazine, (B) monodansylca- daverine, (C) mβcd, and (D) cytochalasin D. Scale bar: 10 µm. cell imaging, which revealed that NP uptake is a very fast process, starting 5 to 10 minutes after exposure to the cells (Figures S2 and S3, Supporting Information File 1). Uptake of 1 µm particles was only observed in J774A.1 macrophages but not in A549 epithelial cells for the given exposure time of 1 hour. A549 cells required a much longer time of 1 hour to internalize 1 µm particles (data not shown). However, since the inhibitors began to induce cell damage after 1 to 1.5 hours, observation time could not be extended. In J774A.1 cells, MDC Endocytotic inhibitors With the knowledge of the presence of specific uptake proteins, it is now feasible to elaborate on the different uptake routes in each cell type by applying different chemical inhibitors. Inhibitors of clathrin- and caveolin-mediated endocytosis or macropinocytosis and phagocytosis were tested for their 1628 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. mediated endocytosis as shown by the lack of intracellular fluo- rescently labelled transferrin and ctx-b in A549 cells. The actin polymerization inhibitor cytochalasin D prevented particle uptake (d = 1 µm) by J774A.1 cells at a concentration of 4 µM. Uptake of transferrin by J774A.1 cells could not be prevented by cytochalasin D (data not shown). In A549 cells, cytocha- lasin D impaired the cell morphology at all tested concentra- tions from 5 µM to 10 µM. A lower concentration of 3 µM did not inhibit particle uptake (data not shown). It is important to mention that inhibition of cells with cytochalasin D cannot distinguish between phagocytosis and macropinocytosis. Figure 3: Investigation of cell morphology after inhibitor treatment. Healthy cells (green inset) retained their cellular structure after inhibitor treatment. Impaired cells (red inset) showed membrane damage, loss of integrity and loss of viability. (A) chlorpromazine, (B) monodansylca- daverine, (C) mβcd, and (D) cytochalasin D. Scale bar: 10 µm. Fluorescence intensity profiles To identify in which protein uptake compartments the NPs (d = 40 nm) are present, it is possible to analyze their regions of fluorescence overlap. Several intensity profiles reveal a distinct overlap (Figure 4A) of clathrin heavy chain and a signal from 40 nm NPs in J774A.1 cells (Figure 4A, region 1). However, cases of dissimilarities between the two fluorescence signals were also recorded (Figure 4A, region 2). Analogous observa- tions (both in agreement and disagreement) of the flotillin-1 fluorescence signal and 40 nm PS NPs were made (Figure 4B, regions 1 and 2). These findings were supported by the resulting Pearson coefficient value found for each region analyzed. Inten- sity profile plots of subcellular events in A549 cells were not performed due to a lower expression of the uptake proteins in these cells compared to the J774A.1 cells. Particle uptake evaluation After 1 hour of incubation, intracellular particles and NP events (either agglomerates or single NPs, which cannot be distin- guished by LSM) were visualized in both J774A.1 and A549 cells (Figure 5 and Figures S2 and S3, Supporting Information File 1). In control cells that were not treated with any inhibitors, uptake of NPs was observed within only a few minutes in both cell types (Figure 5). These observations are supported by live 1629 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. Figure 4: Fluorescence intensity profiles of J774A.1 cells, 40 nm PS NPs with clathrin heavy chain or flotillin-1. (A) and (B) are confocal images of J774A.1 cells treated with 40 nm PS NPs (red) and primary antibodies against uptake proteins (green), respectively. The corresponding relative fluo- rescence profiles of the overlapping signals are shown in the diagrams to the right (regions 1 and 2). Cells are shown in the transmission light channel. The Pearson coefficient (rp) was calculated for each of the regions. (A) 40 nm PS NPs and clathrin heavy chain, (B) 40 nm PS NPs and flotillin-1. The white arrows are 6.8 µm in length. Scale bar = 10 µm. Figure 4: Fluorescence intensity profiles of J774A.1 cells, 40 nm PS NPs with clathrin heavy chain or flotillin-1. (A) and (B) are confocal images of J774A.1 cells treated with 40 nm PS NPs (red) and primary antibodies against uptake proteins (green), respectively. The corresponding relative fluo- rescence profiles of the overlapping signals are shown in the diagrams to the right (regions 1 and 2). Cells are shown in the transmission light channel. The Pearson coefficient (rp) was calculated for each of the regions. (A) 40 nm PS NPs and clathrin heavy chain, (B) 40 nm PS NPs and flotillin-1. The white arrows are 6.8 µm in length. Scale bar = 10 µm. nisms [52-54]. All particle exposure experiments were conducted in serum-free medium in order to avoid binding of serum proteins to the particle surfaces which might induce agglomeration, however, the binding of small molecules or salts in the cell culture medium cannot be excluded. partially inhibited the uptake of 40 nm NPs (Figure 5). A significant fraction of the 40 nm particles was observed outside at the cell membrane (Figure S2, Supporting Information File 1). However, NPs were also detected within the cells (Figure 5). Cytochalasin D also partially blocked the uptake of NPs. Particle uptake evaluation The uptake of 1 µm particles by J774A.1 was completely blocked by cytochalasin D, whereas MDC had no effect. The focus of the present study was on two cell types that have important clearing and barrier functions, namely, macrophages (represented by the mouse J774A.1 macrophage cell line) and epithelial cells (represented by the A549 human alveolar epithe- lium cell line). First, the specificity of the different inhibitors was assessed for both cell types. As a second step, the opti- mized inhibitors were used to study the uptake of the two different particle types. Visualization of the fluorescently tagged particles was done by LSM. LDH measurements revealed no cytotoxicity for the combined inhibitors and endo- cytotic protein markers for both cell types being analyzed. Additionally, inhibitors which negatively affected the cells are summarized in Table 1 and the images in Figure 3 are presented with either red (impairment by inhibitor) or green (no effect by inhibitor) letter insets. Trypan blue staining demonstrated the same outcome. The percentage of dead cells which were treated with cytochalasin D was not as high as expected for A549 cells. This could be due to cytochalasin D blocking actin polymeriza- tion and hence preventing Trypan blue from entering the cells, although the cells are severely affected by this inhibitor. Although most 40 nm NP events were located outside of the cells, intracellular particle events could still be detected in both chlorpromazine-treated and mβcd-treated A549 cells. The 1 µm particles were not observed inside the epithelial cells under any condition (Figure 5). Discussion For any future biomedical application of engineered NPs, it is mandatory to fundamentally understand their interaction with living systems. The cellular uptake pathway of a NP will have direct consequences on its intracellular localization; hence, understanding the overall NP distribution in a specific compart- ment, such as endosomes, lysosomes or others, might provide some interesting suggestions for developing a future drug delivery system. To gain more insight into the uptake mecha- nism(s) of NPs in comparison to larger (i.e., micron-size) parti- cles of the same material, a broad array of chemical inhibitors was used that were shown to inhibit certain endocytotic mecha- 1630 1630 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. Figure 5: Laser scanning microscopy imaging revealed particle uptake in J774A.1 and A549 cells. (A–C) Uptake of 40 nm PS NPs (NP: red, cytosol: grey). (A) Untreated cells with 40 nm NPs. (B) 40 nm NPs and cytochalasin D (cytoD) in J774A.1 and chlorpromazine (cpz) in A549 cells. (C) 40 nm NPs and monodansylcadaverine (mdc) in J774A.1 cells and methyl-β-cyclodextrin (mβcd) in A549 cells. (D) Negative control. (E–G) Uptake of 1 µm PS particles (particles: green, cytosol: grey). (E) Untreated cells with 1 µm particles. (F–G) 1 µm particles with the same adequate inhibitors. Scale bar: 5 µm. White arrows represent intracellular events of 1 µm particles in J774A.1 cells. The negative controls have a scale bar of 10 µm. Figure 5: Laser scanning microscopy imaging revealed particle uptake in J774A.1 and A549 cells. (A–C) Uptake of 40 nm PS NPs (NP: red, cytosol: grey). (A) Untreated cells with 40 nm NPs. (B) 40 nm NPs and cytochalasin D (cytoD) in J774A.1 and chlorpromazine (cpz) in A549 cells. (C) 40 nm NPs and monodansylcadaverine (mdc) in J774A.1 cells and methyl-β-cyclodextrin (mβcd) in A549 cells. (D) Negative control. (E–G) Uptake of 1 µm PS particles (particles: green, cytosol: grey). (E) Untreated cells with 1 µm particles. (F–G) 1 µm particles with the same adequate inhibitors. Scale bar: 5 µm. White arrows represent intracellular events of 1 µm particles in J774A.1 cells. The negative controls have a scale bar of 10 µm. cell types, the exposure time and concentration to achieve optimal inhibition had to be defined. This careful optimization for each inhibitor and each cell type is very often missing in many published studies. Discussion Since cytocha- lasin D did not inhibit the uptake of transferrin and ctx-b, strong evidence is provided that this inhibitor did not severely affect other endocytotic pathways as also observed by others [60]. A decreased uptake of 40 nm NPs and accumulation of NPs at the cell surface in the presence of cytochalasin D was observed. This could also be due to the formation of agglomerates on the cellular surface that would then show similar physio-chemical behavior as micron-size particles. Since accumulation at the cell border of 40 nm NPs was observed prior to actin-driven uptake, we argue that phagocytosis or macropinocytosis is involved in both the uptake of larger aggregates of 40 nm NPs and 1 µm particles. Our findings are in agreement with other studies that showed a reduced uptake of 40 nm carboxylated polystyrene particles in HeLa and 1321N1 cells in the presence of cytocha- lasin A [52] and by cytochalasin D in pulmonary macrophages [59]. In addition, the inhibition of 40 nm NP uptake also occurred in the presence of the clathrin inhibitor MDC in J774A.1 cells. This suggests that J774A.1 macrophages can employ multiple uptake mechanisms for the endocytosis of 40 nm PS NPs by both clathrin-mediated as well as macropinocytosis or phagocytosis. Caveolin-mediated uptake was not observed, since mβcd did not block the uptake of transferrin. Moreover, the related proteins such as caveolin-1 and flotillin-1 were not detected at the cell border. As already known, caveolin-mediated endocytosis is mainly observed in several cell types including capillary endothelium, type I alveolar epithelial cells, smooth muscle cells and fibroblasts [20]. Therefore, this result supports the cell type specific mech- anism of this uptake. In A549 epithelial cells, the macropinocytosis and phagocy- tosis pathways could not be explored, as the uptake of 1 µm particles by these cells was never observed, even under controlled conditions. Surprisingly, these findings are in contra- diction to other studies that have shown uptake of such parti- cles [30,58]. However, these A549 cells were used in a triple cell coculture system combined with macrophages and dendritic cells. Therefore, the results are not directly comparable. In add- ition, the uptake was evaluated only within 1 hour, and phago- cytosis/macropinocytosis might be slower in epithelial cells than in macrophages. Therefore, longer incubation times should be applied in future studies, but also at optimal conditions under which the inhibitors do not induce adverse cell effects. Discussion This balancing act between the desired functional inhibition of a specific pathway and the possible adverse effects on the cells did not always provide an appli- cable result. For instance, for some of the inhibitors the concen- It was shown that the optimal and desired function of the endo- cytotic inhibitors was occasionally different in the two cell types, namely, the A549 epithelial cells and J774A.1 macrophages. The inhibition studies were carried out within 1 hour, since endocytotic processes were very fast [20,55,56] and the inhibitors also began to impair the cells in their morphology after a longer incubation time. For each of the two 1631 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. between the cell types as well as species differences (i.e., the macrophages are a mouse cell line, while the epithelial cells are human) play a role. Hence, a preliminary study to optimize the use of inhibitors with each cell type should be a prerequisite for every future investigation involving endocytotic pathway inhibitors. To summarize, one can say that each cell type reacts differently to the applied inhibitors and possess different uptake routes. trations had to be very high for an efficient inhibition but then in this case they strongly impaired the cells. mβcd also inhib- ited the uptake of transferrin (clathrin-mediated endocytosis) in A549 cells [57], however, mβcd had detrimental effects on the J774A.1 cell morphology and could only be exposed for a limited period of 30 minutes preincubation [32]. In J774A.1 macrophages, macropinocytosis and phagocytosis could be optimally inhibited by cytochalasin D and MDC which could efficiently block clathrin-mediated endocytosis. However, optimal conditions for the inhibition of the lipid raft-mediated pathway in this cell type could not be established. mβcd has been described to be an optimal inhibitor of lipid raft-mediated endocytosis. Therefore it was rather surprising that it did not work in the J774A.1 cells, also because flotillin-1 was highly expressed in all cells as shown by LSM. However, the localization of the protein was mainly intracellular and caveolin-1 was not present at all. This finding should be evalu- ated more thoroughly. In addition, other inhibitors such as statins, filipin or nystatin could be used, however, severe cell damage for those three inhibitors has been observed in earlier studies [32]. Regarding the (nano) particle uptake study, cytochalasin D completely blocked the uptake of 1 µm particles via phagocy- tosis and macropinocytosis in J774A.1cells [59]. Discussion The clathrin-mediated endocytosis was impeded by 100 μM chlor- promazine. Also, 10 mM mβcd was shown to block caveolin- mediated endocytosis with the restriction of a maximum of 30 minutes exposure in the inhibitor. Consequently, this cell line seems to be capable of ingesting particles by clathrin- as well as caveolin-mediated endocytosis. This is underlined by the presence of clathrin heavy chain and caveolin-1 in the cyto- plasm and at the cell membrane. Interestingly, these cells do not show flotillin-1 compared to the J774A.1 cells. Nonetheless, the role of this protein on NP uptake will need to be further evalu- ated in the future. The colocalization of clathrin heavy chain and the 40 nm PS NP fluorescence in the profile plots are also a strong hint for the involvement of a clathrin-mediated pathway in the uptake of 40 nm PS NPs by J774A.1. However, there were regions where 40 nm NPs did not colocalize with the clathrin heavy chain signal. The LSM data showed that J774A.1 cells express flotillin-1, and indeed profile plots of flotillin-1 have shown a colocalization with the 40 nm NPs [61]. It was shown that the phagosome proteome of J774A.1 cells contains high amounts of It is obvious from these results that the inhibitors have different effects depending on the cell type. The reasons for this observa- tion can be many: the genotypic and phenotypic differences 1632 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. flotillin-1 [62]. However, all tested inhibitors for lipid raft- mediated uptake failed with this cell type. In addition, caveolin-1 was not detected. Therefore, the role of this uptake mechanism is still not clear in this cell and for this particle type. It also has been shown that caveolin-1 was only rarely present in human macrophages [62]. Consequently, one can conclude that NPs are taken up by a different mechanism in this cell type, which might also depend on the agglomeration behavior of particles on the cell surface, and thus the “secondary” size of the particles. Neither chlorpromazine nor mβcd (i.e., blocking the clathrin-mediated and the caveolin-mediated pathway) could fully inhibit the uptake of 40 nm NPs in A549 cells, since intra- cellular particle events could still be detected after treatment with those two inhibitors. Thus, A549 may also employ multiple endocytotic pathways, as it is known that different pathways can be used [52]. Discussion In J774A.1 cells, 1 µm particles were engulfed by phagocytosis/ macropinocytosis and 40 nm PS NPs by both clathrin-mediated as well as macropinocytosis or phagocytosis. Therefore, it seems that both cell types take up NPs by different mechanisms, although the mechanisms are not similar for the different cell types. Experimental Cell cultures Human alveolar epithelial type II cells (A549 cell line) and mouse macrophage cells (J774A.1 cell line), both from Amer- ican tissue Type Culture Collection, were cultured in RPMI 1640 with HEPES (Gibco, Luzern, Switzerland) completed with 10% foetal bovine serum (heat inactivated, PAA Laboratories, Austria), 1% L Glutamine (Gibco, Luzern, Switzerland) and 1% penicillin/streptomycin (Gibco, Luzern, Switzerland) and kept at 37 °C and 5% CO2. The A549 epithelial cells were split twice per week using trypsin (0.05% trypsin-EDTA, GIBCO, Switzer- land). J774A.1 cells were sub-cultured using the scraping method, resuspended in the medium and finally centrifuged (2 min, 2000 rpm and 21 °C). A549 cells were seeded at a density of 35 × 103 cells/mL and J774A.1 cells at a density of 25 × 104 cells/mL in BD FalconTM 4 chamber polystyrene vessels with tissue culture treated glass slide with a growth area of 1.7 cm2 (Milian, Geneva, Switzerland). A549 cells were grown to confluence for 7 days prior to exposure experiments and J774A.1 cells for 1 day and allowed to adhere prior to use. Finally, LSM was used (for exact spatial localization) to analyze the interaction of fluorescently labelled particles with single cells. This method is crucial since one can visually distin- guish between extracellular and intracellular particle events. We also have attempted to quantify the uptake of the particles by fluorescence activated cell sorter (FACS). However, it was observed that the use of endocytotic pathway inhibitors resulted in particle agglomerates that were attached to the outer cell membrane which remained even after extensive washing. Therefore, FACS could not be used in combination with inhibitory conditions. Particle characterization and exposure All particles internalized by endocytotic pathways are finally localized in intracellular vesicles. However, some other studies have shown that NPs of different materials were detected in the intracellular space and/or free in the cytoplasm. Since they were not membrane-bound, alternative uptake routes for cellular uptake might exist [22,46,59,63]. Possible uptake mechanisms such as receptor mediated diffusion via membrane pores as well as passive uptake by van der Waals or steric interactions (subsumed as adhesive interactions) [64,65] were proposed by the authors of these studies. The commercially available carboxylate Fluoresbrite™ plain yellow green (cataloged as 1 µm hydrodynamic diameter) and red (cataloged as 40 nm hydrodynamic diameter) polystyrene (PS) particles (Molecular Probes, Luzern, Switzerland) were used for the study. The particles were characterized in terms of size by dynamic light scattering (DLS) and transmission elec- tron microscopy (TEM). The hydrodynamic radius was acquired by DLS (3D LS Spectrometer, LS Instruments AG, Fribourg, Switzerland) based on a viscosity of 0.89 mPas and a refractive index of 1.33. The correlation function was measured at a scattering angle of 90° at T = 25 °C. The data was analyzed with a fitting routine applying a single exponential model and accounting for polydispersity assuming a Schulz–Zimm distrib- ution. Both particles were measured at a concentration of 20 µg/mL in either unsupplemented RPMI medium or dH2O over a time window of 1 hour. Laser scanning microscopy of fixed and living cells For LSM imaging, the cells were fixed with 3% paraformalde- hyde (PFA, Sigma-Aldrich, Switzerland) in PBS for 15 minutes at room temperature. The cells were then washed with 1× PBS, then permeabilized for 15 minutes with 0.2% Triton X and then washed again with 1× PBS. The F-actin cytoskeleton was stained with rhodamine phalloidin (Invitrogen, Luzern, Switzer- land) at a dilution of 1:50 in 1× PBS. Following this step, the cells were then washed and mounted in glycergel mounting medium (C0563, Dako, Baar, Switzerland). Image acquisition was performed with an inverted Zeiss LSM 710 Meta (Axio Observer.Z1, Zeiss, Switzerland) microscope equipped with 405, 488 and 561 nm laser excitation sources. Fields of inter- ests were acquired at 5 different randomly selected areas per glass slide. The experiment was performed using a 63x/N.A 1.4 immersion oil lens. Cellular and morphological information was retrieved using Imaris software (Bitplane 7.4, Zürich, Switzer- land). Endocytotic uptake proteins Antibodies against clathrin heavy chain, flotillin-1 and caveolin-1 (all fluorescently labelled with Alexa Fluor 488, antibodies-online GmbH, Aachen, Germany) were used at a final dilution of 1:20 in 1× PBS on fixed cells. After 1 hour of staining (in a dark room at room temperature) and three washing cycles with 1× PBS, the cells were mounted using medium Glycergel mounting medium (C0563, Dako, Baar, Switzerland). The intensity profiles were performed using Fiji software. The pearson coefficient (rp) reavealed the colocaliza- tion/signal overlap of endocytotic uptake proteins and NPs for all analyzed regions. Inhibition of endocytosis Different endocytotic inhibitors were tested for their optimal concentration, exposure time and cell impairment (Table 1, Figure 3). Inhibition of clathrin-mediated endocytosis in both cell types was tested with chlorpromazine hydrochloride (C8138, Sigma-Aldrich, Switzerland) and monodansylcadav- erine (Dansylcadaverine, D4008, Sigma-Aldrich). Inhibition of caveolin-mediated endocytosis was performed in both cell lines using 10 mM methyl-β-cyclodextrin (mβcd) (C4555, Sigma- Aldrich). A 4 µM cytochalasin D (C8273, Sigma-Aldrich) solu- tion was used to inhibit phagocytosis and macropinocytosis in both cell lines. For live cell imaging, the cells were seeded in a Lab-TekTM II chambered coverglass 4 chamber well (1.5 german coverglass system, NC-155382, Nunc, Milian, Geneva, Switzerland), stained with Cell trackerTM violet BMQC dye and incubated for 1 hour at 37 °C and 5% CO2 followed by three washing steps with 1× PBS. Finally, transparent RPMI 1640 medium (no 1% L-glutamine, no antibiotics, no fetal calf serum and without phenol red pH indicator) was added with either 40 nm or 1 µm polystyrene particles alone or in combination with the inhibitor, and time lapse imaging was started. The live cell imaging ran over a time period of 60 minutes during which the cells were kept in a constant environmental at 37 °C and 5% CO2. Image acquisition was performed as described above. Control experiments Fiji software. Thereby, images were converted to binary images by automated thresholding (default threshold) and analyzed applying an ellipse fit. A total of 982 counts were performed for the NPs and 102 counts were collected for the micron-sized particles. The electrophoretic mobility of the particles was determined by electrophoresis (Brookhaven 90 Plus Instru- ments Corp., Holtsville, USA). The measured particle mobility in the electric field was transformed to the zeta potential by applying the Smoluchowski model. The small particles were measured at a concentration of 60 µg/mL and the larger parti- cles at 20 µg/mL. Alexa fluor 488 coated transferrin (T13342, Invitrogen), dissolved in dH2O containing 1% NaN3 and diluted in RPMI to a final concentration of 120 μg/mL, was used to test the inhibi- tion of clathrin-mediated endocytosis. Cholera toxin subunit-b (ctx-b) labelled with Alexa Fluor 488 conjugate (C34775, Molecular Probes) was used to test inhibition of caveolin-medi- ated endocytosis. Ctx-b was dissolved in 10 mM phosphate- buffered saline (PBS, pH 7.4) containing 1% NaN3 and diluted to a final concentration of 0.6 μg/mL 1× PBS (pH 7.4) in RPMI prior to use. To test macropinocytosis and phagocytosis, 1 µm carboxylate modified fluospheres (molecular probes) were used at a concentration of 20 µg/mL in RPMI. Prior to the cell exposure, all particle suspensions were soni- cated for 2 minutes in order to avoid aggregation. Polystyrene particles were suspended in RPMI 1640 medium and adjusted to a concentration of 20 µg/mL. A 1 mL particle suspension was applied on a growth area of 1.7 cm2 and the cells were exposed for 1 hour in the well. Conclusion It was shown that the particle size is critical in determining which endocytotic uptake route is deployed, and additionally, this process is cell type dependent. Not all inhibitors blocked the related pathway in the two different cell lines in the same way, which is also in agreement with the expected uptake mechanism per cell type. Therefore, each condition must be evaluated with the use of positive markers. A549 cells did not take up any 1 µm PS particles by phagocytosis/macropinocy- tosis over a period of 1 hour, whereas 40 nm NPs were ingested by clathrin- as well as caveolin-endocytotic pathways. The particles were observed with a Hitachi transmission elec- tron microscope (H-7100, Tokyo, Japan) operating at an accel- eration voltage of 75 kV and equipped with a Morada CCD digital camera (Olympus, Tokyo, Japan). Size distributions of both PS particle types were obtained by image analysis using 1633 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. Cell treatment Both cell types were preincubated for 30 minutes and subse- quently exposed to either the control substances (i.e., transferrin or ctx-b) or the PS particles, or in combination with the inhibitor (for continuous inhibition). Exposures were carried out for 1 hour. doi:10.1080/00222930701314932 19.Brandenberger, C.; Mühlfeld, C.; Ali, Z.; Lenz, A.-G.; Schmid, O.; 19.Brandenberger, C.; Mühlfeld, C.; Ali, Z.; Lenz, A. G.; Schmid, O.; Parak, W. J.; Gehr, P.; Rothen-Rutishauser, B. Small 2010, 6, 1669–1678 doi:10 1002/smll 201000528 Parak, W. J.; Gehr, P.; Rothen-Rutishauser, B. Small 2010, 6, Lactate dehydrogenase (LDH) assay A 1 mL sample of the supernatant of each experiment was collected and stored at 4 °C to determine cytotoxicity. Triton X 1634 Beilstein J. Nanotechnol. 2014, 5, 1625–1636. 5. Joshi, H. M.; Bhumkar, D. R.; Joshi, K.; Pokharkar, V.; Sastry, M. 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https://openalex.org/W3199760333	https://opus.bibliothek.uni-augsburg.de/opus4/files/91138/Thummerer_2021_J_Phys%20_Conf_Ser.pdf	English	null	Hybrid modeling of the human cardiovascular system using NeuralFMUs	Journal of physics. Conference series	2,021	cc-by	5,985	PAPER • OPEN ACCESS PAPER • OPEN ACCESS You may also like Interaction between runaway electrons and internal kink in a post-disruption plasma Yueqiang Liu, L. Li, C.C. Kim et al. - HYDRO-PACIAE, a hydrodynamic and transport hybrid model for ultra-relativistic heavy ion collisions Yu-Liang Yan, Yun Cheng, Dai-Mei Zhou et al. - LITHIUM DEPLETION IS A STRONG TEST OF CORE-ENVELOPE RECOUPLING Garrett Somers and Marc H. Pinsonneault - Journal of Physics: Conference Series Journal of Physics: Conference Series You may also like Interaction between runaway electrons and internal kink in a post-disruption plasma Yueqiang Liu, L. Li, C.C. Kim et al. - HYDRO-PACIAE, a hydrodynamic and transport hybrid model for ultra-relativistic heavy ion collisions Yu-Liang Yan, Yun Cheng, Dai-Mei Zhou et al. - LITHIUM DEPLETION IS A STRONG TEST OF CORE-ENVELOPE RECOUPLING Garrett Somers and Marc H. Pinsonneault - To cite this article: Tobias Thummerer et al 2021 J. Phys.: Conf. Ser. 2090 012155 View the article online for updates and enhancements. This content was downloaded from IP address 137.250.100.44 on 06/12/2021 at 08:17 This content was downloaded from IP address 137.250.100.44 on 06/12/2021 at 08:17 This content was downloaded from IP address 137.250.100.44 on 06/12/2021 at 08:17 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 Journal of Physics: Conference Series 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Hybrid modeling of the human cardiovascular system using NeuralFMUs Tobias Thummerer, Johannes Tintenherr and Lars Mikelsons Chair of Mechatronics, Augsburg University, Germany E-mail: {tobias.thummerer@informatik, johannes.tintenherr@student, lars.mikelsons@informatik}.uni-augsburg.de Abstract. Hybrid modeling, the combination of ﬁrst principle and machine learning models, is an emerging research ﬁeld that gathers more and more attention. Even if hybrid models produce formidable results for academic examples, there are still diﬀerent technical challenges that hinder the use of hybrid modeling in real-world applications. By presenting NeuralFMUs, the fusion of a Functional Mock-up Unit (FMU), a numerical ODE solver and an artiﬁcal neural network, we are paving the way for the use of a variety of ﬁrst principle models from diﬀerent modeling tools as parts of hybrid models. This contribution handles the hybrid modeling of a complex, real-world example: Starting with a simpliﬁed 1D-ﬂuid model of the human cardiovascular system (arterial side), the aim is to learn neglected physical eﬀects like arterial elasticity from data. We will show that the hybrid modeling process is more comfortable, needs less system knowledge and is therefore less error-prone compared to modeling solely based on ﬁrst principle. Further, the resulting hybrid model has improved in computation performance, compared to a pure ﬁrst principle white-box model, while still fulﬁlling the requirements regarding accuracy of the considered hemodynamic quantities. The use of the presented techniques is explained in a general manner and the considered use-case can serve as example for other modeling and simulation applications in and beyond the medical domain. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 2 https://github.com/ThummeTo/FMIFlux.jl 1 https://github.com/ThummeTo/FMI.jl 1 https://github.com/ThummeTo/FMI.jl 2 https://github.com/ThummeTo/FMIFlux.jl 1. Introduction h l The structural integration of physical white-box models into artiﬁcal neural networks (ANNs) to retrieve a hybrid model is a growing research ﬁeld, see [1] or [2]. One milestone was the use of state-of-the-art numerical solvers for ordinary diﬀerential equations (ODEs) inside of ANNs instead of residual net structures to reproduce numerical integration and learn dynamic system behavior, like in [3]. The resulting combination of an ANN and an ODE solver - so called NeuralODEs - lead to improvements in model accuracy while also enhancing computation and memory cost. y Based on the idea of NeuralODEs we presented NeuralFMUs in [4], the structural integration of a ﬁrst-principle model in form of a FMU, a modern numerical solver like Tsit5 [5] or CVODE [6] and a feed-forward ANN. Further, we provided the open-source frameworks FMI.jl1 and FMIFlux.jl 2 to allow for the setup and training of NeuralFMUs just like a convenient ANN in the Julia programming language. In this contribution, the considered technologies shall be tested with a model of the human cardiovascular system (s. Sec. 2.1). 1 https://github.com/ThummeTo/FMI.jl 2 https://github.com/ThummeTo/FMIFlux.jl 1 IC-MSQUARE 2021 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series One of the core challenges in hybrid modeling, the fusion of data driven and ﬁrst principle models, is to provide an eﬃcient training process for the resulting heterogeneous structure. For training of ANNs in general, the gradient of the loss function according to the net parameters is needed. This belongs also to hybrid models: The gradient must be determined along the ANN, the numerical solver and the model of the physical system. The diﬀerent jacobians must be determined by diﬀerent methods, because of the availability (or non-availability) of information and interfaces between the NeuralFMU components. For example, the mathematical operations inside of ANNs are known, therefore many methods to retrieve partial derivatives, like automatic diﬀerentiation, are possible to use. Models exported from modeling tools on the other hand need diﬀerent techniques, because they do not necessarily provide the symbolic structure of the model or often actively hide this information because it may contain sensible company knowledge. For FMUs, we suggest the use of the optional built-in function fmi2GetDirectionalDerivative or - if not available - sampling of additional simulation points and partial derivative approximation via ﬁnite (central) diﬀerences. Both approaches are implemented as part of FMIFlux.jl. Finally, if all jacobians over every component are retrieved, the needed diﬀerentiation chains must be deployed to the machine learning framework. For an overview and more detailed insight into the necessary technical steps, see [4]. In the following, some short style explanations about involved tools and standards are given. 1.1. Julia programming language The Julia Programming Language (hereinafter: Julia) is a dynamic typing language, being developed at the Massachusetts Institute of Technology since 2009 and ﬁrst published in 2012 [7]. Julia provides the ability to run code platform-independent and with performance benchmarks similar to native C-implementations. Regardless of the fact that Julia is relatively young (in terms of programming languages), the community is growing rapidly and many other research facilities joined the development of the language and language extensions. 1.2. Modelica The physical modeling was performed in the object-orientated modeling language Modelica (https://modelica.org/). Modelica allows acausal modeling, meaning causalization of the system of equations is handled automatically by the compiler at compile time. This allows for the building of large models, while keeping them human understandable by using an intuitive topology and sub-model enclosures. The two most common tools for graphical supported programming with Modelica are Dymola (by Dassault Syst`emes ®) and OMEdit (open-source), both tools allow for model export as FMU (s. next subsection). 2.1. Reference model f In [8], a model to simulate arterial pulse waves and their changes over aging of healthy patients is introduced. Together with the model itself, simulation results for arterial blood pressure, volume ﬂow, cross section (luminal area) and photoplethysmography (PPG) for 4,374 model patients of ages between 25 and 75 years are published. The model and simulation data was validated with in vivo data from diﬀerent sources. The ﬂuid simulation is implemented as laminar, incompressible, newtonian 1D-ﬂow, extended by a viscoelastic term to model diameter changes in arterial cross sections. The simulation setup and parameterization was done in Matlab (by MathWorks ®), the 1D-ﬂuid simulation was performed with Nektar1D (http://haemod.uk/nektar) using the hemodynamic model from [9]. The arterial system is closed by boundary conditions: The heart is abstracted as predetermined mass ﬂow into the Aorta, the vascular beds (the remaining boundary conditions of the ﬂuid simulation) where modeled as three-element windkessels. Please note, that only the arterial side of the cardiovascular system is modeled: Starting at the heart, the blood ﬂow passes multiple arteries and ﬁnally reaches the vascular beds. 0.6 0.8 [s] Arterial Network Arterial Head Legs Arm Ra Art ow P1D Q1D Q1D , P1D Aorta Inle Diame R 60/HR Sup. Temporal Brachial Digital Femoral Anterior Tibial Sup. Mid. Cerebal Aortic Root Thoracic Aorta Abdominal Aorta Iliac Bifurcation Common Iliac Figure 1: The simpliﬁed structure of the arterial side of the human cardiovascular system (ﬁgure adapted from [8]). The hemodynamics at the 10 selected artery segments (red) will be learned from data. 0.6 0.8 [s] Arterial Network Arterial Head Legs Arm Ra Art ow P1D Q1D Q1D , P1D Aorta Inle Diame R 60/HR Sup. Temporal Brachial Digital Femoral Anterior Tibial Sup. Mid. Cerebal Aortic Root Thoracic Aorta Abdominal Aorta Iliac Bifurcation Common Iliac Sup. Temporal Aortic Root Thoracic Aorta Abdominal Aorta Common Iliac Figure 1: The simpliﬁed structure of the arterial side of the human cardiovascular system (ﬁgure adapted from [8]). The hemodynamics at the 10 selected artery segments (red) will be learned from data. Figure 1: The simpliﬁed structure of the arterial side of the human cardiovascular system (ﬁgure adapted from [8]). The hemodynamics at the 10 selected artery segments (red) will be learned from data. The simulation database of the introduced contribution, further referenced as Pulse Wave Database (PWDB) (https://zenodo.org/record/3275625), serves as an ideal starting point for the presented machine learning task. 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series Journal of Physics: Conference Series This paper further divides into three sections: The presentation of the cardiovascular system model, modeling assumptions and the extension to a hybrid model (s. Sec. 2), followed by training and validation of the hybrid model (s. Sec. 3) and ﬁnally a short conclusion, including current and future work (s. Sec. 4). 1.3. Functional Mock-up Interface p f Functional Mock-up Interface (FMI) allows for the simulation and parameterization of models outside of the original modeling environment in a standardized and platform-independent format. It is possible to generate standard-compliant models, so called FMUs, in two diﬀerent modes: model exchange (ME) and co-simulation (CS). ME-FMUs oﬀer an interface for the system dynamics, meaning the FMU computes a system state derivative for a given system state. In a subsequent step outside of the FMU, the next system state can be derived by numerically integrating the state derivative. CS-FMUs already include a numerical ODE solver, which allows for an even easier simulation. On the other hand, this inclusion prevents manipulation of the system dynamics before the numerical integration and consequently makes this mode unattractive for the aim of this contribution. 2 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 IC-MSQUARE 2021 Journal of Physics: Conference Series • Thoracic Aorta, end of segment 18 • Abdominal Aorta, start of segment 39 • Iliac Bifurcation, end of segment 41 • left Superﬁcial Temporal Artery, end of segment 87 • left Superior Middle Cerebral Artery, end of segment 72 • left Brachial Artery, three quarters along segment 21 • left Digital Arteries, end of segment 112 • left Iliac Artery, half way of segment 44 • left Femoral Artery, half way of segment 46 • left Tibial Artery, end of segment 49 2.1. Reference model The PWDB includes ready-to-use data for 12 arterial segments, the following 10 segments of this set will be used for the later training process: 3 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series Journal of Physics: Conference Series • The diameters of the arterial segments are assumed constant, meaning the arterial cross section does not vary dependent on the system state (pressure). • The diameters of the arterial segments are assumed constant, meaning the arterial cross section does not vary dependent on the system state (pressure). • Finally, the arteries are assumed cylindrical, so the inlet and outlet radius are the same size. To parameterize the pipes with parameter data from the reference model, which assumes a pipe with constant diameter change over length, the mean value of inlet and outlet diameter was used. • Finally, the arteries are assumed cylindrical, so the inlet and outlet radius are the same size. To parameterize the pipes with parameter data from the reference model, which assumes a pipe with constant diameter change over length, the mean value of inlet and outlet diameter was used. (a) Thoracic Aorta (#18) (b) Digital Arteries (#112) Figure 3: Deviation between the Modelica model (green/dashed) and the reference model (red/dotted) for the simulation of a single pressure pulse wave of subject #1, observed at two diﬀerent segments. (a) Thoracic Aorta (#18) (b) Digital Arteries (#112) (b) Digital Arteries (#112) (a) Thoracic Aorta (#18) Figure 3: Deviation between the Modelica model (green/dashed) and the reference model (red/dotted) for the simulation of a single pressure pulse wave of subject #1, observed at two diﬀerent segments. As to expect, these modeling simpliﬁcations lead to a signiﬁcantly diﬀerent simulation result compared to the reference model (s. Fig 3). However, the pulse wave characteristic is roughly visible despite the simpliﬁcations. The main reason for the diﬀerence between reference and simpliﬁed Modelica model is the lack of arterial dynamics: The system pressure inside the simpliﬁed model is the same at any location inside the arterial network, but varies over time with the heart pulse wave. On the other hand, the simpliﬁed model has a much better computational performance. The motivation for hybrid modeling is to retrieve model accuracy close to the reference model, but providing better computational performance at the same time. , p g p p To conclude, the only remaining system dynamics are the pressures over the 46 terminal windkessels, meaning the system state xwk (windkessels) can be uniquely described using a 46 entries state vector. 2.2. First principle model (Modelica) We start by building up a ﬁrst principle model in form of an intuitive, object-orientated Modelica model, using default components from the Modelica Standard Library (MSL) as far as possible. However, the presented procedure is not limited to Modelica models, of course. m m_flow Input 87 41 39 44 46 49 112 72 21 18 Figure 2: The physical model of the human cardiovascular system (arterial side), consisting of 116 static pipes (arterial segments, red/blue) and 46 three-element windkessels (vascular beds, green), in Dymola. From the set of pipes, 10 segments were picked for the machine learning task (red). The mass ﬂow from heart into the Aorta m ﬂow is given as model input. The model was build with components of the MSL. Figure 2: The physical model of the human cardiovascular system (arterial side), consisting of 116 static pipes (arterial segments, red/blue) and 46 three-element windkessels (vascular beds, green), in Dymola. From the set of pipes, 10 segments were picked for the machine learning task (red). The mass ﬂow from heart into the Aorta m ﬂow is given as model input. The model was build with components of the MSL. The ﬁrst principle model of the cardiovascular system is a simpliﬁcation of the reference model concerning the following aspects: The ﬁrst principle model of the cardiovascular system is a simpliﬁcation of the reference model concerning the following aspects: • The arteries are modeled as static pipes (MSL 3.2.3: Modelica.Fluid.Pipes.StaticPipe), meaning mass, momentum and energy balances are assumed steady-state. As a result, the pipe itself does not store any mass or energy, but physical conservation principles are satisﬁed. • The arteries are modeled as static pipes (MSL 3.2.3: Modelica.Fluid.Pipes.StaticPipe), meaning mass, momentum and energy balances are assumed steady-state. As a result, the pipe itself does not store any mass or energy, but physical conservation principles are satisﬁed. 4 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 To retrieve a more exact model that describes a dynamic pressure drop over the arterial segments dependent on arterial cross section change by elasticity, more states are needed. To setup the Modelica model for machine learning, placeholder states xart (arteries) were inserted into the system at the considered artery locations, resulting in the concatenated system state vector x = xwk\|xart. This state space expansion occurs by simply inserting components into the object-orientated model at the desired locations. Two placeholders types are examined: The insertion of ﬂuid capacities with small capacitance (hereinafter: C- placeholders, one state) and the insertion of parallel circuits consisting of a capacitor and an inductance (hereinafter: LC-placeholders, two states). This reﬂects a straightforward model enhancement process: Adding dynamic placeholders to a model at the locations needed, while the correct physical term is learned from data. Note, that the use of a variety of placeholders is possible, like RLC-circuits for example. After this ﬁnal step, both models (one with C- and one with LC-placeholders) were exported as FMUs. 5 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 Journal of Physics: Conference Series 2.3. Hybrid model 2.3. Hybrid model 2 3 ybrid model Hybrid modeling is a technique picked often, if higher modeling accuracy is needed, but conventional ﬁrst principle modeling does not increase precision, is not economically practicable anymore or is simply not possible because of lack of system knowledge. On the other hand, almost any development process in medicine, mechanics or electronics generates data, that can be used to achieve better (more precise or faster computing) hybrid models. During hybrid modeling, we will neglect knowing the reference system and view the problem from a diﬀerent perspective: Starting with the simple Modelica model, we want to improve model accuracy without knowing the physical principles needed for this step. Therefore, the improvement process is done solely on basis of the reference model simulation data, not on physical system knowledge. This mirrors a typical use case: Model accuracy shall be improved, further ﬁrst principle modeling is not an option, but measurement data of a more accurate (or the real) system is available and can be used. ) To start with hybrid modeling, the FMU models exported from Dymola are imported into Julia using the library FMI.jl. With the library extension FMIFlux.jl, we can setup and train NeuralFMUs on basis of the Modelica model FMUs. For the considered use-case, two NeuralFMUs as in Fig. 4 (one with C- and one with LC-placeholders) with layer dimensions as in Tab. 1 were examined. Table 1: Topologies of the considered NeuralFMUs. Layer Type C-placeholders LC-placeholders Activation Inputs Outputs Inputs Outputs #1 state vector separation 56 10 \| 46 66 20 \| 46 none #2 data pre-processing 10 10 20 20 none #3 bias 10 10 20 20 none #4 data post-processing 10 10 20 20 none #5 state vector merging 10 \| 46 56 20 \| 46 66 none #6 FMU 56 56 66 66 none #7 derivative vector separation 56 10 \| 46 66 20 \| 46 none #8 data pre-processing 10 10 20 20 none #9 dense 10 30 20 30 tanh #10 dense 30 10 30 20 none #11 data post-processing 10 10 20 20 none #12 derivative vector merging 10 \| 46 56 20 \| 46 66 none 3. Training & Validation During training, three pulse wave cycles for patient #1 are simulated (2.466 s, varies between subjects because of diﬀerent heart rates). 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series 2.3. Hybrid model The ﬁrst pulse wave is ignored in the training process and allows the system to retrieve a stationary (periodically repeating) state. The training takes place on two cycles to promote learning a time-invariant system behavior, meaning a periodically repeating input (heart) should generate a periodically repeating output (arterial pressure curves) with same period length. Even if the PWDB includes data observed at 500 Hz, the training process is sampled down to 40 Hz because of training performance optimization. To conclude, training takes place on only two pulse waves of one patient, resulting in 66 data points `a 10 Table 1: Topologies of the considered NeuralFMUs. Layer Type C-placeholders LC-placeholders Activation Inputs Outputs Inputs Outputs #1 state vector separation 56 10 \| 46 66 20 \| 46 none #2 data pre-processing 10 10 20 20 none #3 bias 10 10 20 20 none #4 data post-processing 10 10 20 20 none #5 state vector merging 10 \| 46 56 20 \| 46 66 none #6 FMU 56 56 66 66 none #7 derivative vector separation 56 10 \| 46 66 20 \| 46 none #8 data pre-processing 10 10 20 20 none #9 dense 10 30 20 30 tanh #10 dense 30 10 30 20 none #11 data post-processing 10 10 20 20 none #12 derivative vector merging 10 \| 46 56 20 \| 46 66 none Table 1: Topologies of the considered NeuralFMUs. 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 arterial pressure values. Note, that this is a very small training base in the ﬁeld of machine learning. arterial pressure values. Note, that this is a very small training base in the ﬁeld of machine learning. The training results, comparing the original FMU pressure (from the Modelica model), the improved NeuralFMU pressures and the target pressure (reference system data) can be found in Fig. 5. As loss function, a simple mean-squared-error with increasing time horizon between the reference system and NeuralFMU arterial blood pressures was deployed. The loss function rates the deviation only for a random sub-set of three artery segments, while changing the sub-set every training cycle (Batching). The training itself was controlled via parameter freezing: Until reaching a signiﬁcant small error (loss), parameters of the derivative ANN (s. Fig. 4: bottom) were locked to force compensation of state oﬀsets by the state ANN (s. Fig. 4: top) and prevent early corruption of the system dynamics. After reaching a loss threshold, all net parameters were unlocked and trained in parallel. For the C-placeholders, the static pressure wave from the FMU model is vertically scaled and shifted to ﬁt the reference system data as good as possible. The LC-placeholders provide a better ﬁt, because the LC-circuit additionally allows for phase-shifting the original pulse waves. The NeuralFMUs produce less data (only 10 dynamic locations instead of 116) and the output result does only approximate the reference model output, because of the relative small ANN layout and a lack of states to interfere with the system. On the other hand, there is a signiﬁcant gain in performance, which motivates the use of NeuralFMUs for use-cases, that focus on less measurement locations and/or more on fast computations than maximum precision, like embedded hardware or wearable computing. The reference model has a simulation time3 of ≈756 s for 10 pulse waves, resulting in an average ≈75.6 s per pulse waves. The NeuralFMUs on the other hand, have a simulation time4 of only ≈0.20 s per 10 pulse waves, meaning an average of ≈0.02 s per pulse wave5. Even if both simulations were performed on diﬀerent systems, a signiﬁcant performance gain by a factor of ≈3750 is clearly visible. This scope can be used to improve accuracy through deeper and/or wider network topologies or the injection of more and/or more complex state placeholders if necessary. 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 Testing the NeuralFMUs with unobserved data from patient #2 leads to similar ﬁtting pressure curves (s. Fig. 6), however the remaining average error is slightly bigger. Even if the derivative ANN (bottom) did learn a simpliﬁed abstraction of the pressure dynamics, the simpliﬁcation itself was understood in a generalized manner. Please note at this point, that the heart rate diﬀers between the two patients (≈73 bpm for #1 and ≈77 bpm for #2). The state ANN (top) was only able to learn static oﬀsets between the states of the reference system and the ﬁrst principle model parameterized for patient #1, but the oﬀsets in data diﬀer for patient #2. It is visible in the plots, that a small vertical oﬀset between training and testing results exists. These oﬀsets could be reduced by training the state ANN on data of patient #2. A more detailed result validation is omitted at this point, but will be part of a pursuing contribution. 3 AMD RyzenTM 9 3900X on Ubuntu 20.04.2 LTS 4 Intel® CoreTM i7-8565U on Windows 10 Enterprise 20H2 5 Even if the number of states and mathematical complexity of the FMUs diﬀer between C- and LC-placeholders, the diﬀerence in simulation time of the resulting NeuralFMUs was marginal. 6 https://github.com/ThummeTo/FMI.jl 3 AMD RyzenTM 9 3900X on Ubuntu 20.04.2 LTS 4 Intel® CoreTM i7-8565U on Windows 10 Enterprise 20H2 3. Training & Validation 3. Training & Validation 3. Training & Validation During training, three pulse wave cycles for patient #1 are simulated (2.466 s, varies between subjects because of diﬀerent heart rates). The ﬁrst pulse wave is ignored in the training process and allows the system to retrieve a stationary (periodically repeating) state. The training takes place on two cycles to promote learning a time-invariant system behavior, meaning a periodically repeating input (heart) should generate a periodically repeating output (arterial pressure curves) with same period length. Even if the PWDB includes data observed at 500 Hz, the training process is sampled down to 40 Hz because of training performance optimization. To conclude, training takes place on only two pulse waves of one patient, resulting in 66 data points `a 10 g During training, three pulse wave cycles for patient #1 are simulated (2.466 s, varies between subjects because of diﬀerent heart rates). The ﬁrst pulse wave is ignored in the training process and allows the system to retrieve a stationary (periodically repeating) state. The training takes place on two cycles to promote learning a time-invariant system behavior, meaning a periodically repeating input (heart) should generate a periodically repeating output (arterial pressure curves) with same period length. Even if the PWDB includes data observed at 500 Hz, the training process is sampled down to 40 Hz because of training performance optimization. To conclude, training takes place on only two pulse waves of one patient, resulting in 66 data points `a 10 6 6 IC-MSQUARE 2021 Journal of Physics: Conference Series IC-MSQUARE 2021 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 6 https://github.com/ThummeTo/FMI.jl Acknowledgments This work has been partly supported by the ITEA 3 cluster programme for the project UPSIM - Unleash Potentials in Simulation. 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 the library extension FMIFlux.jl 7. Finally, the simulation data was compared to the target values from the PWDB. The resulting NeuralFMU produces, dependent on the requirements of the underlying application, suﬃciently accurate results. Model precision can be further improved by using more or other circuits for the state placeholders or wider and deeper ANN structures. Further, the use of more than one patient during training will improve the NeuralFMU prediction quality on unknown patients. In terms of computational eﬃciency, a signiﬁcant performance gain is visible for the considered example, making the presented approach interesting for performance critical applications. NeuralFMUs have further advantages, e.g. even though they might include black-box models, they are fully diﬀerentiable. This allows for the use of other machine learning techniques or eﬃcient model examination methods, like gradient based algorithms. NeuralFMUs open up to many new interesting use-cases, but bring up many new challenges, too. For example, most physical systems are stable by default, meaning they converge against a physical equilibrium if not disturbed. By manipulating the system dynamics via an ANN, this natural stability is not guaranteed anymore, which may result in a destabilized training process. Best practices for network initialization and stabilization methods during training must be con- sidered and will be part of a pursuing contribution. The corresponding sources for the presented example will be published soon as part of the FMIFlux.jl library repository. 7 https://github.com/ThummeTo/FMIFlux.jl 4. Conclusion & Future Work We highlighted a workﬂow to improve a ﬁrst principle model (at the example of a simple, object-orientated model) using a NeuralFMU without knowing the underlying physical equations necessary. The enhancements were learned solely based on a small set of data, in the presented example generated by a more accurate reference system. Starting with the FMI model export from the modeling tool, the resulting FMU was imported into the Julia programming language using the open-source library FMI.jl6. Inside Julia, a NeuralFMU was set up and trained, using 7 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC MSQUARE 2021 Journal of Physics: Conference Series References [1] Willard J, Jia X, Xu S, Steinbach M and Kumar V 2020 Integrating physics-based modeling with machine learning: A survey [1] Willard J, Jia X, Xu S, Steinbach M and Kumar V 2020 Integrating physics-based modeling with machine learning: A survey [2] Rai R and Sahu C K 2020 Driven by Data or Derived Through Physics? A Review of Hybrid Physics Guided Machine Learning Techniques With Cyber-Physical System (CPS) Focus vol 8 pp 71050–71073 [2] Rai R and Sahu C K 2020 Driven by Data or Derived Through Physics? A Review of Hybrid Physics Guided Machine Learning Techniques With Cyber-Physical System (CPS) Focus vol 8 pp 71050–71073 [3] Chen T Q, Rubanova Y, Bettencourt J and Duvenaud D 2018 N 1806.07366) URL http://arxiv.org/abs/1806.07366 ( p , p ) [5] Tsitouras C 2011 Runge–Kutta pairs of order 5(4) satisfying only the ﬁrst column simplifying assumption vol 62 pp 770–775 URL https://www.sciencedirect.com/science/article/pii/S0898122111004706 [5] Tsitouras C 2011 Runge–Kutta pairs of order 5(4) satisfying only the ﬁrst column simplifying assumption vol 62 pp 770–775 URL https://www.sciencedirect.com/science/article/pii/S0898122111004706 [6] Hindmarsh A C, Serban R, Balos C J, Gardner D J, Reynolds D R and Woodward C S 2021 User documentation for cvode v5.7.0 (sundials v5.7.0) Tech. rep. 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References URL https://computing.llnl.gov/sites/ default/files/cv_guide-5.7.0.pdf g p [7] Bezanson J, Karpinsky S, Shah V B and Edelman A 2012 Julia: A fast dynamic language for technical computing (Preprint 1209.5145) URL http://arxiv.org/abs/1209.5145 [8] Charlton P H, Mariscal Harana J, Vennin S, Li Y, Chowienczyk P and Alastruey J 2019 Modeling arterial pulse waves in healthy aging: a database for in silico evaluation of hemodynamics and pulse wave indexes vol 317 pp H1062–H1085 pMID: 31442381 (Preprint https://doi.org/10.1152/ajpheart.00218.2019) URL https://doi.org/10.1152/ajpheart.00218.2019 [9] Alastruey J, Parker K H and Sherwin S J 2012 Arterial pulse wave haemodynamics (Virtual PiE Led Group) pp 401–443 ISBN 9781855981331 7 https://github.com/ThummeTo/FMIFlux.jl 8 8 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 2090 (2021) 012155 IOP Publishing doi:10.1088/1742-6596/2090/1/012155 IC-MSQUARE 2021 Journal of Physics: Conference Series 𝒙 STATE ANN ME-FMU ሶ𝒙𝑓𝑚𝑢 𝑢𝑓𝑚𝑢 𝑡 ෝ𝒙𝑓𝑚𝑢 DATA PRE-PROCESSING DATA POST-PROCESSING STATE VECTOR SEPARATION STATE VECTOR MERGING 𝒙𝑎𝑟𝑡 ෝ𝒙𝑎𝑟𝑡 DERIVATIVE VECTOR SEPARATION 𝒙𝑤𝑘 DATA PRE-PROCESSING ሶ𝒙𝑎𝑟𝑡 𝒙 STATE ANN ME-FMU ሶ𝒙𝑓𝑚𝑢 𝑢𝑓𝑚𝑢 𝑡 ෝ𝒙𝑓𝑚𝑢 DERIVATIVE ANN DATA PRE-PROCESSING DATA POST-PROCESSING STATE VECTOR SEPARATION STATE VECTOR MERGING 𝒙𝑎𝑟𝑡 ෝ𝒙𝑎𝑟𝑡 ሶ𝒙𝑤𝑘 DERIVATIVE VECTOR SEPARATION ෡ሶ𝒙𝑎𝑟𝑡 NUMERICAL ODE SOLVER න DERIVATIVE VECTOR MERGING ෡ሶ𝒙 𝒙𝑤𝑘 DATA PRE-PROCESSING DATA POST-PROCESSING ሶ𝒙𝑎𝑟𝑡 𝒙(𝑡+ ℎ) ෝ𝒙𝑡+ ℎ Figure 4: The hybrid model structure. From top to bottom: Because not all states of the NeuralFMU shall be part of the training process, the state vector x is separated into the two sub-vectors xwk (the windkessels’ states) and xart (the arterial pressure states). Before being fed into the upper ANN, the arterial state values xart are pre-processed to ensure being inside an ANN-compliant range and are transformed back after the ANN pass. The state ANN at the top of the topology is able to learn and compensate state dependent modeling failures, like static oﬀsets in measurement data in this special case. The new arterial state estimate ˆxart is merged together with the original windkessel states xwk into the modiﬁed system state vector ˆxfmu, that is further passed, together with the system input ufmu and the current simulation time t, to the ME-FMU. Inside the FMU, the current state derivatives ˙xfmu are computed based on the given system state ˆxfmu. DERIVATIVE ANN ሶ𝒙𝑤𝑘 ෡ሶ𝒙𝑎𝑟𝑡 NUMERICAL ODE SOLVER න DERIVATIVE VECTOR MERGING ෡ሶ𝒙 DATA PRE-PROCESSING DATA POST-PROCESSING ሶ𝒙𝑎𝑟𝑡 𝒙(𝑡+ ℎ) ෝ𝒙𝑡+ ℎ f As for the system state, the system state derivative vector is separated into the arterial ˙xart and windkessel state derivative vector ˙xwk. References The arterial pressure dynamics are pre-processed, fed to the ANN, post-processed and the resulting ˆ˙xart is merged back together with ˙xwk into the system state derivative estimate ˆ˙x. The derivative ANN is therefore able to manipulate the system dynamics to retrieve a diﬀerent system behavior. In the considered use case, the pressure drop over the arterial segments can be learned based on data. Because the ANN is learning on basis of state derivatives, generalized learning of the correct physical laws is promoted. ˆ ෡ሶ𝒙 Finally, the system dynamics estimate ˆ˙x is numerically integrated with step size h into the next system state estimate ˆx(t+h) or the next system state x(t+h) respectively. 9 IC-MSQUARE 2021 IOP Publishing 2090 (2021) 012155 g doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series (a) Thoracic Aorta (#18) (b) Abdominal Aorta (#39) (c) Iliac Bifurcation (#41) (d) Sup. Temp. Artery (#87) (e) Mid. Cerebral Artery (#72) (f) Brachial Artery (#21) (g) Digital Arteries (#112) (h) Com. Iliac Artery (#44) (i) Femoral Artery (#46) (j) Ant. Tibial Artery (#49) Figure 5: Training results of the 10 considered arterial segments, based on data of subject #1 of the PWDB from [8]. The horizontal axis labels the simulation time t in seconds, the vertical axis the arterial pressure in Pascals. The plots show the pressure of the Modelica model (black/dotted, clipped), the target reference system pressure (red/dashed), the pressure learned by the NeuralFMU with C-placeholders (blue/solid) and with LC-placeholders (green/solid). (a) Thoracic Aorta (#18) (b) Abdominal Aorta (#39) (c) Iliac Bifurcation (#41) (d) Sup. Temp. Artery (#87) (e) Mid. Cerebral Artery (#72) (f) Brachial Artery (#21) (f) Brachial Artery (#21) (e) Mid. Cerebral Artery (#72) (h) Com. Iliac Artery (#44) (g) Digital Arteries (#112) (i) Femoral Artery (#46) (g) Digital Arteries (#112) (j) Ant. Tibial Artery (#49) (j) Ant. Tibial Artery (#49) Figure 5: Training results of the 10 considered arterial segments, based on data of subject #1 of the PWDB from [8]. The horizontal axis labels the simulation time t in seconds, the vertical axis the arterial pressure in Pascals. The plots show the pressure of the Modelica model (black/dotted, clipped), the target reference system pressure (red/dashed), the pressure learned by the NeuralFMU with C-placeholders (blue/solid) and with LC-placeholders (green/solid). References 10 IC-MSQUARE 2021 IOP Publishing 2090 (2021) 012155 g doi:10.1088/1742-6596/2090/1/012155 Journal of Physics: Conference Series Journal of Physics: Conference Series 2090 (2021) 012155 doi:10.1088/1742-6596/2090/1/012155 (a) Thoracic Aorta (#18) (b) Abdominal Aorta (#39) (c) Iliac Bifurcation (#41) (d) Sup. Temp. Artery (#87) (e) Mid. Cerebral Artery (#72) (f) Brachial Artery (#21) (g) Digital Arteries (#112) (h) Com. Iliac Artery (#44) (i) Femoral Artery (#46) (j) Ant. Tibial Artery (#49) Figure 6: Testing results on the 10 considered arterial segments. Tests where performed against data of (unknown) subject #2 of the PWDB from [8]. The horizontal axis labels the simulation time t in seconds, the vertical axis the arterial pressure in Pascals. The plots show the pressure of the Modelica model (black/dotted, clipped), the target reference system pressure (red/dashed), the pressure learned by the NeuralFMU with C-placeholders (blue/solid) and with LC-placeholders (green/solid). (a) Thoracic Aorta (#18) (b) Abdominal Aorta (#39) (c) Iliac Bifurcation (#41) ( ) ( ) (d) Sup. Temp. Artery (#87) (e) Mid. Cerebral Artery (#72) (f) Brachial Artery (#21) (f) Brachial Artery (#21) (g) Digital Arteries (#112) (i) Femoral Artery (#46) (h) Com. Iliac Artery (#44) (j) Ant. Tibial Artery (#49) (j) Ant. Tibial Artery (#49) Figure 6: Testing results on the 10 considered arterial segments. Tests where performed against data of (unknown) subject #2 of the PWDB from [8]. The horizontal axis labels the simulation time t in seconds, the vertical axis the arterial pressure in Pascals. The plots show the pressure of the Modelica model (black/dotted, clipped), the target reference system pressure (red/dashed), the pressure learned by the NeuralFMU with C-placeholders (blue/solid) and with LC-placeholders (green/solid). 11
https://openalex.org/W1977137657	https://zenodo.org/records/1842049/files/article.pdf	German	null	Über Reaktionen des Methylens. IV: Über die Zersetzung der Ketene bei hoher Temperatur	Berichte der Deutschen Chemischen Gesellschaft	1,913	public-domain	2,933	I) Eventuell riihrt die blaue Firbung, die man anfangs bei der Poly- merisation des Phenylisonitrils beobachtet, von der Bildung des Diphenyl- isonitrils - eines Imenderivates 7 her. Versuche, den K8rper zu isoliereii, hatten keinen Erfolg. (Versuche von Hm. Dr. H. W. Klever.) 9).R. Bauer, B. 40, 2653 [1907]. a) Vorige Mitteilung B. 46, 501 [1912]. V e.r su ch e z u r D a r s t el 1 u ng v o n 0 x a l s ii u r e-p h e n y 1 imi d- bromid u n d -jodid. Diese Kiirper boten Interesse, weil man aus ihnen durch Halogen- abspaltung zu dem dimolekularen Phenylisonitril ') hiitte gelangen miissen, und es ware von Wert, das Verhalten dieses Korpers im Vergleich zum Dikohlenoxyd zu kennen. Bei dem bekannten Oxal- saure-phenylirnidchlorid a) konnte diese Halogenabspaltung wegen der festen Bindung des Chlors nicht durchgefuhrt werden: C:N.CsIIs * ? C:N. Cs Hg -+ .* f- 2 cs Hs .N: c. C (Br) : N . C6 Hs c (Br) : N . c6 I& Die beiden KBrper waren aber nicht zu erbalten. LaBt man Bromwasser- stotf resp. Jodwasserstoff auf Oxelsiure-phenylimidchlorid, das in Benzol ge- last ist, einwirken, so erhglt man nur schmierige Massen; beim Versuch mit Jodwasserstoff tritt starke Jodabspaltung ein. Auch BUS Phosphorpentabromid und Oxanilid kann man nicht zu den1 Oxalshre-phenylimidbromid gelangen; bei tiefer Temperatur tritt keine Re- aktion ein, bei h6herer starke Verschmierung, moglicherweke begiinstigt durch- den Zerfall des Phosphorpentabromids in Phosphortribromid und Brom. p p p Phosphortribromid endlich setzt sich mit Oxanilid nicht um. 170. H. Staudinger und R. Endle: tfber Reaktionen dee Methylens. IV3): ther die Zereetsung der Ketene bei hoher Temperatur. ) [ ] a) Vorige Mitteilung B. 46, 501 [1912]. 1438 Verhalten dieser Korper bei hoher Temperatur kennen zu lernen, urn die Festigkeit der Athylenbindung hier mit der der anderen Verbin- dungen vergleichen zu kiinnen. g g I n friiheren Arbeitenl) ist gezeigt worden, da8 die K e t e n e ent- weder spontan oder bei geringer Temperaturerhohung in polymere Produkte iibergehen und zwar besonders leicht in C y c l o b u t a n - Derivate. Diese Cyclobutane konnen bei weiterer Temperatursteige- rung wieder entpolymerisiert werden, und es fragte sich, wie sich die Ketene nun bei noch hoheren Hitzegraden verhalten. Daruber finden sich in der Literatur scbon einige bemerkenswerte Beobachtungen. So fuhren J. S c h m i d l i n und M. B e r g m a n n a ) an, da8 man bei hohem Erhitzen YOU Aceton, bei Temperaturen iiber 60O0, statt des Ketens K o h l e n o x y d und A t h y l e n erhalte. Das Keten dissoziiert danach in folgender Weise: g 2CHa.CO.CHs -+ 2CHa:CO + 2CH4 --t 2CH2 <+ 2CO -+ CH,:CH,. --t 2CH2 <+ 2CO -+ CH,:CH Sehr auffallend verhalt sich das K o h l e n s u b o x y d : Leitet man seine Dampfe durch eine hoch erhitzte Glasrohre, so scheidet sich darin ein Spiegel ab, der wahrscheinlich aus K o h 1 e n s t of f besteht, und man kann daraus auf folgenden Zerfall schlieden: 0c:c:co --f c -t 2 c o . Die1 s und Wolf3), die diese Beobachtungen machten, weisen darauf hin, da13 sich hier das Kohlensuboxyd genau wie ein Metall- carbonyl, z. B. Nickelcarbonyl, verhalte. Wir untersuchten weiter die Zersetzung des Diphenyl- und Di- methyl-ketens, und zwar erhitzten wir Dampfe dieser Ketene in ver- diinntem Zustand auf hohe Temperatur (650-700°), da unter diesen Bedingungen die Zersetzung besonders glatt verliiuft. g g g g Das D i m e t h y l - k e t e n spaltet dabei K o h l e n o x y d ab, und das un besthdige D i m e t h y 1 - rn e t h y 1 e n geht teils durch Polymerisation in T e t r a m e t hyl-iit h y 1 en, teils unter Wanderung des Wasserstoffs in P r o p y 1 en li ber. 170. H. Staudinger und R. Endle: tfber Reaktionen dee Methylens. IV3): ther die Zereetsung der Ketene bei hoher Temperatur. [Mitteilung aus dem Chemischen Institut der Techn. Hochschule Karlsruhe.] (Eingeg,angen am 12. April 1913.) [Mitteilung aus dem Chemischen Institut der Techn. Hochschule Karlsruhe.] (Eingeg,angen am 12. April 1913.) [Mitteilung aus dem Chemischen Institut der Techn. Hochschule Karlsruhe.] (Eingeg,angen am 12. April 1913.) In der voranstehenden Mitteiluog wurde nacbgewiesen, daB das Di-Kohlenoxyd nicht existenzfiihig ist, sondern unter Sprengung der Athylenbindung in Kohlenoxyd ubergeht, wiihrend sonst die Athylen- derivate auffallend bestandig sind und auch bei hoher Temperatur nicht in hfethylenderivate zerfallen. Zwischen den eigentlichen Athylenkorpern und dern dimolekularen Kohlenoxyd bilden die Ketene gewissermden einen Ubergang, und es war daher von Interesse, das 1) Vergl. u. a. >>Die Ketenea, Chemie in Einzeldarstellungen, wo sich auf )) B. 48, 2821 [1910]. 4) Vergl. den Ubergang von Di azo-bernsteios&ureester in Fumarester (Curtius und Koch, B. 18, 1293 [1885]; A. Darapsky, B. 48, 1095 [1910]); ferner von Diazo-campher in Camphenon (Angeli, B. 87 Ref., 590 [ 18941. S. 38 eine Zusammenstellung befindet. 8) B. 39, 689 [1906]; 40, 355 [1907]. )) B. 48, 2821 [1910]. 4) Vergl. den Ubergang von Di azo-bernsteios&ureester in Fumarester (Curtius und Koch, B. 18, 1293 [1885]; A. Darapsky, B. 48, 1095 [1910]); ferner von Diazo-campher in Camphenon (Angeli, B. 87 Ref., 590 [ 18941. 8) B. 39, 689 [1906]; 40, 355 [1907]. g g [ ] 3 Uber Versuche zur Darstellung von Diphenylmethplen, vergl. Stad- dinger und Kupfer, B. 44, 2203 [1911] usw., aus denen die Unbestandig- keit dieses Methylenderivates deutlich erhellt. 1438 Derartige Umlagqungen von Methylenderivaten sind schon hiiufiger beobachtet worden '): g ) CH1\ ' --t CHa CH,;C: C<CH, CHa . CH3/C'- 1) Vergl. u. a. >>Die Ketenea, Chemie in Einzeldarstellungen, wo sich auf S. 38 eine Zusammenstellung befindet. 1) Vergl. u. a. >>Die Ketenea, Chemie in Einzeldarstellungen, wo sich auf S. 38 eine Zusammenstellung befindet. )) B. 48, 2821 [1910]. g 8) B. 39, 689 [1906]; 40, 355 [1907]. )) [ ] 4) Vergl. den Ubergang von Di azo-bernsteios&ureester in Fumarester (Curtius und Koch, B. 18, 1293 [1885]; A. Darapsky, B. 48, 1095 [1910]); ferner von Diazo-campher in Camphenon (Angeli, B. 87 Ref., 590 [ 18941. , [ ] [ ] l) Engler und Bethge, B. 7, 1128 [1874]. 1439 Das D i p h e n y l - k e t e n wird bei hoher Temperatur auch an der Athylenbindung gespalten, primar in K o h l e n o x y d und D i p h e n y l - methylen. Letzteres polymerisiert sich hierbei nicht zu T e t r a - p h e n y l - a t b y l e n , v i e man es unter anderen Bedingungen beobachtet hat I), sondern zur Absrittigung des Methylen-Kohlenstofles aandern zwei Wasserstoffatome, und es bildet sich F l u o r e n : Nach diesen Resultaten nehmen die Ketene in Bezug auf Be- standigkeit der Athylenbindung in der Tat die erwartete Zwischen- stellung zwischen den Atb ylenk6rpern und dem Dikohlenoxyd ein. Urn die Bestandigkeit der gew6hnlichen Athylenbindung nachzuweisen, wurden eine Reihe Dipbenyl-athylen-Derivate unter gleichen Bedin- gungen wie das Diphenyl-keten auf hohe Temperatur erhitzt; sie blieben dabei vollstandig unverandert. (c6 H5)t C: CH,, bestandig, nicht gespalten (C,Hs)aC:C(CsHs)i, 3 s D (C, H5)a C : C Clt, 3 s s (c6 Hs)9 C: CO, bei hoherer Temperatur unbestrindig - gespalten, OC: CO, nicht existenzfahig. (c6 H5)t C: CH,, bestandig, nicht gespalten (C,Hs)aC:C(CsHs)i, 3 s D (C, H5)a C : C Clt, 3 s s (c6 H5)t C: CH,, bestandig, nicht gespalten (C,Hs)aC:C(CsHs)i, 3 s D (C, H5)a C : C Clt, 3 s s (c6 Hs)9 C: CO, bei hoherer Temperatur unbestrindig - gespalten, OC: CO, nicht existenzfahig. ( , ) , (c6 Hs)9 C: CO, bei hoherer Temperatur unbestrindig - gespalten, OC: CO, nicht existenzfahig. Danach hangt die Bestandigkeit eines Athylenderivates vollstandig yon der Bestandigkeit der sie zusammensetzenden Methylenverbindung ab. Das Dikohlenoxyd zerflllt, weil daraus das bestiindige Kohlen- oxyd entsteht, wlhrend die gewiihnlichen tetrasubstituierten Athylen- derivate bestandig sind, entsprechend der auBerordentlichen Unbestiin- digkeit der disu bstituierten Methylenderivate '). Die Allen-Derivate, deren pyrogene Zersetzung in diesem Zu- sammenhang von Interesse ware, sollen daraufhin nntersucbt werden. P h e n y l i s o c y a n a t und P h e n y l s e n f o l hatten, nach ihrem Bau zu scbliefien, in gleicher Weise wie das Keten zerFallen kiinnen. Aber h i d e KBrper sind unter Bedingungen, unter denen die Ketene die Spaltung erleiden, vollstiindig bestandig: CsHo.N:CO --f CsHs.N:N.CsHs + 2C0, C6Hs.N:CS --t CsHs.N:N.C6H5 + 2CS. l) Engler und Bethge, B. D i p h e n y l- k e ten. Ein Rijhrchen mit 6 g Diphenylketen wurde, nachdem die Ca- pillare geiiffnet war, in das Quarzrohr eingefiihrt, der Apparat zu- sammengestellt, mit Kohlensiiure gefullt, um den Sauerstoff voll- sthdig auszuschlieflen und dann erst nach dem Evakuieren der Ver- such eingeleitet. Unter lebhafter Gasentwicklung gehen Diimpfe uber, die sich griifitenteils in der ersten Vorlage zu einer gelblich-weii3ea Krystallmasse verdichten, die nach dem Umkrystallisieren aus Petrol- iither unter Zusatz von Tierkohle bei 114' schmilzt und nach Miscb- probe mit F l u o r e n identisch ist. Ausbeute an reinem Fluoren ca. 2.2 g. 1439 7, 1128 [1874]. 3 Uber Versuche zur Darstellung von Diphenylmethplen, vergl. Stad- dinger und Kupfer, B. 44, 2203 [1911] usw., aus denen die Unbestandig- keit dieses Methylenderivates deutlich erhellt. V e r s u c h s a n o r d n un g. Zu den Zersetzungen diente eine einseitig zugeschmolzene Quarz- riihre von 40 cm LInge und 2 cm licbter Weite, in deren hinteren Teil die Substanz eingefiillt wurde, wahrend in der Mitte eine Silber- spirale mit Hilfe eines T ecl u-Brenners auf 600 -7OOO erhitzt wurde. Die Temperaturen bestimmten wir bei einigen Versuchen durch ein L e C h a t e l i e r sohes Pyrometer. Als AbsorptionsgefSOe dienten : erst eine grijdere Kugelvorlage, dann zwei Spiralvorlagen, die je nach dem Versuch durch Kaltemischung, Kohlensaure-Ather oder fliissige Luft gekublt wurden. Hieran schlof3 sich ein U-Rohr mit Paraffin01 an, in dern man die Starke der Gasentwichlung bei der Zersetzung beobachten konnte und weiter eine Wasserstrahlluftpumpe, durch die tler Apparat evakuiert wurde. Die Versuche wurden in der Weise ausgefubrt, daB man nach Evakuieren des Apparates die Substanz langsam uber die erhitzte Silberspirale destillierte und die Diimpfe in den Vorlageu kondensierte. 1) Genauere Angaben vergl. R. En dle, Dissertation, Ziirich 1913. 2) Aus Riickstinden der Dimethplketen-Darstellung. 1) Genauere Angaben vergl. R. En dle, Dissertation, Ziirich 1913. 2) Aus Riickstinden der Dimethplketen-Darstellung. D i m e t h y 1- ke t en. Statt des freien Dimethylketens, das ja sehr schwer zu handhaben ist, wurde das relativ leicht zugangliche Te trame t h y 1 -dike to-cpclo b u tan 2, angewandt, welches sich sehr leicht durch Erhitzen in Dimethylketen spaltet. Bei der Zersetzung von 4.5 g Keten tritt sehr starke Gasentwicklung auf, so (la8 das Vakuum von 12 mm auf 50-60 mm zuriickgeht. Zur Kondensation der Zersetzungsprodukte war bei diesem Versnch die erste und zweite Spiral- vorlage mit Kohlensiure-ither, eine dritte mit flilssiger Luft gekiihlt. Zwischen den beiden ersten Spiralvorlagen befand sich ein 20 cm langes Quanrohr von */2 cm lichter Weite, und da in der ersten Spiralvorlage sich 1441 groBere Mengen scheinbar unverhderten Ketens kondensiert hatten, so wurde dieses durch die erhitzte Quarzrtihre weiter durchdestillisrt, urn v6lligen Zer- fall zu erreichen. Bei -180O batten sic% die leichtfliichtigen Teile kondensiert, die aus P r o p yl en bestanden, wie durch Bestimmung des Siedepunktes nach nochmaliger Rektifikation mittels Konstantan-Thermoelementes festgestellt wurde. Ferner wurde es in P r o p y l e n b r o m i d uberge- fuhrt, das bei 12 mm Druck bei 31-33O, unter gewijhnlichem Druck bei 138-139O siedete; Siedepunkt des Propylenbromids 32O bei 13 mm Druck, 141° bei gewohnlichem Druck. Der schwerer fliichtige Teil, der sich in Kohlensaure-Ather kon- densiert hatte, ist trotz seiner schwachgelben Farbe, die von Verun- reinigungen herrii hrte, nicht mehr unverandertes Dimethylketen, sondern T e t r a m e t h y l - a t h y l e n . Es wurde nochmals im Vakuum durch eine gliihende Quarzrohre durchdestilliert und ist dabei im Gegensatz zum Dimethylketen bestandig. Als Tetramethyl-athylen erweist sich das Prodakt durch seinen Siedepunkt 70-740 (Siedepunkt vom reinen Produkt 73'). I) A. 174, 194. ~ ~ ~ ~ . _ _ _ Bnchdruckerei A. W. S c h ad e , Berlin N.. Schulzendorferatr. 26. Sons tige V e rs uc h e. Destilliert man bei ca. 7000 und einem Vakuum von 11 mm Diphenyl-iithylen, Tetrapheoyl-athylen, Diphenyl-dichlor-ath ylen durch den Apparat, so beobachtet man keine Gasentwicklung, und in der Vorlage werden die Produkte unverandert wieder erhalten; nur beim Diphenyl-dichlor-athylen war ganz geringe Zersetzung zu be- nierken. Ferner ging im Vakuum auch Diphenyl-methan vollstandig un- zersetzt uber, wahrend Gra be ') beim Durchleiten der Substanz durch ein rotgluhendes Rohr F l u o r e n erhalten hat. g Aus dem D i p h e n y l - b r o m - m e t h a n erhalt man bei ca. 250° unter Bromwasserstoffabspaltung glatt T e t r a p h e n y 1 - a t h y 1 en, eine Fluorenbildung wurde dabei nicbt beobachtet. Wir versuchten, ob unter den Versuchsbedingungen der Zer- setzung des Dipbenylketens, also bei 7 0 0 0 im Vakuum, das Diphenyl- brom-metban unter Bromwasserstoffabgabe auch in Fluoren ubergehe. Die Silberspirale wurde dabei durch eine Fullung von Tonscherben ersetzt. Wir erhielten aber merkwurdigerweise auch hier das T e t r a - p h e n y l - l t h y l e n und nicht Fluoren, so daB vielleicht, was noch ge- nauer zu untersuchen ist, auch das Kontaktmaterial eine Rolle spielt. Auch die Spaltung des Diphenyl-chlor-rnethans, die in derselben Weise vorgenommen wurde, fubrte ebenfalls zu dem T e t r a p h e n y l - iithylen. P h e o y l i s o c y a n a t und Phenylsenfbl. Beide K6rper destillieren im Apparat iibor die auf ca. 700° erhiizte Silberspirale v6llig nnzersetzt iiber. Ferner wurden die Dampfe dieser Korper langere %eit mit einer gliihenden Platinspirale in Beriihrung gebracht, dadurch daB man in einen Kolben, in dem Phenylisocyanat re+ Phenylsenfd im Vakuum am RiickfluQkiihler kochte, eine durch den elektrischen Strom zum Glihen erhitzte Platinspirale einfiihre. Auch hier trat keine Zersetzung ein. 171. R. Scholl: eber die Desmotropie der Phenole in der Anthracen-Reihe. 171. R. Scholl: eber die Desmotropie der Phenole in der Anthracen-Reihe. (Eingegaogen am 10. Mdrz 1913.) In eiuer Mitteilung iiber Amin-Imin-Desmotropie') bemerken K u r t H. Mey e r undH. Schlosser, daO durch dievon ersterem bewerkstelligtelsolierung der beiden desmotropen Formen des meso-Phenols der Anthracenreihe ge- z e i g t worden sei, daO s i c h d i e l a n g e g e s u c h t e D e s m o t r o p i e d e r Phenole i n d e r Anthracen-Reihe auffindcn laat. Falls mit tliesen Worten der Eindrock erweckt werden soll, als ob K u r t H. M e y e r der Ent- decker dieser Erscheinnng in der Anthracenreihe sei, so entspricht das den Tatsachen nicht. hIan konnte davon absehen, daB dem mit der Antliracen- chemie 1HngerVertrauten schon bekannt war, da13 Anthranol je nach d e n Alter des Priiparates in kalter Lauge in wechselnclem Betrsge 16slich bis ganz unloslich, in heiBer Lauge aber vollkommen loslich sei, und daB ich selbst schon auf Grund diescr Eigenschaften frisch bereitetes Anthranol (gemeint war im Gegeusatz zu alten Sammlungspraparaten) als eine Mischung der beidcn desmotropen Formeln angesprochen habe'). Aber die Tatsache ist nicht zu umgehen. da13 der Namensvetter eines der Genannten, Hans Meyer, schon im Jahre 1909 die Desmotropie des D i a n t h r a n o l s bexw. D i a n t h r o n s aufgefunden hat?. Indem K u r t H. Jleyer und H. SchlBsser diesen Des- motropiefall im weiteren Verlaufe ihrer Arbeit selbst erwahnen, heben sic dcn Pioritatsanspruch zu Bcginn derselben selbst indirekt wieder auf. 1) €3. 46, 29 [1913!. 3) M. 30, 165 [1909]; B. 12, 144 [1909J. 4) Durch ein bedauerliches Verschen ist im lctzten IIefte die Wiedergabe 'J) B. 36, 3416 [1903]. obiger Berichtigung unterblieben. Red. der BBerichteC. 4) Durch ein bedauerliches Verschen ist im lctzten IIefte die Wiedergabe obiger Berichtigung unterblieben. Red. der BBerichteC. 172. H. Fischer: Berichtigung. 172. H. Fischer: Berichtigung. (Eingegangen am 2. Marz4) 1913.) X a n t h o b i 1 i r u bi n s a n r e statt X an t h o p y rr o 1- carbons i ur e. Jahrgang 46, S. 440, Zeile 2 von oben lies: 172. H. Fischer: Berichtigung. (Eingegangen am 2. Marz4) 1913.) X a n t h o b i 1 i r u bi n s a n r e statt X an t h o p y rr o 1- carbons i ur e. Jahrgang 46, S. 440, Zeile 2 von oben lies: 'J) B. 36, 3416 [1903]. 4) Durch ein bedauerliches Verschen ist im lctzten IIefte die Wiedergabe obiger Berichtigung unterblieben. Red. der BBerichteC. 4) Durch ein bedauerliches Verschen ist im lctzten IIefte die Wiedergabe obiger Berichtigung unterblieben. Red. der BBerichteC.
https://openalex.org/W2004031835	https://europepmc.org/articles/pmc3287574?pdf=render	English	null	Mass segmentation using a combined method for cancer detection	BMC systems biology	2,011	cc-by	5,182	* Correspondence: lchuncn@gmail.com; dadatang@yahoo.com 1College of Computer Science and Technology, Wuhan University of Science and Technology, Wuhan, Hubei, China 2Key laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China Full list of author information is available at the end of the article © 2011 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Breast cancer is one of the leading causes of cancer death for women all over the world and mammography is thought of as one of the main tools for early detection of breast cancer. In order to detect the breast cancer, computer aided technology has been introduced. In computer aided cancer detection, the detection and segmentation of mass are very important. The shape of mass can be used as one of the factors to determine whether the mass is malignant or benign. However, many of the current methods are semi-automatic. In this paper, we investigate fully automatic segmentation method. Results: In this paper, a new mass segmentation algorithm is proposed. In the proposed algorithm, a fully automatic marker-controlled watershed transform is proposed to segment the mass region roughly, and then a level set is used to refine the segmentation. For over-segmentation caused by watershed, we also investigated different noise reduction technologies. Images from DDSM were used in the experiments and the results show that the new algorithm can improve the accuracy of mass segmentation. Conclusions: The new algorithm combines the advantages of both methods. The combination of the watershed based segmentation and level set method can improve the efficiency of the segmentation. Besides, the introduction of noise reduction technologies can reduce over-segmentation. Mass segmentation using a combined method for cancer detection From BIOCOMP 2010 - The 2010 International Conference on Bioinformatics and Computational Biology Las Vegas, NV, USA. 12-15 July 2010 RESEARCH Open Access Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 RESEARCH Background to assist the radiologists in the diagnosis of the breast cancer and used to improve the diagnosis accuracy [6]. Breast cancer is one of the leading causes of cancer death for women all over the world [1] and early detec- tion is one of the main ways to reduce the death rate of the human beings with breast cancer [2-4]. One of the ways to detect the breast cancer is to use mammogra- phy. Mammography is thought of as one of the most effective methods to detect early breast cancer. Although mammography is widely used, the rate of cor- rect diagnosis of breast cancer using mammography needs improvement [5]. Thus, in order to improve the diagnosis rate, computer aided diagnosis was proposed In computer aided cancer diagnosis, the detection and segmentation of mass are very important. The shape of mass can be used as one of the factors to determine whether the mass is malignant or benign. In the past, many methods for mass segmentation algorithms have been proposed. These algorithms include manual segmen- tation [7], semi-automatic segmentation [8], and fully automatic segmentation [9]. Although manual segmenta- tion is considered to be the best mass boundary extraction method [10,11], it is time-consuming. Besides, it subjects to intra-observer and inter-observer variation [11]. In [12], Huo et al. developed a semi-automatic region growing approach based on the choice of the starting point by the radiologist. In [13], Kobatake et al. applied a modified Hough transform to extract lines passing near the centre of the mass and automatically selected candidates based on the number of line-skeletons. In [14], Lou et al. Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 2 of 9 proposed an algorithm for mass segmentation and the algorithm is based on the assumption that the trace of intensity values from the breast region to the air-back- ground is a monotonic decreasing function. In [15], Zheng et al. proposed an algorithm using the difference image obtained by subtracting the Gaussian filtered image from the original image. In [16], Petrick et al. proposed a method for mass segmentation. The basic idea of the pro- posed method is to select seeds using local maxima in the original image and generate a gradient image using a fre- quency-weighted Gaussian filtering. With this image, the thresholds of the regions bounded by the edges are extracted. Segmentation evaluation In the past, there have proposed many segmentation evaluation methods, however, segmentation evaluation is still an open topic [25,26]. There are mainly two evalua- tion methods. One is subjective evaluation, the other is objective evaluation. In subjective evaluation, visual check is often adopted while the segmentation obtained by the computer is evaluated against the segmentation obtained by a technician in objective evaluation. In this paper, we adopt objective evaluation. The evaluation measures used in the paper are [25]: Although many other results on mass segmentation have been published, automatic segmentation of mass is still considered difficult because of the ill-defined boundaries and overlapping with fibro-glandular tissue of many masses [11]. In this paper, we study fully auto- matic mass segmentation algorithm. Our basic idea is to combine two segmentation algorithms: watershed based segmentation algorithm and level set based segmenta- tion, As is well known, level set based segmentation methods are powerful image segmentation tools and have been used for image segmentation for long time because they have many advantages, for examples, they can handle any of the concavities, splitting, merging and so on. Thus they are still used in many fields including medical image processing [23]. However, there are sev- eral disadvantages on level set based segmentation methods. One of the main disadvantages is that the computation is costive. Besides, the level set based algo- rithms generally need human interaction. In order to reduce the interaction, this paper proposes an algorithm which combines a fully automatic marker-controlled watershed segmentation method with level set based segmentation. In the combined algorithm, the segmenta- tion results from the watershed are used as the input of the level set segmentation and the level set algorithm is used to refine the boundary. Hitting = TP TP + FN Missing = FN TP + FN OverHitting = FP TP + FN Re lativeHitting = TP TP + FP Re lativeMiss ing = FN TP + FP Kapps = 2 ∗Hitting 2 ∗Hitting + Mis sin g + OverHitting 2 ∗Hitting = 2 ∗Hitting + Mis sin g + OverHitting where TP, FP and FN are True Positives, False Posi- tives, and False Negatives respectively. Figure 2 shows the basic idea of TP, FP and FN of a mass segmentation. In Figure 2, TP represents the intersection of the radiol- ogist and the algorithm, FP represents the segmentation Background In [17], Qi and Snyder proposed a method for mass segmentation. They used B’ezier splines to interpo- late histograms, from which they extracted the region with threshold values at local maxima. In [18], Guliato et al. proposed a pixel based algorithm. The proposed algorithm aims to preserve the transition between masses and nor- mal tissue to segment the mass boundary. In [19], Mudi- gonda et al. used multilevel thresholding to detect closed edges for mass segmentation. Besides the work mentioned above, there is also other work published in [20-22]. proposed algorithm. For reducing computation cost, we resample the original images at a reduced pixel size and 256 gray levels. The mass location was identified by an experienced radiologist and a region of interest (ROI) containing the mass was extracted. The selected samples contain lesions with different breast-tissue density, dif- ferent degrees of subtlety, and different sizes. The distri- butions of the size of malignant and benign masses overlapped. 100 of the dataset are benign and 100 of them are malignant. A program was developed using Matlab to run on all the test images without user intervention. The results show that all cases of segmentation were accurate in comparison with the radiologist-marked on the mam- mograms. Figure 1 shows some mammograms from DDSM and the segmentation results using watershed transform and level set based segmentation method. Experimental materials In the experiments, we selected 200 mammograms ran- domly from the DDSM database [24] to verify the Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 3 of 9 results obtained only by the algorithm and the FN represents the segmentation results obtained only by the radiologist [25]. Hitting denotes the ratio of correct seg- mentation, Missing denotes the ratio of missing mass, OverHitting denotes the ratio of false mass segmente RelativeHitting denotes relative correct ratio against se mentation results, and RelativeMissing denotes relati missing ratio against segmentation results [25]. Figure 1 (a) Original images selected from DDSM; (b) Markers and object boundaries superimposed using watershed algorithm on original images; (c) The final segment results based on improved level set. Figure 1 (a) Original images selected from DDSM; (b) Markers and object boundaries superimposed using watershed algorithm on original images; (c) The final segment results based on improved level set. OverHitting denotes the ratio of false mass segmented, RelativeHitting denotes relative correct ratio against seg- mentation results, and RelativeMissing denotes relative missing ratio against segmentation results [25]. OverHitting denotes the ratio of false mass segmented, RelativeHitting denotes relative correct ratio against seg- mentation results, and RelativeMissing denotes relative missing ratio against segmentation results [25]. results obtained only by the algorithm and the FN represents the segmentation results obtained only by the radiologist [25]. Hitting denotes the ratio of correct seg- mentation, Missing denotes the ratio of missing mass, Page 4 of 9 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Figure 2 True Positives, False Positives, and False Negatives definition. Figure 2 True Positives, False Positives, and False Negatives definition. Segmentation results method usually needs hundreds of iterations to get a good segmentation result. With a good initialization provided by watershed segmentation, the level set method can converge more quickly, thus greatly speed up the whole segmentation procedure. Besides, by using watershed segmentation as the initialization step, we can remove the manual initialization step in general level set segmentation and we can obtain a full automatic seg- mentation algorithm. The comparisons of the segmentation results between the proposed method and the manually segmented image by radiologist are shown in Figure 3. In Figure 3, the black contours are the segmentation results using the proposed algorithm and the green contours are the results obtained by a radiologist. From Figure 3, we can find that the proposed method can obtain good results. We can find that the contours obtained by the proposed algorithm are closed to the contours obtained by the radiologist and it proves that the proposed algorithm is effective. Table 1 and Table 2 show the results of quan- titative analysis and from the results we can also prove the effectiveness of the proposed algorithm. However, the proposed algorithm still has a few lim- itations. In the proposed algorithm, the object to be seg- mented is already ROI images which have been preliminarily cut from the whole mammograms. Thus a mass detection step needs to be merged into the algo- rithm in the future. Although Noise reduction technolo- gies are introduced into the algorithms, over- segmentation still happens on some mammographic images. Over-segmentation affects the efficiency of the algorithm and thus an effective over-segmentation algo- rithm is needed in the future. Another issue is the time complexity of the level set. By using the result from watershed we can save a lot time but much longer com- putation time is still needed to achieve the accurate seg- mentation results. Besides the comparison of the proposed algorithm with the human segmentation, we also compared the effectiveness of different noise reduction technologies for over-segmentation reduction. The comparison results are shown in Figure 4. From Figure 4, we can find that effectiveness of average filter is worse than Gaussian filter while Gaussian filter is worse than aniso- tropic diffusion filter. Anisotropic diffusion filter can reduce the over-segmentation effectively and thus in the proposed algorithm we adopted anisotropic diffusion filter. Discussion (a)The final segment results based on improved level set; (b) The region marked by the radiologist; (c) The Comparison between (a) and (b). segmentation efficiency. Besides, the method has good topological adaptability; it can deal with complex and changing shapes of the segmentation of the mammo- grams well and get high segmentation accuracy. Experimental results show that the proposed segmen- tation method can obtain good results. Discussion In this paper, we have developed a hybrid method to segment the mammograms which used watershed algo- rithm and level set method. We used watershed trans- form to provide a coarse and fast pre-segmentation, and used the resultant segmentation as the initial con- tour for the level set segmentation. Automatic selec- tion of the starting point from watershed transform can reduce the user interaction. The combination of the two segmentation methods speeds up the entire segmentation processing and improves the In this paper, we propose a mass segmentation algo- rithm which combines watershed method and level set method. The new method is divided into two steps: a marker-controlled watershed transform is first used to segment the mass region roughly, and then a level set is used to refine the segmentation. Watershed based segmentation algorithm has many advantages which can overcome the disadvantage in the level set based segmentation. As we know, level set Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 5 of 9 segmentation efficiency. Besides, the method has good topological adaptability; it can deal with complex and changing shapes of the segmentation of the mammo- grams well and get high segmentation accuracy. Experimental results show that the proposed segmen- Method Mass segmentation includes two steps in the proposed algorithm. The first step is to use watershed transform for rough segmentation and the second step is to use level set based method to refine the segmentation Figure 3 Flowchart of the result of segmentation algorithm. (a)The final segment results based on improved level set; (b) The region marked by the radiologist; (c) The Comparison between (a) and (b). segmentation efficiency. Besides, the method has good topological adaptability; it can deal with complex and changing shapes of the segmentation of the mammo- grams well and get high segmentation accuracy. Experimental results show that the proposed segmen Method Mass segmentation includes two steps in the proposed algorithm. The first step is to use watershed transform for rough segmentation and the second step is to use l l t b d th d t fi th t ti Figure 3 Flowchart of the result of segmentation algorithm. (a)The final segment results based on improved level set; (b) The region marked by the radiologist; (c) The Comparison between (a) and (b). Figure 3 Flowchart of the result of segmentation algorithm. Method Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 7 of 9 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 7 of 9 active contour model. This model assumes an image is formed by two homogeneous regions, and can be for- mulated by the following energy functional [29,30]: Figure 5 Watershed. ECV(C, c1, c2) = λ1 inside(C) I0(x, y) −c1 2dxdy+λ2 outside(C) I0(x, y) −c2 2dxdy+μ \|C\| λ1, λ2 ≥0, μ ≥0 (1) ECV(C, c1, c2) = λ1 inside(C) I0(x, y) −c1 2dxdy+λ2 outside(C) I0(x, y) −c2 2dxdy+μ \|C\| λ1, λ2 ≥0, μ ≥0 (1) (1) Where l1, l1, μ, c1, c2 are constants,C is the evolving contour, \|C\| is the length of contour C, inside(C) and outside(C) are the regions inside and outside the contour. Although the proposed level set method could pro- duce successful segmentation, it needs powerful initiali- zation techniques. In order to solve the problem, in the proposed method, we use the contour obtained from watershed segmentation step as the initial contour of the level set. We resolve the drawbacks of the two methods mentioned above by combining them. Figure 5 Watershed. Besides the initialization issue, there is also noise issue. In general, the mammograms have a lot of noise. If the watershed algorithm was applied on the image directly, over-segmentation will happen because the watershed algorithm is very sensitive to noise. To avoid over-segmentation, we need to remove the noise. When the noise is removed, we can get the coarse segmenta- tion using watersheds. The noise reduction methods investigated in the proposed paper include average filter, Gaussian filter and anisotropic diffusion [31]. Anisotro- pic diffusion was introduced by Perona and Malik [31] and it uses the gradient between the image area to con- trol diffusion degree. Anisotropic diffusion can eliminate the noise effectively while preserve the edge of the image. The anisotropic diffusion used in the proposed algorithm is the method developed in the [32]. image obtained from c. In order to segment the objects in the image, the foreground markers will be computed for the objects. After the markers are obtained, the flood waves will propagate from the set of markers to cover the topographic surface \|\|∇c\|\| [27]. When the water reaches the maximum gray value, the edges of the union of all dams come into being the watershed seg- mentation. Method Mass segmentation includes two steps in the proposed algorithm. The first step is to use watershed transform for rough segmentation and the second step is to use level set based method to refine the segmentation obtained by watershed transform. Watershed based Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Page 6 of 9 Table 1 The different part Data (pixels) of Fig.3 CaseNo TP FP FN 0046 4517 635 825 0051 3235 370 179 0069 2913 1475 140 0074 12912 2611 4654 0123 7419 1452 2566 0161 4339 2050 858 0226 18834 890 575 0274 1583 704 80 Table 1 The different part Data (pixels) of Fig.3 algorithms are mathematical morphology methods for image segmentation and they have many advantages in comparison with other image segmentation methods. For example, watershed transform based segmentation methods generally have high computation speed and can obtain closed contour lines and accurate position. Besides, watershed based image segmentation algorithms can handle weak edges very well [27]. The basic idea of watershed can be described as fol- lows [27]: let c be a gray image, \|\|∇c\|\| is the gradient Table 2 Validation measure Data (percent) of Fig 3 CaseNo Hitting Missing OverHitting RelativeHitting RelativeMissing Kappa 0046 0.85 0.15 0.12 0.88 0.16 0.86 0051 0.95 0.05 0.11 0.90 0.05 0.92 0069 0.95 0.05 0.48 0.66 0.03 0.78 0074 0.74 0.26 0.15 0.83 0.30 0.78 0123 0.74 0.26 0.15 0.84 0.29 0.79 0161 0.83 0.17 0.39 0.68 0.13 0.75 0226 0.97 0.03 0.05 0.95 0.03 0.96 0274 0.95 0.05 0.42 0.69 0.03 0.80 Figure 4 (a) The result after different filter; (b) The segment results based on (a). Table 2 Validation measure Data (percent) of Fig 3 CaseNo Hitting Missing OverHitting RelativeHitting RelativeMissing Kappa 0046 0.85 0.15 0.12 0.88 0.16 0.86 0051 0.95 0.05 0.11 0.90 0.05 0.92 0069 0.95 0.05 0.48 0.66 0.03 0.78 0074 0.74 0.26 0.15 0.83 0.30 0.78 0123 0.74 0.26 0.15 0.84 0.29 0.79 0161 0.83 0.17 0.39 0.68 0.13 0.75 0226 0.97 0.03 0.05 0.95 0.03 0.96 0274 0.95 0.05 0.42 0.69 0.03 0.80 Figure 4 (a) The result after different filter; (b) The segment results based on (a). Table 2 Validation measure Data (percent) of Fig 3 Figure 4 (a) The result after different filter; (b) The segment results based on (a). igure 4 (a) The result after different filter; (b) The segment results based on (a). References Guliato D, de Carvalho JD, Rangayyan RM, Santiago SA: Feature extraction from a signature based on the turning angle function for the classification of breast tumors. J Digit Imaging 2008, 21(2):129-144. 11. Guliato D, de Carvalho JD, Rangayyan RM, Santiago SA: Feature extraction from a signature based on the turning angle function for the classification of breast tumors. J Digit Imaging 2008, 21(2):129-144. Figure 7 Flowchart of the segmentation algorithm. 12. Huo Z, Giger ML, Vyborny CJ, Bick U, Lu P, Wolverton DE, Schmidt RA: Analysis of spiculation in the computerized classification of mammographic masses. Med Phys 1995, 22(10):1569-1579. 12. Huo Z, Giger ML, Vyborny CJ, Bick U, Lu P, Wolverton DE, Schmidt RA: Analysis of spiculation in the computerized classification of mammographic masses. Med Phys 1995, 22(10):1569-1579. 13. Kobatake H, Yoshinaga Y: Detection of spicules on mammogram based on skeleton analysis. IEEE Trans Med Imaging 1996, 15(3):235-245. needs a certain number of iterations, which produces the final, highly accurate, smooth results. g p y 13. Kobatake H, Yoshinaga Y: Detection of spicules on mammogram based on skeleton analysis. IEEE Trans Med Imaging 1996, 15(3):235-245. 14. Lou SL, Lin HD, Lin KP, Hoogstrate D: Automatic breast region extraction from digital mammograms for PACS and telemammography applications. Comput Med Imaging Graph 2000, 24(4):205-220. Acknowledgements 15. Zheng B, Good WF, Armfield DR, Cohen C, Hertzberg T, Sumkin JH, Gur D: Performance change of mammographic CAD schemes optimized with most-recent and prior image databases. Acad Radiol 2003, 10(3):283-288. The paper is supported by NSFC 61100055, NSF of Hubei Province (NO. 2008CDB345), Educational Commission of Hubei Province (NO.Q20101101) Department of Science and Technology of Hubei Province (NO. D20091102), and Science Foundation of Wuhan University of Science and Technology Project 2011xz019. This article has been published as part of BMC Systems Biology Volume 5 Supplement 3, 2011: BIOCOMP 2010 - The 2010 International Conference on Bioinformatics & Computational Biology: Systems Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/1752-0509/5?issue=S3. 16. Petrick N, Chan HP, Sahiner B, Helvie MA: Combined adaptive enhancement and region-growing segmentation of breast masses on digitized mammograms. Med Phys 1999, 26(8):1642-1654. 17. Qi H, Snyder WE: Lesion detection and characterization in digital mammography by Bezier histograms. 1999: IEEE 1999, 1022:1021-1024. 17. Qi H, Snyder WE: Lesion detection and characterization in digital mammography by Bezier histograms. 1999: IEEE 1999, 1022:1021-1024. 17. Qi H, Snyder WE: Lesion detection and characterization in digital mammography by Bezier histograms. 1999: IEEE 1999, 1022:1021-1024. 18. Guliato D, Rangayyan RM, Carnielli WA, Zuffo JA, Desautels JEL: Segmentation of breast tumors in mammograms by fuzzy region growing. 1998: IEEE 1998, 1002:1002-1005. mammography by Bezier histograms. 1999: IEEE 1999, 1022:1021 1024. 18. Guliato D, Rangayyan RM, Carnielli WA, Zuffo JA, Desautels JEL: Segmentation of breast tumors in mammograms by fuzzy region growing. 1998: IEEE 1998, 1002:1002-1005. 18. Guliato D, Rangayyan RM, Carnielli WA, Zuffo JA, Desautels JEL: Segmentation of breast tumors in mammograms by fuzzy region growing. 1998: IEEE 1998, 1002:1002-1005. p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Method Figure 5 shows the definition of watershed. In the implementation of the watershed algorithm, if we only use gradient of watershed for segmentation, there are too many ridgelines which will cause over-seg- mentation (see Figure 6(b)). In order to reduce the over- segmentation, marker-controller watershed is used to reduce over-segmentation. In mark based watershed method, markers are connected through the component. After the marker-based watershed applied, we can get Figure 6(c). The proposed algorithm is shown in Figure 7. It is com- posed of several steps, the original image will be prepro- cessed and then used as the input of the watershed segmentation and the rough segmentation is obtained. The rough segmentation will be used as the start contour for the level set segmentation. This approach combines the advantages of the two methods and overcome the dis- advantages of each single method: marker-based watershed is rough but fast and the level set segmentation After the image is segmented using watershed trans- form, we will use the resultant contour as the initial contour for a level set based method to refine the seg- mentation. The level set algorithm used for the segmen- tation in the proposed algorithm is from [28]. The level set algorithm proposed in [28] is based on region based Figure 6 (a) Original image; (b) Gradient based watershed method; (c) Marker based watershed. Figure 6 (a) Original image; (b) Gradient based watershed method; (c) Marker based watershed. Page 8 of 9 Page 8 of 9 Page 8 of 9 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Figure 7 Flowchart of the segmentation algorithm. Authors’ contributions JL, XL, LC and JC developed the algorithm using watershed and level let and wrote the original the paper. JT proposed the investigation of over- segmentation issue and revised the paper. YD did data analysis. All authors read and approved the final manuscript. References 1. American Cancer S: Breast cancer facts & figures 2007-2008. American Cancer Society Atlanta, GA; 2007. 2. Tang J, Rangayyan RM, Xu J, El Naqa I, Yang Y: Computer-aided detection and diagnosis of breast cancer with mammography: recent advances. IEEE Trans Inf Technol Biomed 2009, 13(2):236-251. 3. Elter M, Horsch A: CADx of mammographic masses and clustered microcalcifications: a review. Med Phys 2009, 36(6):2052-2068. 4. Liu X, Tang J, Zhang X: A multiscale image enhancement method for calcification detection in screening mammograms. 2009: IEEE 2009, 677-680. 5. Chan HP, Sahiner B, Helvie MA, Petrick N, Roubidoux MA, Wilson TE, Adler DD, Paramagul C, Newman JS, Sanjay-Gopal S: Improvement of radiologists’ characterization of mammographic masses by using computer-aided diagnosis: an ROC study. Radiology 1999, 212(3):817-827 6. Sahiner B, Petrick N, Chan HP, Hadjiiski LM, Paramagul C, Helvie MA, Gurcan MN: Computer-aided characterization of mammographic masses: accuracy of mass segmentation and its effects on characterization. IEEE Trans Med Imaging 2001, 20(12):1275-1284. g g 7. Mudigonda NR, Rangayyan RM, Desautels JE: Gradient and texture analysis for the classification of mammographic masses. IEEE Trans Med Imaging 2000, 19(10):1032-1043. 7. Mudigonda NR, Rangayyan RM, Desautels JE: Gradient and texture analysis for the classification of mammographic masses. IEEE Trans Med Imaging 2000, 19(10):1032-1043. 8. Kilday J, Palmieri F, Fox MD: Classifying mammographic lesions using computerized image analysis. IEEE Trans Med Imaging 1993, 12(4):664-669. 8. Kilday J, Palmieri F, Fox MD: Classifying mammographic lesions using computerized image analysis. IEEE Trans Med Imaging 1993, 12(4):664-669. 9. Shi J, Sahiner B, Chan HP, Ge J, Hadjiiski L, Helvie MA, Nees A, Wu YT, Wei J, Zhou C, et al: Characterization of mammographic masses based on level set segmentation with new image features and patient information. Med Phys 2008, 35(1):280-290. 9. Shi J, Sahiner B, Chan HP, Ge J, Hadjiiski L, Helvie MA, Nees A, Wu YT, Wei J, Zhou C, et al: Characterization of mammographic masses based on level set segmentation with new image features and patient information. Med Phys 2008, 35(1):280-290. y 10. Rangayyan RM, Mudigonda NR, Desautels JE: Boundary modelling and shape analysis methods for classification of mammographic masses. Med Biol Eng Comput 2000, 38(5):487-496. 10. Rangayyan RM, Mudigonda NR, Desautels JE: Boundary modelling and shape analysis methods for classification of mammographic masses. Med Biol Eng Comput 2000, 38(5):487-496. 11. Author details 1 19. Mudigonda NR, Rangayyan RM, Desautels JE: Detection of breast masses in mammograms by density slicing and texture flow-field analysis. IEEE Trans Med Imaging 2001, 20(12):1215-1227. 1College of Computer Science and Technology, Wuhan University of Science and Technology, Wuhan, Hubei, China. 2Key laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China. 3Rush University Cancer Center, Rush University Medical Center, Chicago, Illinois, USA. 20. Dominguez RA, Nandi A: Toward breast cancer diagnosis based on automated segmentation of masses in mammograms. Pattern Recognition 2009, 42(6):1138-1148. Page 9 of 9 Page 9 of 9 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 Liu et al. BMC Systems Biology 2011, 5(Suppl 3):S6 http://www.biomedcentral.com/1752-0509/5/S3/S6 21. Song E, Jiang L, Jin R, Zhang L, Yuan Y, Li Q: Breast mass segmentation in mammography using plane fitting and dynamic programming. Acad Radiol 2008, 16(7):826-835. 22. Chu Y, Li L, Clark R: Graph-based region growing for mass-segmentation in digital mammography. Proceedings of SPIE 2002, 4684:1690-1697. in digital mammography. Proceedings of SPIE 2002, 4684:1690-1697. 23. Malladi R, Sethian JA, Vemuri BC: Shape modeling with front propagation: a level set approach. IEEE Trans Patt Anal Mach Intell 1995, 17(2):158-175. 23. Malladi R, Sethian JA, Vemuri BC: Shape modeling with front propagation: a level set approach. IEEE Trans Patt Anal Mach Intell 1995, 17(2):158-175. 24. Heath M, Bowyer K, Kopans D, Moore R, Kegelmeyer P: The digital database for screening mammography. Medical Physics Publishing; 2001, 212-218. database for screening mammography. Medical Physics Publishing; 2001, 212-218. 25. Li X: Automatic image segmentation based on level set approach: application to brain tumor segmentation in MR images. Université de Reims Champagne-Ardenne; 2009. g 26. Zhang H, Fritts JE, Goldman SA: Image segmentation evaluation: a survey of unsupervised methods. Computer Vision and Image Understanding 2008, 110(2):260-280. 26. Zhang H, Fritts JE, Goldman SA: Image segmentation evaluation: a survey of unsupervised methods. Computer Vision and Image Understanding 2008, 110(2):260-280. 27. Vincent L, Soille P: Watersheds in digital spaces: an efficient algorithm based on immersion simulations. IEEE Trans Patt Anal Mach Intell 1991, 13(6):583-598. 27. Vincent L, Soille P: Watersheds in digital spaces: an efficient algorithm based on immersion simulations. IEEE Trans Patt Anal Mach Intell 1991, 13(6):583-598. 28. Author details 1 Tang J, Liu X: Classification of mass in mammography with an improved level set segmentation by combining morphological features and texture features. In Multi Modality State-of-the-Art Medical Image Segmentation and Registration Methodologies. Volume 2. Springer Verlag;. 29. Chan T, Vese L: An Active Contour Model without Edges.Scale-Space Theories in Computer Vision . In Lecture Notes in Computer Science. Volume 1682. Springer; 1999:(1999):141-151. 30. Chan TF, Vese LA: Active contours without edges. IEEE Trans Image Process 2001, 10(2):266-277. 30. Chan TF, Vese LA: Active contours without edges. IEEE Trans Image Process 2001, 10(2):266-277. 31. Perona P, Malik J: Scale-space and edge detection using anisotropic diffusion. IEEE Trans Patt Anal Mach Intell 1990, 12(7):629-639. 31. Perona P, Malik J: Scale-space and edge detection using anisotro diffusion. IEEE Trans Patt Anal Mach Intell 1990, 12(7):629-639. diffusion. IEEE Trans Patt Anal Mach Intell 1990, 12(7):629-639. 32. Tang J: A Multi-direction GVF snake for the segmentation of skin cancer images. Pattern Recognition 2009, 42(6):1172-1179. 32. Tang J: A Multi-direction GVF snake for the segmentation of skin cancer images. Pattern Recognition 2009, 42(6):1172-1179. doi:10.1186/1752-0509-5-S3-S6 Cite this article as: Liu et al.: Mass segmentation using a combined method for cancer detection. BMC Systems Biology 2011 5(Suppl 3):S6. doi:10.1186/1752-0509-5-S3-S6 Cite this article as: Liu et al.: Mass segmentation using a combined method for cancer detection. BMC Systems Biology 2011 5(Suppl 3):S6. 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https://openalex.org/W2104808528	https://zenodo.org/records/2475827/files/article.pdf	German	null	Über Verbindungen des Pyridins mit den Alkalimetallen. III. Mitteilung	Berichte der Deutschen Chemischen Gesellschaft	1,917	public-domain	1,986	I ) B. 47, 2598 [1914]; 49, 1060 [1916]. 31 6. Bruno Emmert: Ober Verbindungen dea Pyridins mit den Alkalimetallen. 111. Mitteilung. [Aus dem Chemiscben Institut der Universitat Wiirzburg.] (Eingegangen am 4. November 1916.) I n zwei fruheren Publikationen') wurde gezeigt, daI3 die Alkali- rnetalle durch Addition von Pyridin zwei Reihen tiefgefirbter, selbst- entziindlicher Verbindungen geben, denen die Forrneln Me(C5 Hs N)a und MeCsHsN zukornrnen. Urn Einblick in die Art der Bindung des Pyridins an das Metall zu erhalten, wurden nunmehr die Urnsetzun- gen des Mo n o p yrid in- N a t r i u m s naher studiert. Bei Einwirkung von Wasser (feucbtem Ather) auf Monopgridin- Natriurn entsteht ohne wahrnehrnbare WasserstofF-Entwicklung neben Natriumbydroxyd ein atherlosliches, sehr reaktionsf%higes, autoxy- dables Gemenge, dessen Zusammensetzung der Formel Clo HI, NS ent- spricht. Es war von vornherein wahrscheinlich, da13 bier ein Gemisch von T e t r a h y d r o - d i p y r i d y l e n vorliege, die sich nach der Gleichung: 2NaCsHsN + 2Hz0 = (C5HgN)a + 2NaOH gebildet haben mochten. Uber die Orte der Binduogen zwischen den Dihydro-pyridin-Ringen war zunachst nichts auszusagen. Der Trednung des Gemisches standen infolge der Veranderlichkeit der Substanz groRe Schwierigkeiten entgegen. Ich versuchte daher, aus der Unter- suchung der Autoxydationsprodukte einen RiickschluB auf die Natur seiner Bestandteile zu errnoglichen. Bei der Oxydation in feuchtem Ather entstand neben einem gelben Niederschlag ein in Ather 16s- liches Produkt, welches sich als fast reines 7,T-Dipyridyl erwies. Seine Bildung beweist, daI3 ein Teil des urspriinglichen Reaktions- produktes aus T e t r a h y d ro-y,y-dipy r i d y 1 (Formel I) bestand. Der erwahnte gelbe Niederschlag schwankte in seiner Zusammen- setzung zwischen den Formeln C ~ ~ H I S ON3 und CloI-112 ONi, HaO. Ich babe auch hier auf eine Isolierung der Komponenten des Gemisches verzichtet. Es wurde vielmehr die Substanz zur Wasserabspsltung und Dehydrierung mit konzentrierter Schwefelsaure erhitzt und dabei in allerdings kleinen Mengen a , a - D i p y r i d y l erhalten, so daI3 auch das Vorhandeneein von T e t r a h y d r o - c!, a -d i p y rid y 1 (Formel 11) im I ) B. 47, 2598 [1914]; 49, 1060 [1916]. Ausgangsmaterial erwiesen scheint. Ein Hinweis auE noch andere Tetrahydro-dipyridyle wurde nicht beobachtet. Den ifbergang der Tetrahydro-Korper in Dipyridyle wird man sich so zu deuten haben, da8 durch die Autoxydation Korper niit Hydroxylgruppen entstehen, und diese spontan oder bei Einwirkung tler konzentrierten Schwefel- saure Wasser abspalten. Die Rildung der Tetrahydro-dipyridyle aus dem hlonopyridin- Natrium kann durch die Annahme erklart werden, daB das Natrium bei Anlagerung an das Pyridin sich der Valenz des Stickstoffs, welche nach der zentrischen Formel in das Innere des Pyridinkerns gerichtet zii denken ist, bemachtigt, wobei dann gleichzeitig eine Valenz des y- oder a-Kohlenstoffatornu frei wird. Mit den freien Valenzen k6nnen sich zwei Pyridinkerne zu Verbindungen von den Formeln I11 und I V vereinigen. Jlit Wasser werden diese die entsprechenden Tetrahydro- dipyridyle liefern. Ein gleicher Vorgang, bei dem die beiden Pyridin kerne in @-Stellung verkettet werden, laBt sich ohne Annahme von Diagonalbindungen im Sechsring nicht formulieren. Solche Bindungen sind an sich recht unwahrscheinlich, womit die Tatsache iiberein- stimmt, daB unter den Umwandlungsprodukten der Pyridin-Natrium- Terbindung P-Dipyridyle nicht aufgefunden wurden. Die bisher besprochene Formulierung sieht irn Monopyridin-Na- trium ein echtes Tetrahydro-dipyridyl-Derisat. Sie vermag wohl die Umsetzung der Nntriumverbindung mit Wasser zu erklaren, aber sie gibt keinen Anhaltspunkt fur die tiefe Farbung und die auaerordent- liche Selbstentziindlichkeit der Verbindung. Eine volle Erklarung wiirde durch die Annahrne gegeben sein, daB das Natriumatom nur mit einer N e b e n valenz an den Pyridin kern gebnnden ist, und ebenso ,die beiden Pyridinkerne sich durch Nebenvalenz in y , p und a, u-Stel- lung binden, wie es die Formeln V und V I zeigen. Wird durch Um- setzung mit Wasser das Natrium durch ein Wasserstoffatom ersetzt, so beansprucht dieses eine volle Valenz des Stickstoffatoms, woditrch dann eine Bindung der Pyridinkerne durch Hauptvalenz an der Stelle veranlaBt wird, wo vorher Bindung durch Nebenvalenz statt hatte. 1lurch diesen Wechsel der Hindungsart wiirde auch erklart, waruni das Zersetzungsprodukt des Monopyridin-Natriums durch Wasser itu Gegensntz zum urspriinglichen Kiirper kaum oder nicht gefilrbt ist, und weshalb an Stelle cler Selbstentzundlichkeit nur eine Neigung zur Autosydation getreten ist. I n den oben stehenden Porrnelu wirtl eine besonders nahe 13ezie- hung des Nntriuniatorns zum Stickstoffatorn angenommeii. Diese konnte experimentell dadurch bestHtigt werden , daf3 bei Einwirkung von trockneni Kohlendioxyd auf hZonopyridin-Natriutii sich unter Ent- fiirbung ein in Wasser leicht liisliches Salz bildet, welches durch SRuren unter I(ohlensHure-E:ntwicklung zerlegt wird, also in seinen Icigenschaften eineni carbatninsauren Salz entspricht. B. 47. ?GOO [lgl-l]: 49, 1061 [191G\. Berichte d. D. Chetn. Gesellschaft. Jahry. 1.. A m dem zu- niichst wasserloslichen Zersetzungsprodukt der Carbaminsiiure scheidet sich wiederurn durch Oxydatiou at1 der Luft das Gernisch der unliis- lichen gelben Kbrper von der Zusamrnensetzuug CloHt~ ON? u n d CloHl2 OK2, IT20 ab. 1; s 11 e r i m e n t e 1 1 e s. U a r s t e 1 1 u n g d e s G e ni i s c h e s d e r T e t r a h y d r o - d i py r i d y le Die Darstellung des Munopyridin-Natriums war die gleiche, wie sie in friiheren Abhnndlungen ') beschrieben wurde. Nur wurde iedesmal von 10 g gepulvertem Natrium susgegangen und dernent- sprechend die Ilealction in eineni Literkolben vorgenornnien. Das Ab(lestillieren des iiberschiissigen Pyridins geschah im Ta- Luum zuniichst bei einer Olbad-Temperatur uon 40°, dann murde die Temperatur aiif 135O gesteigert, urn dns entstandene Dipyridin-Natriuni in hlonopyridin-iVatriulri iiberzufuhren. Nach dem Erkalteu wurtle der Kolben durch .Einsaugen mit feuchtern Ather gefiillt. Die Reaktion trat langsani unter schwacher Erwiirmung ein. Es w a d e desbalh zeitweise mit IGswasser gekuhlt. Wasserstoff wurde nicht entwickelt. Am nichsten Tage wurden wenige ccm Wasser in den Kolben ge- bracht und unter zeitweisem, kriiftigem Urnschiitteln 4-5 Tage stehen gelassen. Der grijl3te Teil des Kolbeninhnlts hntte sich n u n im -4ther mit gelber Fnrbe geliist. Es wurtle jetzt soviel Wasser zugefugt, tlaO dns Natriurnhydrosyd in Liisung ging. Ungeliist Llieben 8-9 g IIarz. I.)ie wlifirige Liisr1ng wurde rnehrmals mit :ither ausgeschiittelt iintl tler gesamte ;ither unter LiiEtabschlurJ kurze Zeit iiber Kali getrocknet. B ~ i m Abrlestillieren des athers in einer ~~rasserstoff-AtmosphHre hinter- hlirb eine ziihe, gelbliche Masse (% g). Zur Analyse wiirde sie 7 7 34 34 unter Durchleiten von WasserstoIE in1 Vakuum bei looo yon den letzten Resten Ather befreit. Sie zeigte dann die Zusammensetzung des T e t r a h y d ro- d i p y r i d y Is. y p y y 0.1365 g Sbst.: 0.3754 g COs, 0.0907 g HsO. y p y y 0.1365 g Sbst.: 0.3754 g COs, 0.0907 g HsO. CloHl2N,. Ber. C 74.94, I1 7.55. Gef. 75.01, * 7.44. O x y d a t i o n d e s Tetrahpdro-dipyrid\-l-(;emisches u u d I s o l i e r u n g d e s ;,,y-Dipyridyls. Es wurde, wie oben, eine 5therische Losung des Tetrahydro-di- pyridyl-Gemisches hergestellt, wobei wiederum von 10 g Natrium aus- gegangen wurde. Auch hier bildeteu sich 8 g harziger, unloslicher Substanzen, von denen abfiltriert wurde. Die Losung trubt sich bein1 Stehen an der Luft rnsch durch Ospdation. l ) J. pr. r2] 44, 107 [1891]. ?) X. 3, S5l [ISSB]. 1; s 11 e r i m e n t e 1 1 e s. Um die nufgenommeue Sauerstoffmenge zu bestimrnen, wurde die Flasche, welche den Ather enthielt, niit einem Sauerstoff-Gasometer verbunden. Die Gasaufnahnie giug bei zeitweisem Schutteln nur langsam vonstatten und konute erst nnch drei Wochen als beendet gelten. Reduziert auf O0 und ‘iG0 mm Druck, wurtlen 2730 ccni Sauerstoff aufgenommen , was der zur Bildung eines Geniisches von I’,yDipyridyl und des Korpers CloH1? ON1 berechneten Menge etwa entspricht. Wahrend der OH?- dation hatten sich reichliche Mengeri gelber Flockeu abgeschieden. Sie wurden abfiltriert; ihr Gewicht betrug nach dem Trocknen im Vakuum 12 g. Die ltherische Losung wurde mit Kali getrocknet nntl verdampft. Der Ruckstand (15 g) erstarrte beini Abkuhlen krystalli- nisch. Bei der Destillation ging nach einem kleinen Vorlauf (l/? g), der geringe Mengen Pyridin z u euthalten schieu, bei 290-305O an- nHhernd reines 7,y-Dipyridyl (I2 g) uber. Im Kolben blieb ein geringer Ruckstand, der sich bei liohereo Teinperaturen unter Eiit- wicklung brauner Dgrnpfe zersetzte. ])as Uestillat wurde nus Ligroiri unikrystallisiert uud noch inehrmals destilliert. Schliip. 110-1 12”, Sdp. 302O (korr.). H e u s e r und Stohrl) geben als Schnielzpunkt des T,y-Dipyridyls 111-112°, W e i d e l und R u s s o ? ) ala Siedepunkt 304.8O (korr.) an. W (dBo, 744 mm). 0.2124 g Sbst.: 0.5974 g CO?, 0.0996 g 1120. - 0.1057 Shst.: 16.S CI’III W (dBo, 744 mm). 0.2124 g Sbst.: 0.5974 g CO?, 0.0996 g 1120. - 0.1057 Shst.: 16.S CI’III CloHsN?. Ber. C 7ti.89, H L I G , N 17.95. GeE. n 76.71, )) 5.25, )) 18.00. 1) Der Siedcpuukt komitc wcgen der I<lciulir:it drr SaLstxnznienge uichi scliarE festgestcsllt wertlen. 2; 11. 10, 396 [1899]. Is o 1 i e r u n g d e s (L, u- I? i p y rid y 1 s. Die i m vorigen -Abschnitt erwkhnte gelbe, bei der Osydatiou it? 1:locken ausfallende Substxnz wurde m r Reinigung mehrere Ma1 init - l ) J. pr. r2] 44, 107 [1891]. ?) X. 3, S5l [ISSB]. kaltem Wasser dnrchgeschiittelt, getrocknet, in warmem, absolutem Alkohol gel6st und durch langsamen Zusatz von trocknem Ather ge- fillt. Dieses Verfahren wurde nochmals wiederholt. Die Substanz war jetzt nur noch schwach gelblich. Zur Vakuum iibw Schwefelsaure getrocknet. Analyse wurde sie im 0.1854 g Sbst.: 0.4535 g co2, 0.1 1s2 g I120 C ~ ~ H ~ ? O N P . B c ~ . C 68.14, H CluHlr02Nz. Ecr. )) GI.SI, ;) Gef. ,) 66.71, 6.8i. 7.". i . ! X 0.1854 g Sbst.: 0.4535 g co2, 0.1 1s2 g I120 C ~ ~ H ~ ? O N P . B c ~ . C 68.14, H CluHlr02Nz. Ecr. )) GI.SI, ;) Gef. ,) 66.71, 6.8i. 7.". i . ! X 20 g der gereinigten Substanz wurden in 80 ccni konzentrierter Schwefelslure gelost und 8 Stdn. irn Olbad auf 380' erhitzt. Nach den1 Erknlten wurde mit Eis verdunnt, von kohligen Resten filtriert, mit Nstronlauge ubersiittigt und ausgeiithert. Der Ather hinterliefl beim Verdampfen 3 g Ruckstand, der bald in derben Krystallen er- starrte. Zur Reinigung wurde mit Wasserdampf ubergetrieben, noch- rnnls mit Ather extrahiert, der Atker verdunstet und der Riickstand destilliert I). Die Substanz zeigte den von B l a u ') angegebenen Schmp. 69.5O. Selbst in groBer Verdunnung gab sie mit Ferrosulfat die fur das a, cl-Dipyridyl so aufierordentlich charakteristische, inten- sire Rotfarbung. 0.2150 g Sbst.: 0.6067 g COz, 0.1019 g HsO. CloHsN~. Bet-. C 76.89, H 5.16. Gef. B 76.90, )) .5.30. g g CloHsN~. Bet-. C 76.89, H 5.16. Gef. B 76.90, )) .5.30. 2; 11. 10, 396 [1899]. 6. Emil Votoicek: tfber einige Derivate der Rhodeose (Abbau der Rhodeose). (Eingegarigcn am 30. Oktoher 1916.) 6. Emil Votoicek: tfber einige Derivate der Rhodeose (Abbau der Rhodeose). (Eingegarigcn am 30. Oktoher 1916.) (Eingegarigcn am 30. Oktoher 1916.) (Eingegarigcn am 30. Oktoher 1916.) Seioerzeit habe ich yon den stickstofflialtigen Abkiimmlingen der V O I I inir entdeckten Nethyl-pentose Rhodeose nur eine Reihe voi) Elydrazonen und Osazonen beschrielen, da eben Hydrazinderivate fur die Erkennung bezw. Trennung reduzierender Zuckerarten wichtiger sirid, ala alle mderen. Knchdem ich im yorigen Jahre wieder in Hesitz einer relativ grofien Menge (etwa 20 g) dieses kostbaren Zuckers ge- Iangte, konnte ich uuuinehr nuch das bisher noch fehlentle 0 s i m . s!iwie einige seiner Uiii\\andlungsprodiIkte darstellen, iiiiinlich diejenigeu. wrlche bei tler energischen Acetylierung nnch dent M'o hlschen Ver- . I 1
https://openalex.org/W4382049840	https://www.researchsquare.com/article/rs-3053572/latest.pdf	English	null	Predictive value for prognosis of sepsis based on the Light Gradient Boosting machine algorithm model	Research Square (Research Square)	2,023	cc-by	5,384	Predictive value for prognosis of sepsis based on the Light Gradient Boosting machine algorithm model Shengyue Chen 1Dalian Medical University Changjie Ke 2Hangzhou Dianzi University Mingwei Zhai Zhai 2Hangzhou Dianzi University Maofeng Wang 3Dongyang People's Hospital Fangfang Sun 2Hangzhou Dianzi University Yong Yang 1Dalian Medical University Jianping Chen 3Dongyang People's Hospital Article Keywords: Posted Date: June 26th, 2023 DOI: https://doi.org/10.21203/rs.3 License:   This work is license Read Full License Additional Declarations: No comp Shengyue Chen 1Dalian Medical University Changjie Ke 2Hangzhou Dianzi University Mingwei Zhai Zhai 2Hangzhou Dianzi University Maofeng Wang 3Dongyang People's Hospital Fangfang Sun 2Hangzhou Dianzi University Yong Yang 1Dalian Medical University Jianping Chen Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/18 Page 1/18 Abstract Sepsis is one of the leading causes of death in the critical care unit. The latest data that over 19 million patients every year in the world suffer from severe sepsis indicates it of great significance to evaluate the development tendency of sepsis and to investigate the prediction value of prognosis. Based on the Light Gradient Boosting (LGB) machine learning algorithm, we have now developed and tested an LGB prediction model by using the data source from the Medical Information Mart for Intensive Care-IV database for the model construction and validation, thus to predict the prognosis of sepsis. Additionally, we established three more models including the Logistic regression, the Random Forest and the K-Nearest Neighbor based prediction model and made comprehensive comparison with the LGB prediction model in the indicators involving 8 aspects, obtaining an area under the curve (AUC) of LGB prediction model at 0.998, which demonstrates it of strong reliability to exhibit high accuracy for predicting the prognosis of sepsis patients. Our findings support the LGB prediction model as a preferred machine learning model for predicting the prognosis of patients with sepsis. 2.2 Research subjects and data collection The study identified over 20,000 patients with sepsis using command queries from the MIMIC-IV database. Inclusion and exclusion criteria were applied based on the actual situation of the study. Inclusion criteria were (1) meeting the diagnostic criteria of Sepsis 3.0 [11] and (2) age ≥ 18 [12]. Exclusion criteria were (1) automatic discharge or abandonment of treatment, (2) brain death, (3) immune function diseases and immune regulation problems (4) presence of tumors, (5) pregnant women who had not yet given birth, and (6) newborns or miscarriages. Based on these criteria, a total of 5716 patients were included in the study, each with 33 clinical information variables, including demographic information, laboratory test results, and clinical diagnosis or predictive score. All the patients who died among the 5716 patients were in-hospital deaths. 2.1 Database and ethics The samples in the study come from the Medical Information Mart for Intensive Care (MIMIC)-IV database, a critical care medical database that is open to global users free of charge [10]. MIMIC was established in 2003 by several well-known medical institutions and research units worldwide as an important source of clinical medical research data. In addition, all the patient information used in this study have been identified in the MIMIC-IV critical care database, therefore, there is no need to obtain each patient's personal consent. 1. Introduction Sepsis is a syndrome of systemic inflammatory response due to infection [1–2], characterized by rapid development, high mortality rate, and high cost [3]. It has become one of the leading causes of death in critically ill patients [4]. Therefore, sepsis prognosis prediction through early identification of sepsis and use of the patient's early vital sign information is considered, after which human interventions can start as early as possible to reduce patient mortality based on the prediction results. Currently, in clinical practice, scoring systems are the main tool used to predict the prognosis of sepsis, including the Simplified Acute Physiology Score(SAPS II)[5], the Acute Physiology and Chronic Health Evaluation (APACHE II) [6, 7], and the Sequential Organ Failure Assessment (SOFA) [8]. However, most of these scoring systems involve users in a cumbersome process, and their specificity and sensitivity need to be improved. Even though, for example, the qSOFA score involves fewer vital sign parameters, its predictive and diagnostic validities are significantly reduced to the point where it no longer meets the timeliness requirements of clinical diagnosis or the reliability needs of scientific decision-making [9]. The accumulation of Big Data and development of data processing technology provide a mature basis for the application of machine learning algorithms in disease prediction. Therefore, this study attempts to explore the predictive value of 1 Dalian Medical University, Dalian, Liaoning, China. 2Hangzhou Dianzi University, Hangzhou, Zhejiang, China. 3Dongyang People's Hospital, Jinhua, Zhejiang, China. 4These authors contributed equally to this work: Shengyue Chen, Changjie Ke. *Email: yyang@hdu.edu.cn;13750997616 @163.com. the LightLlight Gradient Boosting machine(LGB) algorithm for the prognosis of sepsis and to provide a reliable mathematical basis for clinicians to develop scientific prediction models. Page 2/18 Page 2/18 2.5 Construction of the prediction model After data preprocessing, the study randomly selected 572 patients with sepsis from the original sample of 5716 patients at a ratio of 9:1 for model validation using Python 3.8. The remaining 5144 sepsis patients were divided into a training set and testing set at a ratio of 7:3. The study used the LGB algorithm and other machine learning algorithms available in the Sklearn library to build and train the prediction model. The study also used the "importance" function to calculate the importance of each feature variable, with larger values indicating greater importance [14]. The LGB algorithm originated from two papers published by Microsoft Asia Research Institute (based in Beijing and Shanghai,China)on NIPS in 2016 and 2017 [15]. The main idea behind LGB is to use the techniques of gradient-based one-side sampling and exclusive feature bundling to address the time- consuming problem of estimating information gain for all possible split points by scanning all data instances in gradient-boosting decision trees. Gradient-based one-side sampling and exclusive feature bundling together constitute the LGB model in the sample and feature dimensions. Another significant speed optimization in the LGB model is the use of the Histogram algorithm [16, 17, 18]. Current applications of LGB mainly use Histogram as the basis of the LGB model. 2.3 Data preprocessing First, the collected patient data were checked for outliers in each parameter using box plots based on Stata(version 16.0, Stata, StataCorp LLC, College Station, Texas, USA). If outliers were found, the study used capping methods to handle them, and special outliers were handled on a case-by-case basis. The study then used Stata 16.0 to check for missing data in each parameter. Due to excessive missing data (over 30%) in alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and total bilirubin, these parameters were removed using deletion methods. For parameters with a small amount of missing data, mean or mode imputation was used, and multiple imputation methods were used to fill in missing values. The final sample included 5716 patients with sepsis, each with 26 vital sign variables and three scoring systems (SOFA, SAPS II, and APS III). To avoid overfitting in the machine learning model, the study used Z-score normalization to standardize the data. An important basis for Z-score standardization is the formula that transforms the original sample data x into new data by combining the mean and the standard deviation (std) of the sample through formulaic transformation [13]: x， x，= (x −mean)/std 1 Page 3/18 Page 3/18 In addition, the study discretized the floating-point data using quartile-based binning into four categories (a, b, c, and d). Zero to 25% represents category a, 25–50% represents category b, 50–75% represents category c, and 75–100% represents category d. This approach has several benefits, including facilitating rapid model iteration, avoiding overfitting, and enhancing model robustness. 2.4 Algorithm modeling process In this study, the algorithm flowchart shown in Fig. 1 was used to build the machine learning model. Multiple indicators were also calculated to perform a comprehensive evaluation of prediction model performance. 2.6 Evaluation of the prediction model The study calculated the accuracy, sensitivity, and specificity of the prediction model on the test set using a combination of Python (version 3.8, Python, California, USA)3.8 and SPSS (version 26.0, SPSS, Chicago, Ill., USA). The receiver operating characteristic curve (ROC) [19] and the area under the curve (AUC) were calculated to evaluate the LGB prediction model. In addition, the study used learning curves to measure the fitting degree of the prediction model during data training. To ensure comparability, the study also built prediction models using the logistic regression (LR) [20, 21], random forest (RF) [22, 23], and k-nearest neighbor (KNN) [24, 25, 26]algorithms. The study compared the performance of these models on the same sample. Page 4/18 Page 4/18 2.7 Statistical methods The general statistical data analysis was implemented using SPSS. The study first conducted a normality test on the data. If the sample was normally distributed, the study used the mean ± standard deviation ( The general statistical data analysis was implemented using SPSS. The study first conducted a normality est on the data. If the sample was normally distributed, the study used the mean ± standard deviation ( ) for statistical analysis and independent sample t-tests for between-group comparisons [27]. If the −x ±s ) for statistical analysis and independent sample t-tests for between-group comparisons [27]. If the sample was not normally distributed, the study used the median (interquartile range) for representation and the Mann-Whitney test for between-group comparisons. In this study, a p-value less than 0.05 was considered statistically significant in all statistical analyses. The general statistical information on the 5144 patients in this study is shown in Table 1. The sample was divided into a death group and a survival group based on the patients' mortality status. −x ±s Page 5/18 Table 1 Table 1 General information on the study subjects Clinical Information Survival Group Death Group P-Value Numbers (male/female) 4320(2442/1878) 824(431/393) 0.158 Age 64.62 ± 15.57 68.41 ± 14.72 0.507 Blood pH 7.37 ± 0.09 7.34 ± 0.11 < 0.05 TotalCO2 25.3 ± 6.33 23.0 ± 6.65 < 0.01 Aniongap 14.28 ± 4.01 15(6.0) < 0.01 Bicarbonate 25.12 ± 5.08 22.65 ± 5.68 < 0.01 Chloride 102.0(7.0) 102.71 ± 7.23 < 0.05 Creatinine 1.0(1.0) 1.4(1.5) < 0.01 PCO2 40(12.0) 40.51 ± 10.96 0.239 PO2 84(76) 86(63.75) 0.941 Bun 22(23.0) 32(36.75) < 0.01 Sodium 138.69 ± 5.06 138.52 ± 5.84 < 0.05 WBC(×109/L)b 8.9(6.5) 11.4(9.7) < 0.01 Glucose 117(54) 138.52 ± 5.85 0.177 Hemoglobin 9.7(3.0) 9.3(2.65) 0.400 Hematocrit 30.81 ± 5.96 29.72 ± 5.84 < 0.05 MCV 92.0(9.0) 93.14 ± 8.17 < 0.05 MCHC 32.37 ± 1.60 32.52 ± 1.61 0.280 MCH 29.69 ± 2.83 30.29 ± 2.96 < 0.01 Potassium 4.10(0.80) 4.22 ± 0.68 0.285 Platelet 212(161) 164(160) < 0.01 RBC 3.40 ± 0.72 3.22 ± 0.69 0.163 RDW 15.6(3.1) 16.4(3.93) < 0.01 CKD 0.0(1.0) 0.0(1.0) < 0.01 MDRD_est 1.00(0.24) 0.99(0.25) < 0.05 GCS 15(1.0) 15(1.0) < 0.01 Page 6/18 Page 6/18 Clinical Information Survival Group Death Group P-Value SOFA 3.0(3.0) 4(3.0) < 0.01 APSIII 55.0(32.0) 83.33 ± 29.77 < 0.01 SAPSII 41.46 ± 14.20 52.25 ± 15.78 < 0.01 3. Results 3.1 Correlation comparison of the four machine learning algorithms Python was used in this study to calculate the correlations between the LR, RF, KNN, and LGB classification algorithms. The correlation matrix shown in Fig. 2 represents the specific situation. Figure 2 shows that LGB has the highest correlation with RF, with a correlation of 0.82. The correlations of LGB with KNN and LR are 0.75 and 0.64 respectively, both of which are higher than 0.60. The correlations between the algorithms were all greater than 0.60, indicating that they had a significant impact on each other. When considering whether to use ensemble learning to improve prediction accuracy and model performance, the high correlation between the algorithms indicated that there was no need to consider ensemble learning. 3.3 Evaluation of multiple prediction models 3.3 Evaluation of multiple prediction models Table 3 and Fig. 3 show that among the four machine learning prediction models and the three traditional scoring system prediction models, the machine learning prediction models are one level higher than the traditional scoring prediction models. Among all prediction models, the AUC value of the LGB prediction model was the highest at 0.982, with specificity and sensitivity of 1.00 and 0.96, respectively. Table 3 Comparison of results of multiple prediction models. Item AUC P 95%CI Sensitivity Specificity PPV NPV Prediction Accuracy LGB 0.982 < 0.01 0.974 ~ 0.988 1.000 0.96 82.1% 100% 96.6% RF 0.933 < 0.01 0.907 ~ 0.958 0.866 1.00 100% 97.6% 97.9% KNN 0.874 < 0.01 0.841 ~ 0.907 0.748 1.00 100% 95.6% 96.1% LR 0.777 < 0.01 0.737 ~ 0.818 0.555 1.00 100% 92.5% 93.1% APSIII 0.733 < 0.01 0.714 ~ 0.753 0.624 0.73 30.9% 91.1% 71.6% SAPSII 0.697 < 0.01 0.677 ~ 0.716 0.674 0.63 25.6% 91.0% 63.5% SOFA 0.601 < 0.01 0.579 ~ 0.623 0.458 0.71 23.0% 87.3% 66.8% 3.2 Test Results of the LGB Prediction Model In this study, LR,RF,KNN and LGB classification algorithms prediction models were built based on 3600 patients from training set data using the Anaconda self-contained Python. Based on the test set of 1544 sepsis patients (each with 26 pieces of clinical information as characteristic variables), the status (survival or death) of the 1544 patients was generated based on the prediction model. The predicted values generated by each prediction model (“0” represents survival, “1” represents death) were then compared one by one with the true labels of the test set. The ROC curves and accuracy recall curves of the four prediction models shown in Fig. 3 were obtained using the SPSS statistical software. At the same time, the sensitivity and specificity of the four prediction models were obtained based on the coordinates of the ROC curve. Based on the sensitivity and specificity values as well as the actual situation of the 1544 sepsis patients in the test set, of which 238 died and 1306 survived, the accuracy, PositivePredictive Value (PPV), and Negative Predictive Value (NPV) were calculated. The specific results are shown in Tables 2 and 3. As shown in Table 2, the test results of the LGB prediction model indicate that out of 1544 patients, 1492 were predicted correctly, resulting in an accuracy of 96.6%. Page 7/18 Table 2 Confusion matrix of the LGB prediction model test results Result Positive Negative Prediction Accuracy Positive 238 52 96.6% Negative 0 1254 Table 2 Confusion matrix of the LGB prediction model test results Result Positive Negative Prediction Accuracy Positive 238 52 96.6% Negative 0 1254 Table 2 Confusion matrix of the LGB prediction model test results Result Positive Negative Prediction Accuracy Positive 238 52 96.6% Negative 0 1254 3.5 Learning curves of the four machine learning models In this study, learning curves were introduced to evaluate the fitting degree of the four machine learning algorithms, LR, RF, KNN, and LGB, during modeling and to obtain their predictive abilities on sample data. The learning curve introduces training sets and cross validations that change synchronously with the sample during model training. The curve’s starting point is the degree of deviation between the two curves and the size of the variance between the curves. Through these, the fitting situation of the two curves can be judged intuitively [28]. Figure 5 shows that none of the four machine learning algorithms showed high bias during modeling, or in other words, that none of the four prediction models showed underfitting. The figure reveals that the LR and LGB prediction models had the best fitting degrees, but the LR prediction model showed a small deviation at the beginning of training, with the fitting degree improving as the sample size increased. The LGB prediction model showed neither overfitting nor underfitting throughout the process, but maintained the fitting state. Therefore, the fitting degree of the LGB prediction model may be better than that of the LR prediction model. 3.4 Ranking of feature variable importance To investigate which feature variables have the greatest impact on the target prediction value in the model, a ranking of feature variable importance was performed for the RF and LGB prediction models. The specific information is shown in Fig. 3. The ranking of feature variable importance shows the top 20 feature variables in importance. Figure 4 shows that in the RF prediction model, the top five feature variables in importance were platelet, white blood cell count (WBC), bicarbonate, mean corpusular volume (MCV), and creatinine. In the LGB prediction model, the top five feature variables in importance were WBC, platelet, age,blood urea nitrogen(bun), and anion gap. Page 8/18 3.6 Independent sample validation To further verify the effectiveness of these models, this study used independent samples to validate the predictive performance of the four algorithms. A total of 576 patients were selected as the independent sample for model validation. The results are shown in Table 4. Table 4 shows that the LR, KNN, RF, and LGB models had a prediction accuracy of over 95% for the 572 patients. The prediction accuracies of the RF and LGB models were similar; the LR and KNN models also had high prediction accuracy, which may have been related to the sample size of the independent validation. Regarding the AUC, the AUCs of the LR, KNN, RF, and LGB predictive models were all above 0.90. Compared with the AUC of the training set model, the AUC values of the LR and KNN models were significantly improved in the independent validation, which may have been related to the smaller sample size. Meanwhile, the AUCs of the RF and LGB models remained high in the validation of the 572 patients, demonstrating the stability and reliability of the models. Page 9/18 Page 9/18 Table 4 Comparison of validation results of the LR, RF, KNN, and LGB classification algorithms. Item AUC P 95%CI Sensitivity Specificity PPV NPV Prediction Accuracy LR 0.916 < 0.01 0.869 ~ 0.962 0.831 1.00 100% 97.0% 97.4% KNN 0.955 < 0.01 0.920 ~ 0.990 0.910 1.00 100% 98.4% 98.6% RF 0.978 < 0.01 0.952 ~ 1.000 0.955 1.00 100% 99.2% 99.3% LGB 0.998 < 0.01 0.995 ~ 1.000 1.000 0.996 97.8% 100% 99.6% Table 4 4. Discussion The LGB algorithm has been widely used in the field of machine learning and data mining due to its advantages of simplicity, strong robustness, efficiency, rapidity, and high accuracy. Its application in the field of medical data mining is also relatively mature. However, there is less research on its application in sepsis prognosis prediction modeling. In this study, the AUC value of the LGB algorithm model on the test set data was 0.982, the accuracy rate was 96.6%, and the sensitivity and specificity were 1.00 and 0.96 respectively. The learning curve fit during the training process was also good, indicating that the LGB prediction model has good model performance and predictive ability. In addition, this study also investigated the effectiveness of traditional scoring prediction models such as SOFA, SAPS II, and APS III, as well as three other machine learning algorithm models. Table 2 and Fig. 2 indicate that the LGB prediction model has better predictive capability for sepsis prognosis. Moreover, the independent sample validation indicates that the LGB prediction model has better predictive credibility. This study also explored the importance of the feature variables to the research objectives. Due to the large number of feature variables, the two top-ranked variable features were considered: platelet count and WBC. In a recent study, Vardon et al. proposed that decreased platelet counts are significantly associated with poor prognosis in sepsis. Decreased platelet counts are common in sepsis patients entering ICU wards. Although numerous factors cause platelet count to decrease, the level of platelets can serve as a warning for the prognosis of sepsis [29]. Numerous medical studies have shown that significantly elevated levels of WBC expression can give an early warning of poor prognosis in sepsis. WBC is an immune marker in the organism that plays an important role in the nonspecific immune system by producing antibodies to defend against pathogen invasion and other pathogenic microorganisms [30]. The WBC levels in the serum of sepsis patients can give an early warning of sepsis prognosis. Recently, Prof. Islam et al. noted that machine learning methods performed better than sepsis scoring systems, with an AUC value of 0.89 in a meta-analysis using machine learning methods to predict outcomes for sepsis patients [31]. In a study by Xin Zhao et al., the LGB and XGBoost algorithms were Page 10/18 Page 10/18 separately modeled to predict the prognosis of sepsis. 4. Discussion The results showed that the LGB algorithm had more advantages than the XGBoost algorithm in predicting sepsis prognosis [32]. In this study, the LGB algorithm model was compared with LR, KNN, and RF, and the final results showed that the accuracy, specificity, sensitivity, and AUC value of the LGB algorithm model in this study were higher than those of Xin Zhao et al. The main factors in the variability between these two include different data sources; different sample sizes used for modeling; different feature variables and the number of feature variables; and even different tuning parameters for the training models. Combined with current research, the present study provides a more reliable basis for modeling and predicting the prognosis of sepsis using machine learning algorithms, which adds a certain feasibility to the prediction model of the LGB algorithm used in this study. Therefore, it is appropriate to use prediction models to predict outcomes for patients in clinical practice. According to the prediction results, certain human interventions can be carried out on patients to prevent their condition from deteriorating. In addition, this study has some limitations, mainly in the lack of practical clinical application experience. The basic research of medical mathematics and physics in this study would need to be gradually transformed into a practical application platform in follow-up work. Therefore, the LGB algorithm model has a better model effect and predictive ability in predicting sepsis prognosis, which can provide an attractive mathematical and physical basis for clinical practice modeling. Declarations Data availability The data that support the findings of this study are available at the Medical Information Mart for Intensive Care-IV database. Acknowledgements This work was partially supported by the National Natural Science Foundation of China (Grant No. 81671038). The authors are grateful for the funding. Author contributions Sy-C, Cj-K, Y-Y, and Jp-C contributed to the conception and design of the work. All authors contributed to the acquisition, analysis, and interpretation of data for the work. Sy-C and Cj-K contributed to drafting the work. All authors contributed to revising it critically for important intellectual content. All authors contributed to the final approval of the version to be published. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Competing interests Competing interests Additional information Correspondence and requests for materials should be addressed to Cj-K or Jp-C. Correspondence and requests for materials should be addressed to Cj-K or Jp-C. References 1. Singger M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definition for Sepsis and Septic Shock (Sepsis-3). JAMA. 315, 801–810 (2016). 1. Singger M, Deutschman CS, Seymour CW, et al. The Third International Consensus Definition for Sepsis and Septic Shock (Sepsis-3). JAMA. 315, 801–810 (2016). 2. Dawulieti J, Sun M, Zhao Y, et al. Treatment of severe sepsis with nanoparticulate cell-free DNA scavengers. Science advances. 6, 7148 (2020). 2. Dawulieti J, Sun M, Zhao Y, et al. 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Figures Figure 1 Machine learning modeling flowchart Figure 1 Machine learning modeling flowchart Page 14/18 Figure 2 Correlation matrix of the four machine learning algorithms Figure 2 Correlation matrix of the four machine learning algorithms Correlation matrix of the four machine learning algorithms Correlation matrix of the four machine learning algorithms Page 15/18 Figure 3 ROC f lti l di ti d l Figure 3 ROC curves of multiple prediction models Figure 3 ROC curves of multiple prediction models Page 16/18 Figure 4 Ranking of feature variable importance for the RF and LGB prediction models. Figure 4 Ranking of feature variable importance for the RF and LGB prediction models. Ranking of feature variable importance for the RF and LGB prediction models. Page 17/18 Figure 5 Learning curves of prediction models: LR, RF, KNN, and LGB. Figure 5 Figure 5 Learning curves of prediction models: LR, RF, KNN, and LGB. Page 18/18
https://openalex.org/W4288044218	https://eajbsc.journals.ekb.eg/article_250681_a53857013c1a08c85f0ab3451a12720c.pdf	Arabic	null	Ecophysiological Studies on the Oxidative Stress Responses of Phlomis aurea, Ballota undulata and Nepeta septemcrenata Endemic Plants in Saint Katherine Mountain, Egypt	Egyptian Academic Journal of Biological Sciences. C, Physiology and Molecular Biology/Egyptian Academic Journal of Biological Sciences. C, Physiology and Molecular Biology	2,022	cc-by	5,363	Citation: Egypt.Acad.J.Biolog.Sci. ( C.Physiology and Molecular biology ) Vol. 14(2) pp13-23 (2022) DOI: 10.21608/EAJBSC.2022.250681 ABSTRACT ARTICLE INFO Article History Received:30/5/2022 Accepted:12/7/2022 Available:16/7/2022 Responses to antioxidants are reported in this research. Some indigenous plants have catalase (CAT), superoxide dismutase (SOD), and peroxidase activity (POD) as scavengers of reactive oxygen species (ROS) for mitigating environmental circumstances. In May 2019, physicochemical parameters such as EC, TDS, TSS, pH, HCO3, CO3-, Cl-, and SO4 were measured. Five locations in Egypt's Saint Katherine Mountain were chosen. During the sampling process, A statistical analysis of Physico-chemical components discovered considerable variations in numerous chemical and physical soil variables among diverse wadis. Changes in HCO3 were found to be non-significant, but substantial and highly-significant variations in other chemical and physical property measures were discovered among the five wadis examined. The current study found that in a stressed ecosystem, the plants chosen overcome the stress by altering their stress enzyme activities, implying evidence of adaptive mechanisms to thrive in a stressful environment and the suitability of three plant species—Phlomis aurea, Ballota undulata, and Nepeta Septemcrenata for environmental matrices (as indicators), particularly under altered climatic conditions. Keywords: P. aurea, B. undulata, N. Septem crenata, Environmental stress, Physico-chemical parameters, Antioxidant enzyme, catalase (CAT), superoxide dismutase (SOD) and peroxidase activity (POD). Citation: Egypt.Acad.J.Biolog.Sci. ( C.Physiology and Molecular biology ) Vol. 14(2) pp13-23 (2022) DOI: 10.21608/EAJBSC.2022.250681 . Vol. 14 No. 2 (2022) . Vol. 14 No. 2 (2022) Vol. 14 No. 2 (2022) Vol. 14 No. 2 (2022) Egypt. Acad. J. Biolog. Sci., 14(2):13-23 (2022) Egyptian Academic Journal of Biological Sciences C. Physiology & Molecular Biology ISSN 2090-0767 www.eajbsc.journals.ekb.eg Egypt. Acad. J. Biolog. Sci., 14(2):13-23 (2022) Egyptian Academic Journal of Biological Sciences C. Physiology & Molecular Biology ISSN 2090-0767 www.eajbsc.journals.ekb.eg www.eajbsc.journals.ekb.eg Ecophysiological Studies on the Oxidative Stress Responses of Phlomis aurea, Ballota undulata and Nepeta septemcrenata Endemic Plants in Saint Katherine Mountain, Egypt Ecophysiological Studies on the Oxidative Stress Responses of Phlomis aurea, Ballota Citation: Egypt.Acad.J.Biolog.Sci. ( C.Physiology and Molecular biology ) Vol DOI: 10.21608/EAJBSC.2022.250681 Salwan H. Dawood, et al. 14 SKP is home to 44 percent of Egypt's indigenous plant species, making it one of the most floristically varied places in the Middle East (Tackholm 1974). and qualitative variations in secondary compound production among plants and population groups (Julkunen-Tiitto et al.1996). These factors include altitude, high/low temperature, drought, and light conditions (Gong et al.2018., Gea-Izquierdo et al.2012). Increased reactive oxygen species (ROS) production is caused by a combination of high-altitude environmental stress, which increases the risk of oxidative damage (Polle et al 1999., Soliman et al.2020). Sinai has over 1285 species, with about 800 of them found in its southern portion (El-Esawi et al., 2018). Several indigenous species in Egyptian flora have been found in previous investigations (Shaltout et al., 2019). In Egypt, there are about 60 endemic plant species, with 31 of them on the Sinai Peninsula (which accounts for 51.6 percent of Egyptian endemism) (Boulos 1995). Twenty-four of these are unique to South Sinai and are recognized for their therapeutic characteristics as well as their usage in traditional medicine and treatments (El-Demerdash 2020). Antioxidative enzymes such as ascorbate peroxidase, catalase, superoxide dismutase, and glutathione reductase may scavenge various ROS such as superoxide, hydroxyl radicals, and singlet oxygen (Conklin 2001 and Zamin et al. 2020). High mountain plant species have previously been shown to be adapted to high irradiance and cooling stress (Streb et al.2003). The synchronized action of enzymatic and non- enzymatic antioxidants in plants regulates the level of reactive oxygen species (ROS). Catalase (CAT), superoxide dismutase (SOD), and peroxidase activity (POD) are examples of enzymatic antioxidants that are important ROS scavengers (Chai and Wong 2012). Because of SKP's high elevation, severe and complicated climatic conditions limit plant to spread and variety (Shaltout et al., 2015). Reduced O2 and CO2, strong winds, high sun irradiation, shallow rocky soils, cold temperatures, and poor water and nutrient content are some of the harsh circumstances (Roupioz et al., 2016). Even though these stress conditions may slow plant development, many species have survived and evolved distinct adaptation mechanisms in response to them (Wonsick and Pinker 2014). The antioxidant defense capability of plant samples from diverse habitats was explored in this study, as well as the eco- physiological reactions of some indigenous species as a result of their exposure to stressors existing at various habitats on Saint Katherine Mountain under varying conditions. Study Area: Saint Katherine Mountain, Egypt's highest mountain, was selected for this research (2641 meters above sea level) It is situated in the heart of the southern Sinai triangle. Shaq Musa and Wadi Garagneia are two big deep canyons, and three mountains of black volcanic granite stand out starkly against the surrounding cliffs. The Katherine pluton is part of a Precambrian basement INTRODUCTION The mountains are known for their diverse biodiversity. As compared to lowland plants, most plant species at high elevations are isolated and have a limited number of niche habitats (Williams et al., 2009). As a result, mountain plant populations exhibit a larger percentage of endemism than lowland plant populations (Alhaithloul 2019, Habib et al., 2020) Because of their narrow altitudinal distribution ranges, endemic species are more commonly endangered at high elevations (Grabherr et al., 1994, Moustafa-Farag et al., 2019). Most plants grow only near their climatic boundaries (El-Keblawy & Khedr 2017, Elkelish et al., 2019) and climate restrictions largely influence high-altitude habitats. The area over 2800 meters above sea level (a. s. l.) in the Saint Katherine protectorate (SKP), Sinai Peninsula, is remarkable not only for its magnificent sceneries but also for its richness of medicinal plants of national and international significance (Moustafa and Klopatek 1995., Abdel-Azeem et al., 2019). As a result of various climatic conditions, elevations, and geography, SKP has a diverse range of ecosystems (Ayyad et al., 2000). Salwan H. Dawood, et al. Several determinants affect the number of secondary metabolites in plants, including age, season, and nutrition status (Batiha et al. 2020., Rashad et al.2020). Similarly, environmental factors such as altitude, high/low temperature, drought, and light conditions cause significant quantitative complex that contains acid plutonic and volcanic rocks in the southern Sinai Peninsula. complex that contains acid plutonic and volcanic rocks in the southern Sinai Peninsula. Soils: A stainless-steel auger was used to obtain soil samples from 20 cm deep at each elevation. The samples were gathered at random from three locations and then pooled, sealed, and labeled in plastic bags. Soil samples were dried at 40°C in an air- forced oven. The dried materials were sieved to remove stones and plant residues before being processed in a stainless-steel mill and sieved at 2-mm intervals. The sieved soils were collected and kept until they could be analyzed further. Chemical tests of the soil were carried out according to Jackson's methodology Jackson (1967). Variations in the average soil chemical characteristics among the five wadis were recorded as follows: Maximum values in PH, EC, TDS, CaCO3, Na+, K+, and Mg++ were found in Musa's Gorge, with values of 8.1, 158.81, 348.22, 31.30, 24.16, 74.44, and 5.16, respectively, whereas current minimum values in water content were found at 0.20. Plant Sample Gathering: Plant samples were collected from five wadies (Wadi Gebal, Wadi Graginya, Wadi El-Arbae'en, and Wadi Abu-Tuweita) and Musa's Gorge in May 2019, for three plant species Phlomis aurea, Ballota undulata, and Nepeta Septem crenata. Soils: Physico-Chemical Properties of Soils: Physico-Chemical Properties of Ecophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants Ecophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants 15 non-significant difference. The most common forms of texture were loamy sand in Wadi Gebal and Musa's Gorge, and sandy clay loam in Wadi Graginya and Wadi El- Arbae'en, while the soil texture of W.AbuTuweita is sandy loam (Table 1). Certain Enzyme Extraction and Assaying Activities: Certain Enzyme Extraction and Assaying Activities: Wadi Gebal has high water content, Cl, and HCO3 values of 1.41, 18.50, and 14.47, respectively, whereas it has low EC, TDS, CaCO3, Na, and SO4 values of 107.15, 225.21, 12.08, 16.52, and 59.86. Superoxide dismutase (SOD) was assayed according to the method of Dhindsa et al. (1980). Peroxidase (POD) was measured as described by Polle et al. (1994). Catalase (CAT) was determined by the procedure described by Aebi (1984). With a score of 5.93, Wadi Graginya showed the highest result in organic matter percent. Wadi AbuTuweita reported a maximum value of 28 for Ca and the lowest values of 6.28, 2.58, 11.51, and 12.87 for PH, Mg, Cl, and HCO3, respectively. Wadi El- Arbae'en, a high value of 70.37 was observed in SO4, while the lowest values of 4.33, 21.19, and 18.5 were reported in organic matter, K, and Ca. Each statistic represents the average of 3 replicates with standard errors. * = significance at P < 0.001. Statistical Analyses: At the 0.05, 0.01, and 0.001 levels of probability, all data were statistically examined using one-way ANOVA and post hoc-LSD tests (the least significant difference) (Snedecor and Cochran 1982). The biochemical analysis data is the average of three replicates. A statistical investigation of chemical and physical soil parameters indicated substantial variances in several chemical and physical soil variables among various wadis. Non-significant changes in HCO3 were found, while significant and high-significant variances in other chemical and physical property parameters were found across the five wadis tested. RESULTS RESULTS Variation in Soil Properties of among Different habitats. Variation in Soil Properties of among Different habitats. Tables 1, 2, and 3 show the mean soil parameters of the five different habitats that sustain the sampled plants. The means of soil characteristics reveal a highly significant variance in soil physical attributes (Table 1), a moderate significance difference, and a Table 1: A one-way analysis of variance (ANOVA) was used to compare the physical properties of soil in diverse habitats. Each statistic represents the average of 3 replicates with standard errors. * = significance at P < 0.001. Salwan H. Dawood, et al. 16 Table 2: A one-way analysis of variance (ANOVA) was used to investigate the chemical properties of soil in diverse habitats Each statistic represents the average of 3 replicates with standard errors. = significance at P< 0. 01, * = significance at P< 0.001. Table 2: A one-way analysis of variance (ANOVA) was used to investigate the chemical properties of soil in diverse habitats Each statistic represents the average of 3 replicates with standard errors. = significance at P< 0. 01, * = significance at P< 0.001. Table 3: A one-way analysis of variance (ANOVA) was used to investigate the chemical properties (water-soluble ions) of soil in diverse habitats. Table 3: A one-way analysis of variance (ANOVA) was used to investigate the chemical properties (water-soluble ions) of soil in diverse habitats. Each statistic is based on the average of 3 replicates with standard errors. NS = non-significant, whereas * = significant at P< 0.001. Each statistic is based on the average of 3 replicates with standard errors. NS = non-significant, whereas * = significant at P< 0.001. U/ mg-1 protein). W. Gebal plants had the highest value (80.43 U/ mg-1 protein) while W. Abu Tuweita plants had the lowest value (62.73 U/ mg-1 protein). Near W. El- Arbaeen, Nepeta septemcrenata plants had the highest value (67.04 U/ mg-1 protein) and the lowest value (54.88 U/mg-1 protein) at Musa's Gorge. Antioxidant Enzymes: Figure 1(a,b and c) depict the mean of antioxidant enzyme activity. These findings suggest that the largest value of superoxide dismutase (SOD) was found in phlomis aurea plants in W. El-Arbaeen (fig 1). Phlomis aurea plants exhibited the highest maximum value (518.4 U/g-1) in W. El-Arbaeen and the lowest minimum value (388.42 U/g-1) at Musa's Gorge at the species level. W. Graginya had the highest value (501.7U/g-1) while Musa's Gorge had the lowest value (398.98U/g-1). Plants of Nepeta septemcrenata had the highest value (473.2 U/g-1) at Musa's Gorge and the lowest value (323.7 U/g-1) at W.AbuTuweita. Phlomis aurea plant samples collected in W.AbuTuweita had a maximum value of (CAT) 51.45 U/ mg-1 protein and a minimum value of 40.77 U/ mg-1 protein. At Musa's Gorge, Ballota undulata plants had the highest value (47.94 U/mg-1 protein) and the lowest value (37.92 U/ mg-1 protein), whereas, at W.AbuTuweita, had the lowest value (37.92 U/ mg-1 protein). At Musa's Gorge, plants of Nepeta septemcrenata exhibited the highest value (52.01 U /mg-1 protein) and the lowest value (39.42 U/ mg-1 protein) (Fig.1c). In phlomis aurea plants, peroxidase (POD) had the highest value in W.AbuTuweita (72.45 /U mg-1 protein) and the lowest value in W. El-Arbaeen (63.13 Ecophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants 17 ophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants 17 Ecophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants 17 Figs.1. Variation in antioxidant enzyme activity (SOD), (POD), and (CAT) of plant species collected from Saint Katherine's five habitats. Figs.1. Variation in antioxidant enzyme activity (SOD), (POD), and (CAT) of plant species collected from Saint Katherine's five habitats. DISCUSSION them to adapt to adverse conditions such as drought and strong light (Kramer 1983., Tanaka-Oda et al. 2010). Despite their ecological importance and numerous intriguing qualities, these unique species' ecophysiology has received little attention, particularly at the SKP high elevations (Hegazy 2016., Helal et al. 2020). As a result, understanding how such adaptations affect plant development is important for researchers who want to forecast how endemic species may respond to climate change in the future. Plants in high mountain environments are subjected to unfavorable or even dangerous abiotic conditions, which change growth dynamics and endanger their survival. This is especially true for species that are endangered or endemic El-Keblawy and Khedr (2017). In the SKP Mountains, there is a significant contrast between habitats. The spread and intensity of plant growth are reduced (Moustafa and Zaghloul 1996). This might be ascribed to the preponderance of adverse climatic conditions in the highest region of the SKP Mountains. The current study's results (Table 2) for chemical and physical soil parameters indicated considerable variations amongst wadis in a variety of chemical and physical soil variables. Changes in HCO3 were found to be non-significant, but there were considerable and extremely significant differences in other chemical and physical property parameters across the five wadis studied. Low temperature and oxygen Elevation affects landscape topography, geology, rainfall volume, and, as a result, soil moisture and texture, ground- water depth, hydrology, evaporation, soil type, and vegetation itself (Knoop and Walker 1985). According to the findings, the majority, if not all, of the metabolic components evaluated were significantly altered by changes in elevation rankings and habitats. The metabolic components of the plants differed substantially throughout the elevation ranks of the three species studied. Several other researchers obtained similar results. Plants in semi-arid environments have many physiological features that allow Salwan H. Dawood, et al. 18 deficiency (Neina 2019., Santiago 2000). water precipitation, light intensity, and UV radiation (Soethe et al. 2008., Sharma et al. 2014). are additional adverse biotic features of the SKP as a high elevation habitat. In general, a complex interplay between abiotic stresses and plant development has resulted in numerous physiological features through adaptation, acclimation, and speciation, which may differ between plant species (Chen et al. 2019., Pandey et al. 2017). DISCUSSION current study (Table 1) suggested that the highest value of superoxide dismutase (SOD) was discovered in phlomis aurea plants, indicating a reversal trend of change in the activity level of antioxidant enzymes (catalase, superoxide dismutase, and peroxidase) in all species. In W. El-Arbaeen, Phlomis aurea plants had the highest maximum value (518.4 U/g-1). W. Gebal had the highest value (80.43 U/ mg-1 protein) of Ballota undulata plants in this regard. Plants of Nepeta septemcrenata also had the highest value (52.01 U /mg-1 protein) in Musa's Gorge. Following these findings, Yang (Srivastava 2008). discovered that plant cells and tissues exhibit a variety of metabolic reactions to environmental stress, some of which may be adaptive (El-Esawi 2017). The simultaneous activity of enzymatic and non- enzymatic antioxidants regulates the number of reactive oxygen species (ROS) in plants. Catalase (CAT), superoxide dismutase (SOD), and peroxidase activity (POD) are three enzymatic antioxidants that are essential ROS scavengers (Chai and Wong 2012). The mechanisms of action of various antioxidants in the alleviation of oxidative stress vary (Elkeilsh et al. 2020). Abiotic stress disrupts cellular homeostasis in the plant species under consideration, resulting in the generation of free radicals, which can cause oxidative damage (Kusvuran 2012, Kusvuran et al. 2016). Furthermore, high light intensity and UV-B radiation are among the most aggressive stressors in the SKP's high alpine area (El-Ghani et al. 2017). Under these circumstances, electrons released from excited chlorophylls may be transported from photosystem I of the photosynthetic process to O2 to produce superoxide radicals, triggering a chain reaction of free radical liberation (Logan 2007). At low concentrations, hydrogen peroxide acts as a signaling molecule to induce the defense responses of plants under stress, but at high concentrations, it can cause significant disruption in metabolism through damage to lipids of membranes and nucleic acids, conformational changes of enzymic proteins, destruction of thiol-containing compounds, and so on (Sharma and Dubey 2007). Environmental differences (such as light, temperature, soil qualities, and altitude) significantly contribute to the antioxidant activity of indigenous medicinal plants (Liu et al. 2016). Many substantial differences in the antioxidant activity and soil chemical characteristics of three indigenous targeted species gathered from five distinct environments were discovered in this investigation. To respond to high abiotic stress, Phlomis aurea relies mostly on raising the activity of its antioxidant enzymes. ( y ) All sorts of abiotic or biotic stressors enhance the formation of hazardous oxygen derivatives. DISCUSSION Plants that thrive under oxidative stress actively activate antioxidant systems, which aid in adaptability. Through the scavenging of free radicals, antioxidant defense mechanisms can counteract oxidative stress (Sharma et al., 2012) The results (Figures 1–3) showed that plant species grown in diverse environments, where plants thrive under relatively low- stress conditions, rely on antioxidant enzymes and antioxidant chemicals to scavenge free radicals (Hasanuzzaman et al. 2019., Gong et al. 2018). The findings of the y Conclusions Environmental factors play a significant role in variation in vegetation distribution and plant community structure, with this variation in plant community structure producing a noticeable difference in most environmental factors such as gradients in elevation, temperature, and rainfall between different locations. It was revealed that poor environmental conditions Ecophysiological Studies on the Oxidative Stress Responses of P. aurea, B. undulata and N. septemcrenata Endemic Plants 1 19 Dhindsa, R.S., P.P., Dhindsa & Thorpe, T.A. (1980). Leaf senescence correlated with increased levels of membrane permeability and lipidperoxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany,32: 93-101. induced by abiotic factors including moisture, salinity, and mineral elements can limit plant growth and development, as well as cause physiological and biochemical abnormalities. 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Response of plant secondary ARABIC SUMMARY دراسات فسيولوجية بيئية حول استجابات اإلجهاد التأكسدي للنباتات المستوطنة (العورور و القاصه و )الزيتيه في جبل سانت ،كاترين مصر. سلوان حسين،داود محمد متولي،مرسي عبد المنعم علي،شرف حسام مهدي فوده قسم النبات والمايكروبيولوجي– كلية العلوم– جامعة االزهر : البريد األلكتروني للباحث الرئيسي, salwanhusin@gmail.com , MMMoursy@azhar.edu.eg hossamfouda2016@azhar.edu.eg في هذا البحث تم تقييم مضادات األكسدة االنزيمي وعالقتها بالضروف الكيميائية والفيزيائيه لثالث من النباتات المتوطنm في منطقة سانت كاترين في.مصر في هذا البحث تم تقييم نشاط انزيمات الكاتاليز والديزميوتيز وكذلك نشاط انزيم البيروكسيديز كمضادات للشوارد الحره لالوكسجين للتخفيف من الظروف البيئية . تم قياس الخواص الكيميائية والفيزيائية مثلEC ، TDS ، TSS ، pH ، HCO3 ، CO3- ، Cl- ، SO4 . تم اختيار خمسة مواقع في جبل سانت .كاترين في مصر أث ناء عملية أخذ،العينات بينت التحاليل االحصائيه للمكونات الفيزيائية والكيميائية اختالفات كبيرة في العديد من متغيرات التربة الكيميائية والفيزيائية بين الوديان المتنوعة( المواقع الخمس سابقة الذكر) . كانت التغييرات في HCO3 غير عالية القيمه المعنويه ، بينما لوحظ اختالفات جوهرية وعالية األهمية في مقاييس الخواص الكيميائية والفيزيائية األخرى بين الوديان الخمسة التي تم فحصها. وجدت الدراسة الحالية أنه في النظام البيئي،المجهد تتغلب النباتات المختارة على اإلجهاد عن طريق تغيير أنشطة إنزيمات،اإلجهاد مما يدل على و جود ميكانيكية التكيف للنمو في بيئة مجهدة ومالئمة لثالثة أنواع نباتية هي (العورور والقاصة والزيتيه) للمصفوفات البيئية،كمؤشرات السيما في ظل الظروف المناخية.المتغيرة الكلمات المفتاحية: ,العورور ,القاصه الزيتيه , مضاد االكسده ,العوامل الفيزيائية والك يميائية
https://openalex.org/W2082964214	https://europepmc.org/articles/pmc4402170?pdf=render	English	null	Mast Cell and Autoimmune Diseases	Mediators of inflammation	2,015	cc-by	7,009	Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 246126, 8 pages http://dx.doi.org/10.1155/2015/246126 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 246126, 8 pages http://dx.doi.org/10.1155/2015/246126 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2015, Article ID 246126, 8 pages http://dx.doi.org/10.1155/2015/246126 Yunzhi Xu and Guangjie Chen Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Immunology, Shanghai 200025, China Correspondence should be addressed to Guangjie Chen; guangjie chen@163.com Received 16 December 2014; Revised 25 March 2015; Accepted 25 March 2015 Academic Editor: Teresa Zelante Copyright © 2015 Y. Xu and G. Chen. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mast cells are important in innate immune system. They have been appreciated as potent contributors to allergic reaction. However, increasing evidence implicates the important role of mast cells in autoimmune disease like rheumatoid arthritis and multiple sclerosis. Here we review the current stage of knowledge about mast cells in autoimmune diseases. 1. Introduction or prostaglandins or a variety of cytokines, chemokines, and growth factors [16].h If the immune system fails to recognize self- from non- self-molecules, self-reactive lymphocytes can be activated by innate immune cells and lead to an autoimmune response [1]. Genetics, hormonal influences, and environment play important roles in autoimmune diseases. Some of the factors have been identified [2–4]. However, the specific determi- nants that initiate an autoimmune response and allow it to be sustained and cause pathology are still unknown. Autoimmune diseases and allergic diseases share important features. Both of them are the result of “hypersensitive” immune responses directed toward inherently harmless anti- gens [5]. Besides, many diseases models that we now know are regarded as autoimmune diseases, such as “experimental allergic” neuritis, encephalomyelitis, orchitis, uveitis, and glomerulonephritis [6]. It is accepted that the cells of the adaptive immune system are the directors of autoimmune responses [7]. In addition, innate immune cells are critical for sustaining the response that leads to pathology [8–13].i g The idea that MCs are involved in the initiation and sustaining events of autoimmunity is based on abundant data from studies of both human disease and animal models [17– 19]. 2. Mast Cells MCs were discovered by Friedrich von Recklinghausen in 1863 and named by Paul Ehrlich in 1878 [20]. Connective tissue is derived from undifferentiated mesenchymal cells. During the first 100 years after the discovery, it was believed that MCs were a component of connective tissue, functioned, and died within connective tissue [21]. Furthermore, MCs complete differentiation in connective tissue [21]. Until the 1980s, in vivo and in vitro evidence showed that MCs originate from hematopoietic stem cells, but the mast cell-committed precursors (MCPs) have not been identified [21, 22]. In the work of Chen et al., MCPs in the bone marrow of adult mice were identified. They are identified by the phenotype Lin−c-Kit+ Sca-1−Ly6c−Fc𝜀RI𝛼−CD27−𝛽7+ T1/ST2+ [23]. In addition, the experiment strongly suggests that MCPs are the progeny of multipotential progenitors (MPPs) other than common myeloid progenitors or granulocyte/macrophage progenitors [23]. Mast cells (MCs) are first described by Paul Ehrlich in 1878 [1]. They have been viewed as effectors in IgE-mediated allergic or antiparasitic responses; however, researches in the last two decades have found that MCs are also involved in innate immunity and inflammation by releasing a large array of inflammatory mediators [14, 15]. These mediators include compounds such as histamine and MC specific proteases prestored in cytoplasmic secretory granules (SGs) and newly synthesized lipid mediators such as leukotrienes Development of MCs from MPPs does not need cell division [21]. It is known that MCs leave the bone marrow as immature cells and they mature via abundant cytokines 2 Mediators of Inflammation Mast cell OX40L Treg OX40 Inhibits MC degranulation, suppresses Treg and, promotes Th17 Th17 Treg IL-6 Differentiation Th17 T cell Mast cell CD4+ TGF𝛽, IL-21, and IL-23 Figure 1: Direct cell interaction between mast cells and T cells. TGF𝛽, IL-21, and IL-23 TGF𝛽, IL-21, and IL-23 Inhibits MC degranulation, suppresses Treg and, promotes Th17 Inhibits MC degranulation, suppresses Treg and, promotes Th17 OX40L Differentiation Figure 1: Direct cell interaction between mast cells and T cells. between MCs and Treg cells needs to be further explored in autoimmunity.h in the local tissue microenvironment [20, 24]. For example, nerve growth factor (NGF) is well known as an important MCs growth factor [25]. However, MCs show plasticity [20]. Moreover, mature MCs show extensive proliferation potential [21].h Th17 cells are CD4+ T cells. At the meantime, they are defined by the expression of the transcription factor ROR𝛾t and cytokines IL-17. 2. Mast Cells As Th1 cells, Th17 cells are involved in the mouse models of MS and RA. The combination of TGF𝛽, IL-6, IL-21, IL-23, and IL-1𝛽contributes to the differentiation of Th17 from a na¨ıve CD4+ T cell. TGF𝛽is essential for the development of Treg cells, but it is inhibited by IL-6. MCs can express TGF𝛽, IL-6, IL-21, and IL-23 under some condition and promote Treg and Th17 cell differentiation and plasticity [30]. It is interesting that MCs counteract Treg cells suppression through IL-6 and OX40-OX40L axis towards Th17 cell differentiation [33] (Figure 1). The granules of MCs can be stained metachromatically purple with Toluidine Blue and it is routine staining for the demonstration of MCs [20, 26]. MCs are defined as connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) by the histamine, cytokines, and proteolytic enzyme which MCs store [20]. In addition to innate and acquired immunity, MCs play important role in bacterial infection and autoimmunity [24, 27, 28]. MCs can secrete the contents of preformed cytoplasmic secretory granules (SGs) while encountering certain stimulants. For MCs, this process is fundamental to their role in innate and acquired immunity [29]. Various molecules are able to activate MCs. 3.2. Interaction between Mast Cells and B Cell. MCs express a variety of B cell-modulating molecules and immunoglobulin (Ig) receptors [30]. MC FcRs include IgE and IgG receptors [34]. Depending upon the type of MCs, IgG-antigen com- plexes may activate MCs [34]. Conversely, the coengagement of IgG and IgE receptors inhibits cells activation [34]. Increas- ing data has been established indicating that MCs play critical roles in IgG-dependent tissue-specific autoimmune diseases [34]. Low amounts of MCs are effective in influencing B cell survival and proliferation in vitro through cell-cell contact and MC-derived IL-6 expression whatever state the MC activation is in [35]. Furthermore, MCs can promote B cells to differentiate into CD138+ plasma cells secreting IgA and it is dependent on CD40-CD40L expressed on B cells and MCs, respectively [35] (Figure 2). 3. Interactions between Mast Cells and Other Cells MCs can work with other cells like T and B lymphocytes to enhance activation and migration by cell-cell interactions or secreted products [18, 19]. Recently, the role of the inter- actions between mast cells and other cells in autoimmune diseases is becoming apparent [30]. 3.1. Interaction among Mast Cells, T Regulatory Cell (Treg), and Th17 Cells. Treg cells are defined as CD4+CD25+FoxP3+ and are known to suppress T effector cell response. Thus Treg cells can induce tolerance and control autoimmunity. MCs and Treg cells constitutively express OX40L and OX40, respectively. Therefore, mast cell-Treg cell interactions are in an OX40-dependent way. Gri et al. found that Treg cells directly inhibited Fc𝜀RI-dependent MC degranulation through cell-cell contact requiring OX40-OX40L interaction [31] (Figure 1). Kashyap’s group showed that coculture with Treg enhanced cytokines production by MCs [32]. In addition, MCs can also suppress Treg activity in an OX40L-independent way [30]. However, the relationship 4. MCs and Autoimmune Diseases It is well known that T cells are important in directing and initiating the immune response in the target tissues [30]. In addition, other cells also play an important role in aggravat- ing the inflammatory damage [30]. Furthermore, there are several examples of MCs association with autoimmune dis- eases including multiple sclerosis (MS), rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), bullous 3 Mediators of Inflammation 3 CD40L CD40 B cell Mast cell FcRs IgG IgE Differentiation CD138+ plasma cells IgE binds without antigens and MCs are activated Figure 2: Direct cell interaction between mast cells and B cells. Differentiation Figure 2: Direct cell interaction between mast cells and B cells. pemphigoid, chronic idiopathic urticaria, and experimental vasculitis [36–39]. Here we take MS, RA, IDDM, and chronic urticaria (CU) for example and summarize the role of MCs in the autoimmune diseases. Kruger et al. have observed MCs within the demyelinated plaques in the brains of 7 patients with MS [26]. Moreover, MCs were found mostly located in close connection with small vessels [26]. The data suggest that MCs playing a role in MS have continued to accumulate [30]. It is reported that mast cell deficient mice fail to develop EAE [52]. As in MS, an increase of MCs is also found at sites of inflammatory demyelination in the brain and spinal fluid of EAE [53]. MCs are associated with Fc𝜀R, the histamine-1 (H1) receptor, and tryptase [24]. Elevated levels of tryptase are present in the cerebrospinal fluid of MS patients and gene array analyses of MS reveal overexpression of genes encoding Fc𝜀R, H1 recep- tor, and tryptase [24, 54]. BMCs do not express their surface growth factor (c-kit) receptor normally but do so during EAE [55]. Several studies reveal that mast cell-derived mediators can increase BBB permeability [56, 57]. Products produced by MCs can enter neurons and this indicates a new brain- immune system [58]. Rat BMCs can produce tumor necrosis factor (TNF) and TNF take part in both brain inflammation and increased vascular permeability [59, 60]. An increased mast cell tryptase in the cerebrospinal fluid (CSF) of MS patients can activate peripheral mononuclear cells to secrete TNF, IL-6, and IL-1 and stimulate protease-activated receptor (PAR) which leads to microvascular leakage and widespread inflammation [54, 61, 62]. Besides, human MCs will secrete matrix metalloproteinase- (MMP-) 9 and IL-6 while contact- ing activated T cells [63]. 4. MCs and Autoimmune Diseases So we proposed that MCs may be an underestimated contributor to the demyelinating process of MS. 4.1. MCs and MS. Mostly, the interest in the role of MCs in the initiation and propagation of autoimmune disease comes from studies on MS [40]. MS is a progressive demyelinating disease. Widespread inflammatory lesions present in the brain and spinal cord of patients with MS [30]. The symptoms of MS contain visual disturbances, bowel and bladder incontinence, and sensory and motor dysfunction [30]. Furthermore, patients with MS are found to lose memory, impair attention, and slow information processing [41, 42]. Experimental autoimmune encephalomyelitis (EAE) is a murine model of MS. Similar to MS, the symptoms of EAE resulted from breach of the blood-brain barrier (BBB) which allows inflammatory cells to infiltrate into the central nervous system (CNS) and destruct myelin and oligodendrocytes [30]. CD4+ T cells, including IFN-𝛾-secreting T helper 1 cells (Th1), IL-17-producing T helper 17 cells (Th17), and IL-9-producing T helper 9 cells (Th9), contribute to the pathogenic autoimmune response in EAE [43]. However, the roles of these cells in MS are still unclear [44].h There are MCs in the leptomeninges, the choroid plexus, thalamus, hypothalamus, and median eminence [24]. Similar to CTMCs and MMCs, brain mast cells (BMCs) can be identified morphologically by Toluidine Blue staining mostly. Moreover, histamine fluorescence with o-phthaldialdehyde is able to show BMCs in the leptomeninges, thalamus, and hypothalamus. And histamine immunohistochemistry can show BMCs in the median eminence [45–48]. However, many BMCs are stained with Sudan Black which is distinct from CTMCs or MMCs [20]. Additionally, the ultrastructural appearance of activated BMCs is different from that of CTMCs because it is primarily characterized by intragranular changes without typical compound exocytosis [49, 50]. They may regulate vascular permeability and inflammatory cell entry in the brain parenchyma [51]. Moreover, there is interaction between functional MCs and neuron in the brain and it can mediate neuroinflammation. All in all, MCs participate in the pathogenesis of MS in many different ways [24]. Firstly, they release cytokines/ chemokines to recruit and activate T cell/macrophage after stimulation. Secondly, MCs present myelin antigen to T cell. Furthermore, MCs disrupt the BBB to allow activated T cells to infiltrate to brain and target in myelin basic protein (MBP). What is more, MCs damage myelin and then release fragments resulting in stimulating secretion of tryptase. 4. MCs and Autoimmune Diseases found that W/Wv and Sl/Sld, which are deficient in MCs, were resistant to development of joint inflammation. They proposed that MCs may serve as a cellular link among numerous components in inflammatory arthritis [68]. What is interesting is that MCs are normally expressed in the synovial compartments of healthy people but increased in RA patients [69]. The number of MCs increases 5- to 24- fold in affected joints in human RA when compared to the number of those in normal joints [69]. It is also found that MCs number expand more than 3-fold in multiple animal models of RA [70–72]. Besides, the cytokines and proteases which are produced by MCs are involved in the pathogenic process of RA, particularly TNF, IL-1𝛽, IL-17, and tryptase [30, 73]. Tryptase is a preformed mast cell-specific protease and is thought to lead to the inflammatory response by working with heparin to induce the neutrophils and synovial fibroblasts to release cytokines [74]. Tryptase can also directly activate synovial fibroblasts by interacting with the protease- activated receptor 2 (PAR2) to express more proteases that degrade cartilage and bone [75, 76]. Normally, MCs locate within the pancreatic ducts and are close to the pancreatic islets [86]. A lot of studies have found a striking increase in the frequency of MCs in the acinar parenchyma in inflammatory disease of pancreas [86–88]. Besides, MCs produce various mediators which are able to affect the development of IDDM. For example, leukotriene B4 (LTB4), which is released by MCs and may be important for recruitment or retention of autoreactive T cells in the target organ, is found increased in type I diabetes [89]. What is the most important is that Geoffrey et al. discovered more MCs in the pancreatic lymph nodes of lymphopenic diabetic BB rats before disease onset [36]. As a result, there is suspicion that MCs are involved in IDDM. Matsumoto and Staub’s group found that RA may be asso- ciated with the enzyme glucose-6-phosphate isomerase (GPI) [77]. K/BxN mice produce autoantibodies that can recognize GPI. The antibodies aggregate with GPI, and then immune complex is deposed on the surface of the articular cavity to initiate a signaling cascade including MCs. Cytokines such as IL-1 and IL-17A are also involved [73, 78]. 4. MCs and Autoimmune Diseases In turn, it enhances demyelination and induces further inflammation through stimulation of PAR possibly. As a result, MCs can be a possible therapeutic target for MS. In vitro, on one hand, mast cell proteases degrade myelin protein, while on the other hand, myelin stimulates mast 4 Mediators of Inflammation cell degranulation directly [64, 65]. Therefore, treatment with inhibitors of mast cell degranulation may be a good way to inhibit MS. Dimitriadou et al. found that hydroxyzine was able to inhibit EAE [66]. can precipitate the initiation of inflammation within the joint through the production and release of IL-1 [68, 80]. Stem cell factor (SCF) is essential for mast cell survival and development in vitro [1]. Furthermore, TNF-𝛼derived from MCs can induce fibroblasts to produce SCF, the ligand for the CD117/c-Kit receptor [81, 82]. SCF increases the recruitment of MCs and creates an amplification loop [81, 82]. 4.2. MCs and RA. RA is a systemic and chronic inflammatory disease that affects about 1% of the population worldwide [30, 67]. After decades of research, we have found that T and B lymphocytes, neutrophils, monocytes, and vascular endothe- lium play the roles in RA [67]. However, the pathogenesis and mechanism of RA are still unclear [67]. Rodent models of autoimmune diseases are of great use to study the pathogenic process of diseases. There are a number of models of RA including K/BxN, adjuvant-induced and pristane models, but the streptococcal cell wall (SCW) arthritis in rat and the collagen-induced arthritis (CIA) in mice are the most widely used [67]. 4.3. MCs and IDDM. Insulin-dependent diabetes mellitus (IDDM) is also called type I diabetes. IDDM is a chronic metabolic disorder that develops in two discrete phases and is mediated in part by CD8+ T cells [19, 83]. In the process of IDDM, various leukocytes invade the pancreatic islets and lead to insulitis. Then the insulin-producing 𝛽cells of the pancreas are destructed and lead to hyperglycemia [19]. Furthermore, IDDM is commonly associated with immune- mediated damage [84]. There are several rodent models of IDDM. In susceptible rodents, small dose of streptozotocin induces insulinopenic diabetes in which immune destruction plays the role, as in human type I diabetes [85]. In addition, the nonobese diabetic (NOD) mouse and biobreeding (BB) rat are the two most commonly used animals that sponta- neously develop diseases with similarities to human type I diabetes [85]. Lee et al. 4. MCs and Autoimmune Diseases The serum from K/BxN mouse causes similar inflammatory arthritis in a wide range of mouse strains, but KitW/W-v mouse deficient in MCs resistant to autoimmune inflammatory arthritis was induced by injection of sera from K/BxN mouse. If the MCs are recon- stituted, the sensitivity would be restored [68]. KitW-sh mice deficient in MCs are sensitive to autoimmune inflammatory arthritis induced by injection of sera from K/BxN mouse and mast cell-reconstituted KitW-sh mice are still susceptible to arthritis induced by sera from K/BxN mouse [79].l 4.4. MCs and CU. Chronic urticaria (CU) is a distressing disorder that adversely impacts the quality of life, but its pathogenesis is not delineated well [90]. An autoimmune subset of chronic spontaneous urticaria is increasingly being recognized internationally based on laboratory and clinical evidence that has accrued over the last 20 years [91]. In 1983, Leznoff et al. suggested that urticaria should be considered autoimmune [92]. Gruber et al. detected functional anti- IgE antibodies and proposed that these could be the cause of urticarial wheals [93]. And now it is well recognized that about 30–50% CU patients have circulating functional autoantibodies against the high-affinity IgE receptor or against IgE [94]. Besides, CU is associated with various autoimmune diseases [95]. Urticaria is triggered by inappropriate activation and degranulation of dermal mast cells. And the cellular contents released by MCs prime the immediate phase of inflamma- tion, resulting in a lymphocyte and granulocyte mediated hypersensitivity reaction [96]. In turn, the infiltrating inflam- matory cells produce more proinflammatory mediators to MCs accumulate in the synovial tissues and fluids of patients with rheumatoid arthritis and produce inflamma- tory mediators [1]. 4. MCs and Autoimmune Diseases In addition to the degranulation in the articulate cavity after antibody administration, the activation of MCs through the IgG immune complex receptor Fc𝛾RIII Mediators of Inflammation 5 CIA: Collagen-induced arthritis CNS: Central nervous system CSF: Cerebrospinal fluid CTMCs: Connective tissue mast cells CU: Chronic urticaria EAE: Experimental autoimmune encephalomyelitis GPI: Glucose-6-phosphate isomerase H1: Histamine-1 IDDM: Insulin-dependent diabetes mellitus Ig: Immunoglobulin LTB4: Leukotriene B4 MBP: Myelin basic protein MCPs: Mast cell-committed precursors MCs: Mast cells MMCs: Mucosal mast cells MMP: Matrix metalloproteinase MPPs: Multipotential progenitors MS: Multiple sclerosis NGF: Nerve growth factor NOD: Nonobese diabetic mouse PAR: Protease-activated receptor PAR2: Protease-activated receptor 2 RA: Rheumatoid arthritis SGs: Secretory granules SCW: Streptococcal cell wall Th1: T helper 1 cells Th17: T helper 17 cells Th9: T helper 9 cells TNF: Tumor necrosis factor Treg: T regulatory cell. recruit and activate other cells and extend the host response [96]. It lowers the reactive threshold of MCs to induce stimuli and promotes the maintenance of susceptibility to urticaria [90]. It provides an explanation for Smith’s discovery that MCs numbers remain unaltered [97]. Bossi et al. evaluated permeabilizing activity of sera from CU patients and healthy people by measuring serum-induced degranulation of two MC lines (LDA2 and HMC-1) [98]. They discovered that almost all the CU patients sera promoted degranulation of MCs and 17/19 mast cell supernatant from HMC-1 and SNs from LAD2 incubated with CU sera increased endothelia permeability [98]. It is said that histamine released from MCs is the major effector on pathogenesis [94]. Bossi et al. also found that endothelial cell leakage was prevented by antihistamine [98]. Acknowledgments This work was supported by grants from National Nature Science Foundation of China (81373208), Shanghai Com- mission of Science and Technology (11JC1411602), Shanghai Municipal Education Commission (12ZZ103), and Shanghai Board of Health Foundation (2011177). i Cpa3Cre/+ mice are a strain deficient in MCs. In spite of a great deal of evidence of the involvement of MCs in the autoimmune disease models, using Cpa3Cre/+ mice in study did not find an active role of MCs in both the K/BxN serum transfer model of RA and the EAE model of MS [102]. Besides, Gutierrez et al. found that IDDM in NOD mice was unaffected by mast cell deficiency [103]. Therefore, the research about the roles of MCs in autoimmune diseases remains a matter of great debate and ought to be further studied, which is important for creating new MC targeted therapies [5]. References [1] E. Z. da Silva, M. C. Jamur, and C. 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https://openalex.org/W4283522146	https://dergipark.org.tr/tr/download/article-file/2375545	Turkish	null	SADAKAT BİR ERDEM MİDİR?	Dokuz Eylül Üniversitesi Sosyal Bilimler Enstitüsü dergisi/Dokuz eylül üniversitesi sosyal bilimler enstitüsü dergisi	2,022	cc-by	5,168	Öz Bu makalede sadakat kavramının kendi başına bir erdem olup olamayacağı sorgulanmaktadır. André Comte-Sponville’in görüşlerinden Hannah Arendt’in görüşlerine uzanan bir değerlendirme ile sadakat kavramının bir erdem olacaksa bile hangi durumlarda erdem olacağı ele alınmaktadır. Sadakat sözcüğü her ne kadar kendi başına bir erdem olarak görülse de aslında kendisine nesne olarak aldığı şey aracılığı ile bir erdem olabilir. Bunu sadakatin farkı anlamlarına bakarak kavrayabiliriz. Sadakat bazen vefa, bağlılık gibi anlamlara gelirken bazen de itaati meşrulaştırmaktadır. Özellikle totalitarizmin egemen olduğu toplumlarda mutlak bir sadakate dayalı olarak kitle toplumunun itaat etmesi sağlanmaktadır. Bu anlamıyla sadakat kendisine sadece kendisini nesne almaktadır. Yani sadakat, kendisi için söz konusu olmakta ve bu durumda itaati doğurmaktadır. Hannah Arendt’in totalitarizm eleştirisi ile birlikte, sadakatin içeriğinin etik olmaktan ziyade politik olduğu söylenebilir. Bununla birlikte sadakatin Nazi ideolojisi de dahil olmak üzere pek çok ideolojik söylemin aracı olmaktan daha fazlası olup olamayacağı da tartışmaya açıktır. Yine Hannah Arendt’in geçmişle hesaplaşma ve hakikate sadakat kavramları ele alındığında sadakate etik bir temel kazandırma imkânının onun politik içeriğini açmaktan geçtiği anlaşılacaktır. Sadakati toplumlar için bir zehir olmanın yanısıra panzehir haline getiren şey, onun geçmişi hatırlama ile olan ilişkisidir. Geçmişle yüzleşme bir hakikatin dile getirilmesiyle ve dünya hakkında yargı verebilmek ile ilgilidir. Hakikat sonrası çağ diye adlandırılan bu çağda gelecek ufkunun yeniden biçimlendirilmesi için geçmişle kurulacak sadakat bağı etkili olabilir. Anahtar Kelimeler: Sadakat, Arendt, Eichmann, Totalitarizm, Hafıza.  Doç. Dr., Dokuz Eylül Üniversitesi, Edebiyat Fakültesi, Felsefe Bölümü, ORCID: 0000-0001- 7941-3198, ozlemduva@gmail.com. Dokuz Eylül Üniversitesi Sosyal Bilimler Enstitüsü Dergisi Cilt: 24, Sayı: 2, Yıl: 2022, Sayfa: 487-499 ISSN: 1302-3284 E-ISSN: 1308-0911 Dokuz Eylül Üniversitesi Sosyal Bilimler Enstitüsü Dergisi Cilt: 24, Sayı: 2, Yıl: 2022, Sayfa: 487-499 ISSN: 1302-3284 E-ISSN: 1308-0911 Yayın Geliş Tarihi: 14.04.2022 Yayına Kabul Tarihi: 10.05.2022 Online Yayın Tarihi: 30.06.2022 http://dx.doi.org/10.16953/deusosbil.1103787 Yayın Geliş Tarihi: 14.04.2022 Yayına Kabul Tarihi: 10.05.2022 Online Yayın Tarihi: 30.06.2022 http://dx.doi.org/10.16953/deusosbil.1103787 Araştırma Makalesi Duva Kaya, Ö. (2022). Sadakat bir erdem midir?. Dokuz Eylül Üniversitesi Sosyal Bilimler Enstitüsü Dergisi, 24 (2), 487-499. Bu makale için önerilen kaynak gösterimi (APA 6. Sürüm): SADAKAT BİR ERDEM MİDİR? Özlem DUVA KAYA Özlem DUVA KAYA Öz Anahtar Kelimeler: Sadakat, Arendt, Eichmann, Totalitarizm, Hafıza. IS LOYALTY A VIRTUE? Abstract This article aims to examine whether the concept of loyalty can be a virtue on its own. Combining André Comte-Sponville’s views with Hannah Arendt’s, it discusses conditions in which loyalty can be regarded as a virtue. Although “loyalty” is understood as a virtue in itself, it actually becomes a real virtue through the objects which loyalty makes its own object. We can identify this distinction by analyzing the different meanings of loyalty. 487 DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 Keywords: Loyalty, Arendt, Eichmann, Totalitarianism, Memory. Keywords: Loyalty, Arendt, Eichmann, Totalitarianism, Memory. Duva Kaya, Ö. Loyalty sometimes means faithfulness and devotion, whereas sometimes it justifies obedience. In mass societies especially where totalitarianism is dominant, obedience is ensured through absolute loyalty. In this sense, loyalty only makes itself as an object. In other words, loyalty is a problem for itself, and in this case, obedience easily arises. Through Hannah Arendt's critique of totalitarianism, it can be asserted that the content of loyalty is political rather than ethical. However, it is disputable whether loyalty can be more than a tool in many ideological discourses, including Nazi ideology. Considering Hannah Arendt's concepts of "confronting past" and loyalty, it becomes clear that the possibility of giving an ethical basis to loyalty lies in amplifying its political content. Loyalty is both toxic and antitoxic for societies due to its connection to memories . Confronting the past is about expressing the truth and being able to develop judgments about the world. In our age, which is called the post-truth era, the bond of loyalty established with the past can be effective in reshaping the horizon for the future. Keywords: Loyalty, Arendt, Eichmann, Totalitarianism, Memory. GİRİŞ Sadakat kavramının sözlük anlamı “içten bağlılık ve sağlam, güçlü dostluk”tur (TDK). Kavramın Batı dillerindeki kökeninde “efendiye bağlılık yemini” gibi anlamlar da bulunmaktadır (Stanford Felsefe Ansiklopedisi). Ancak bu tanımlar sadakatin içeriğini tam olarak yansıtmamaktadır. Sadakatin kelime olarak anlamını ortaya koyabilmek için bile onunla akraba kavramlara başvurmak gerekmektedir; şükran, minnet, vefa, adanma gibi; ya da bağlanma, hakikate yaslanma, ihanet etmeme gibi. Diğer yandan kavramı açıklamak için kendisine başvurduğumuz her bir referans kavramın, sadakat kavramının içeriğini farklı bağlamlara taşıyacağı da aşikârdır. Eğer sadakat kavramını bir eylem içerisinde tanımlayacak olursak, bu defa da bu eylemlerin niteliklerinin birbirlerinden farklılaşmaları sonucunda karşımıza çıkan bir anlam çokluğu ile karşılaşırız. Örneğin “sadık kalma” eylemi bize neyi ifade etmektedir? “Kendi gerçeğinden, hakikatinden ayrı kalmamak mı, kendi hakikatine bağlı kalmak mı, sadık kalınması gerekene ihanet etmemek mi, yoksa herhangi bir şeye/kimseye vefa göstermek” mi? Bununla birlikte gündelik kullanımı içerisinde sadakatin, bilincimizde yer eden farklı çağrışımları da vardır. Bu yüzden sadakatin doğasını açığa çıkarmak adına bir anlatıya başvurduğumuzda, gerçek bir sadakat olarak kabul edilen şeyin, değerli olan veya onayımızı hak eden bir şey olacağını ortaya koymaya çalışırız. Ancak yine de, sadakatin belirli türden bağımsız değerlendirme standartlarına cevap veren bir şey olduğunu veya böyle görülmesi gerektiğini iddia edemeyiz. Sadık olabileceğiniz farklı şeyler bulunabileceği gibi, sadık olmanın farklı yolları da mümkündür. Örneğin, bir kişinin partnerine karşı hissetme ve davranış biçimi ile, her ikisi de sadakatin tezahürleri olsa bile, muhtemelen en sevdiği yazarın yapıtlarına ilişkin hissetme ve davranış biçimi birbirinden oldukça farklıdır. Diğer yandan sadakatten bazen, belirli bir kişinin cesur ve zeki, ve aynı zamanda sadık olduğunu söylediğimizde olduğu gibi, bir karakter özelliği olarak da bahsedilir. Bir ilke veya ideal olarak da sadakatten söz edebiliriz. Sadakat hakkındaki tüm bu düşünme biçimlerinin arkasında ise, bireyler ve sadık oldukları şeyler arasındaki belirli bir tür 488 DEU Journal of GSSS, Vol: 24, Issue: 2 Sadakat Bir Erdem... ilişkinin (özellikle aidiyet ve adanma gibi bir ilişkinin) olduğu fikri yatar. Sadakatin içerisinde sadık olan bir özne ve onun sadık olduğu bir nesne/hatıra/kişi mevcuttur. Bu nedenle sadakatin ne ifade ettiğini anlamak için dikkatimizi, tüm bağlılıkların ortak noktasının ne olduğuna ve bağlılıkların sadakat olarak görülürken aldığı nesnelere ve biçimlere göre nasıl farklılaşabildiğine yöneltmeliyiz. André Comte Sponville, sadakat kavramının anlam içeriğinin belirlenmesinde ortaya çıkan bu belirsizlikler ve güçlükler nedeniyle “sadakatin değerini yaratan şeyin, onun nesnesi olduğunu” ifade eder (Sponville, 2004, s. 19). GİRİŞ Dolayısıyla sadakati “kendisi için” ve “kendinde” anlam ve değer taşıyan bir kavram olarak değil, kendisine nesne aldığı kavramlarla ilişkisinde yeni anlamlar kazanabilen değişken bir kavram olarak ele almak durumunda olduğumuzu öne sürer. Özellikle değerlerle ilişkisinde sadakat, kendisine nesne olarak aldığı şeyin değeriyle belirlenmiş bir değer içeriğine sahip olmalıdır. Sponville’in bu belirlemeleri, sadakat kavramı ile ilgili temel bir tartışmaya bağlanır: Sadakat bir erdem olabilir mi? Etik ve Politika Kavşağında Sadakat Sadakatin bir erdem olup olamayacağı tartışması, onun kendi başına övgüye değer olup olmadığı sorusunu da içerir. Böyle bir sorgulama ise bizi etik bağlamdan politik bağlama taşıyarak sadakatin erdemlerle ilişkisini konu edinmeyi gerektirir ve onun “iyi ya da kötü” olup olmadığı sorusunu temel alan etik belirlemeleri; sadakatin ne zaman iyi, ne zaman kötü olduğu, hangi koşullar ve tecrübeler içerisinde iyi ya da kötü kabul edileceği, sadakat duyulan kişi, kurum ya da pratiklerin sadakatin anlamını nasıl değiştirip dönüştüreceği gibi politik sorulara doğru yöneltir. Daha da ötesi sadakat, belki de politik içeriği konu edinilmeksizin etik içeriği anlaşılabilecek bir kavram da değildir. Antik dönem filozofları bu nedenle sadakati, cesaret, ölçülülük, adalet gibi kendi başına bir erdem olarak tanımlamaktan kaçınmışlardır. Onu bir erdem haline getiren şey, içine yerleştiği politik bağlam veya kendisine payanda olmuş değerlerdir. Bu nedenle Antik Yunan kültürü içerisinde sadakat, her zaman bir gerilim içerisinde sunulmuştur. Oresteia’da Klyteimnestra’nın Iphigenia’ya karşı sadakatinin Agamemnon’a olan sadakatsizliği ile karşı karşıya gelmesi, Antigone’nin törelere sadakati ile Kreon’un buyruklarına sadakatsizliği arasındaki gerilim, sadakate ilişkin olarak Antik dönem filozoflarının görüşlerine sirayet eden bir ikilem içerisinde, felsefe tarihi boyunca bu kararsız tutumla ele alınmaya devam etmiştir. Diğer yandan sadakat ve sadakatsizliğin aynı politik eylem içerisinde sürdürülebiliyor oluşu, sadakatin cesaret ya da bilgelik gibi kendi içinde tutarlı ve bütünlüklü bir doğaya sahip olmaktan ziyade gerilimli ve ikircikli bir doğaya sahip olduğunu gösterir. O halde yeni bir soru ile daha karşı karşıya kalmaktayız: Sadakat kendisi yoluyla iyi ve adil bir hayatın sürdürülebileceği bir araç mıdır yoksa koşulsuz bir bağlanma mıdır; dahası koşulsuz bağlanma olarak sadakat iyi ve adil bir hayatın (iyi hayatın) imkânını yaratır mı yoksa tüketir mi? Sadakati bazen bir erdem, bazen 489 Duva Kaya, Ö. Ancak iyi niyetin ölçülemezliği, her türlü eylemin meşrulaştırıcısı olarak kullanılabilme potansiyeli ve dahası, iyi niyet ile kötülüğe sebep olan eylemler 490 DEU Journal of GSSS, Vol: 24, Issue: 2 Sadakat Bir Erdem... arasında kıyaslanabilir bir mesafenin tesis edilemezliği vb. sorunlar sadakatin iyi niyet ve bağlılık eksenindeki anlamına dayalı içeriğinin, onu bir erdem olarak tanımlamaya yetip yetmeyeceği konusunu daha da tartışmalı hale getirmektedir. Platon’un da işaret ettiği üzere tirana duyulan sadakat ile insanlar arasında güven veren ve olumlanan bir sadakate yüklenen anlam arasındaki farklılık, beraberinde başka soruları da getirir: Acaba sadakat bir yandan philia ile, dostluk ve bağlılıkla anlaşılabilen bir kavram iken, diğer yandan aynı zamanda çıkarlarımızın ve korkularımızın örtüsü de olabilir mi? Törelere, yöneticilere veya kurumlara olan sadakatin gerisinde otorite karşısındaki korku kalıntılarını yahut belki de bizatihi kendisini bulabilir miyiz? Ya da belki bu kadarından daha fazlası olabilir mi sadakat? Machiavelli, korkunun tek başına sadakati sağlayamayacağını ifade ediyor ve sadakat için başka yöntemlere başvurulması gerektiğini vurguluyordu. Prens adlı eserinde O, devletin güvenliği tehlikedeyse ya da tebaanın varlığı zarar görüyorsa, hükümdarın erdemli davranmaktan kaçınabileceğini, halka yalan söyleyebileceğini ifade ediyordu. Ancak iktidarı korumanın ve egemenliğin devamlılığını sağlamanın tek yolunun sadakati kalıcı ve hatta mutlak hale getirmek olduğunu da buna ekliyordu (Machiavelli, 1999, s. 37). O halde sadakat kendisi aracılığıyla erdemlere ulaşabildiğimiz birşey olduğu gibi erdemsizliklerin meşrulaştırılmasının bir yolu da olabilir. Nitekim totaliter yönetimlerin kendilerini meşrulaştırdığı en güçlü dayanaklardan birinin sadakat olduğunu görüyoruz: Kökenlere, soya, belirli bir tarihsel deneyime, ulus bütünlüğüne, liderlere, kimliklere sadakat çoğu zaman gayrimeşru yönetimlerin kendilerini meşrulaştırmalarının en güçlü dayanak noktası olmuştur. Bu meşrulaştırıcı söylemlerin bir malzemesi olarak sadakat, üzerinde herhangi bir ortak bağın kurulamayacağı ölçüde parçalanmış, ortak değerleri yahut normatif referansları kalmamış toplumları bir arada tutacak bir yapıştırıcı işlevini üstlenmiştir. Kimi zaman sadakatin nesnesi bile belirsizdir; bireyin sadakat duyduğu şey ile kendi benliği ve tercihleri arasında aşılmaz bir mesafe ve boşluk vardır; sadakat artık birşeye değil, “sadakat” e duyulan bir sadakat haline gelmiştir. Böylece artık değerlerin yerini alabilen ama kendisini asla aksiyolojik biçimde ortaya koyamayacak olan bir edime dönüşmüştür sadakat. Böylesi bir durumda sadakat, kendisine nesne aldığı herhangi bir unsurun ortadan kalkması durumunda işlevsizleşip biçim değiştirebilecek bir şey haline gelmiştir. Bu yüzden kolayca bütün erdemsizliklerin meşrulaştırılacağı bir gerekçe olarak da sunulabilir. Sadakatin bu işlevi, Hannah Arendt’in Eichmann davası üzerine söyledikleri, yani onun Kant etiğine, yasaya ve ahlakî buyruklara koşulsuz itaate olan eleştirisi yoluyla açıklık kazanabilir. Duva Kaya, Ö. DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 ise erdemsizlik olarak görmemizi sağlayan şey nedir? Zannediyorum ki bu sorular, etik soruların politik sorulara bağlandığı kavşaklarda anlaşılabilir. Platon Devlet’te tiranlığın ilk aşamalarından itibaren bir toplumda nasıl aşama kaydettiğini açıklarken, tam da bu kavşakta anlaşılabilecek bir meseleye işaret etmekteydi; tiranın başlangıçta arazi bağışı, borç affı vb. gibi vaadlerde bulunarak yurttaşların kendisine olan sadakatini perçinlediğini, ancak bunu öncelikle kendi “sadık kadrosuna” uyguladığını belirtirken O, sadakat kavramının bu gerilimli doğasını iki tür sadakati ayırt ederek açığa çıkarıyordu. Tiran, kendi egemenliğini sağlamlaştırmak için kendisine karşı sadakat gösteren’leri ödüllendirerek toplumu, kendisini eleştirenlerden, aslında sitenin iyisini düşünenlerden, yani siteye sadakatle bağlı olanlar’dan arındırır (Platon, 2005, s. 206 [563a, 564]). Gerek duyduğu takdirde kendisine biat eden uyrukların sadakatlerini sağlamlaştırmak üzere köleleri satın alıp azat edebilir; böylece özgürleşen köle, kendisini azat eden efendisine eskisinden de çok sadakat göstermeye başlar. Bu yüzden Platon tragedya şairlerinin tiranların bilgelerle düşüp kalkarak akıllı olacakları yönündeki ifadelerinin gerçeği yansıtmadığını, bir tiranın ancak koşulsuz sadakatin taşıyıcısı olan kölelerle düşüp kalkabileceğini söyler: “Bu insanlar iktidara gelmeden hemen önce kişisel (özel) hayatlarında şöyle davranırlar: Önce kendilerine yağcılık edecek ya da her türlü hizmete hazır olan kimseler ile ilişki kurmaya çalışır ya da kendilerinin bir şeye ihtiyaçları olduğunda, itaatkâr biri olup çıkar, sırf dost görünebilmek için her şeyi yaparlar; ancak bir kez amaçlarına ulaştılar mı, ötekileri tanımazlar bile!” “Aynen böyledirler!” “Ömür boyu dostları olmaz onların, her zaman bir başkasının ya efendisi ya da kölesidirler; çünkü hakiki dostluk, tiran karakterinin özüne her zaman aykırıdır.” (…) “Haklı olarak böyle insanlara ‘güvenilmez, sadakatten yoksun insan’ deriz!” “Nasıl demeyelim ki?” (Platon, 2005, s. 216 [ 576a,576b]). Bu ifadelerden de anlaşıldığı gibi Platon “tirana duyulan sadakat” ile “güven veren sadakat” arasında bir gerilim ve hatta bir karşıtlık olduğunu belirtmektedir. Sadakat, politik eylemin doğasına bağlı olarak erdemli ya da erdemsiz sadakat olarak ayrılabilir ve tam da bu nedenle biri, kolaylıkla diğerine dönüşebilir. Bu da sadakati diğer erdemlerle ilişkisinde ele alsak bile her zaman erdem zemininde ele alamayacağımızı gösterir. Burada artık Sponville’in salt erdemlerle ilişkisinde anlamaya çalıştığı sadakat kavramının etik bağlamından uzaklaşarak başka türlü bir anlama zeminine ihtiyaç duymakta olduğumuzu belirtelim. Zira Sponville’in yorumuna göre sadakat, samimiyet, samimiyete dayalı ilişkiler ve iyi niyet çabasıyla ilintilidir ve iyi niyet için çabalamanın kendisi bir erdemdir (Sponville, 2004: 258). Duva Kaya, Ö. DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 Duva Kaya, Ö. Dahası Arendt’in sadakat ile ilgili görüşleri, sadakatin hem zehirleyici hem de iyileştirici yanlarını anlamada bize yol göstermektedir. Bu nedenle sadakat kavramını ele alırken Sponville’in yorumundan uzaklaşarak, ancak onun; sadakatin kendi içinde bir erdem olmayıp, kendisi aracılığıyla erdemlerin anlaşılabileceği ve bu nedenle birbirine zıt çok farklı görünümler kazanabileceği yönündeki tespitini de akılda tutarak, sadakatin içeriğini anlamada bu makalenin sınırları açısından ikinci durak olan Hannah Arendt’e dönmek faydalı olacaktır. 491 Hannah Arendt, Kötülüğün Sıradanlığı ve Sadakat Hannah Arendt, Kötülüğün Sıradanlığı ve Sadakat Hannah Arendt’in “Kötülüğün Sıradanlığı: Eichmann Kudüs’te” adlı eseri, Nazi Almanyası’nda kendisine verilen emirleri istisnasız uygulayan ve Hitler’e vermiş olduğu sadakat sözünü yerine getiren Adolf Eichmann’ın Kudüs’te çıktığı duruşmadaki ifadelerinin derlenmesini ve yorumlanmasını içerir. Eichmann ve o dönemdeki Nazi subayları, eylemleri konusunda sorguya çekildiklerinde, kendi eylemlerini “ yasalara sadakat” ile açıklamakta ve kendi savunmalarını pür bir sadakat ilkesi üzerinde temellendirerek, eylemlerinin sonuçlarından sorumlu tutulmaktan kaçınmaya çalışmaktadırlar. Eichmann ise kendisinin mahkemeye çıkarılmasından evvel yargılamalarda yavaş yavaş belirginleşmeye başlayan bu “sadakat” gerekçesini daha da somut bir hale getirerek, görevlerine sadık bir subay olarak bu eylemler nedeniyle olsa olsa “ yardım ve yataklıktan” suçlanabileceğini ama ölümlerden sorumlu tutulamayacağını ifade etmekte, böylece bu kavramı, hukukî bir karar anına tahvil ederek sorumluluktan kaçmaya çalışmaktadır. Bu olayda, o zamana dek genellikle etikle ilişkisinde konu edinilen sadakatin, yargı kararlarına sızan politik doğasını açık bir biçimde görmekle birlikte-Arendt’in bakış açısından açıklayacak olursak- etiğin, politikanın imkânını tüketen açmazlarına da tanık olmaktayız. Arendt’e göre eğer yasalara koşulsuz itaat, mevcut yasalara ve görevlere sadakatle bağlı olmak gibi bir durumla uyuşabiliyorsa, o zaman Kantçı kategorik imperatifin doğasını sorgulamak gerekir. Burada etik yasa, adaleti bireylerin vicdanlarına terk edilmiş bir bir uygunluk formu gibidir ve bireyler, a priori verili bir yasaya uygunluk ile vicdanlarını test ettiklerinde, tıpkı Eichmann’ın söylemlerinde olduğu gibi bütün adaletsizlikleri yasaya ya da otoriteye sadakat ile meşrulaştırabilmektedirler : “Adolf Eichmann’ın vicdanı kuşkusuz karmaşık olmakla beraber bu konuda yalnız değildi, uzaktan da olsa Alman generallerinin vicdanlarıyla benzerlik gösteriyordu. Bu Alman generallerinden biri, Nürnberg’de kendisine yöneltilen ‘Siz onurlu generaller nasıl oldu da sorgusuz sualsiz bir sadakatle bir katile hizmet etmeye devam edebildiniz ?’ sorusuna ‘Başkomunatınını yargılamak askerin işi değildir. Bırakın da bu işi tarih veya yukarıdaki Tanrı yapsın.’ cevabını vermişti. (Nitekim general Alfred Jodl Nürnberg’de asıldı). Pek zeki sayılmasa da, sözünü etmeye değecek bir eğitimi olmasa da Eichmann en azından herkesi suçlu hale getirenin bir emir değil de kanun olduğunu az çok sezmişti. Führer’in ağzından çıkan sözler, sıradan emirden farklıydı; bu sözlerin geçerliliği zaman ve mekânla sınırlı değilken, bu sınırlama, sıradan emrin en göze çarpan özelliği idi (Arendt, 2012b, s. 156). “Adolf Eichmann’ın vicdanı kuşkusuz karmaşık olmakla beraber bu konuda yalnız değildi, uzaktan da olsa Alman generallerinin vicdanlarıyla benzerlik gösteriyordu. Bu Alman generallerinden biri, Nürnberg’de kendisine yöneltilen ‘Siz onurlu generaller nasıl oldu da sorgusuz sualsiz bir sadakatle bir katile hizmet etmeye devam edebildiniz ?’ sorusuna ‘Başkomunatınını yargılamak askerin işi değildir. Sadakat Bir Erdem... DEU Journal of GSSS, Vol: 24, Issue: 2 Çağın düşünürlerinin birçoğunun ele aldığı gibi Arendt de İkinci Dünya Savaşı sırasında yaşananları kötülük ile ilişkisinde ele almakta ve Nazi toplama kamplarında yaşananları kötülüğün en radikal biçimi olarak tarif etmektedir. Bu noktada daha önce Kant tarafından kullanılan “ radikal kötülük” kavramının anlamını açıklaştırmaya çalışan Arendt, Kant’ın bu terime yüklediği anlamın kötülüğü açık bir biçimde yansıtmadığını öne sürer. Onun bu analizlerine yakından bakıldığında sadakatin, Arendt’in radikal kötülükten ne anladığını ortaya koyan anahtar kavramlardan birisi haline geldiği görülür. Aslında radikal kötülükler hepimizin içinde yer alan ve kendisini bir “ödeve saygı”da, “görev bilinci”nde yahut “otoritelere sadakat”te meşrulaştıran “sıradan kötülükler” ile ilgilidir. Kötülüğü radikalleştirip marjinalleştirmeden, son derece sıradan bir görev bilinci ve sadakat edimi ile açıklamak, radikal ve kitlesel bir kötülüğün nasıl ortaya çıkabildiğini anlamımızı sağlayabilir. Totalitarizmin Kaynakları adlı eserinde Arendt, Kantçı etik kavramlar da dahil olmak üzere o güne değin bilinen tüm standartları çökertip insanlığı yıkıcı bir gerçeklikle karşı karşıya bırakan Auschwitz olayının ancak, koşulsuz bir sadakati tartışmasız biçimde meşrulaştırmanın zemini olan “totalitarizm” olgusu ile anlaşılabileceğini belirtir. Totalitarizm, parçalanmış ve yalnız bireylerin kitle haline getirilmesine, bireylerin aynılaştırılıp birbirine benzetilmesine ve donuklaştırılmasına neden olan bir harekettir ve temelde tüm üyelerinden total bir sadakat talep eder. Söz konusu bu sadakat ise, kitle toplumu üyelerinin, totaliter hareketler sayesinde “dünyada herhangi bir yere sahip oldukları hissinin” yaratılmasına neden olur ve bu yanılsama üzerinden kitleleri mobilize eder (Arendt, 1976, ss. 323-324). Böylece totalitarizm insanları giderek kendi ait oldukları insanlık hallerinden uzaklaştırır, onları birer otomata dönüştürür (Berktay, 2012, s. 75). Bu akılyürütmeleri ile Arendt, Eichmann’ın eylemlerinin ve bu eylemlerin radikal bir kötülüğe yol açan karakterinin “ kötü niyet” tarafından belirlenmediğine, daha ziyade rejime istisnasız ve sorgulamaksızın bağlılık, otomatlaşma ve bir bütüne aynı biçimde dahil olma ihtiyacından kaynaklandığına dikkat çekmiştir. Bu nedenle Eichmann’ın duruşma boyunca kendisini “ görevine sadakatle bağlı bir asker” olarak aklamaya çalışması, “verilen kararlara riayet ettiğini” bildirmesi onun söylemlerinde, hayatı boyunca Kant'ın “koşulsuz buyruk felsefesine” uygun yaşamaya çalıştığını vurgulamaya çalışmasına neden olmaktadır. Arendt, Eichmann'ın Kant'tan yanlış bir ders çıkarttığının söylenebileceğini, ama Kantçı etik temellendirmelerden bu tür sonuçlar çıkarmanın da mümkün olduğunu, bu yüzden radikal kötülüklerin ve “kötülüğe” sadakatin etik bir yaklaşımla engellenemeyeceğini düşünmektedir. Bu noktada Arendt, Kant etiğinin temeli olan koşulsuz buyruğun, örtük kalmış bir “karşılıklılık ilkesi”ne sahip olması ve a priori verili bir yasaya “bağlılık” fikrinin bu türden yanlış sonuçlar çıkarmaya müsait olması nedeniyle problemli olduğunu öne sürmektedir. Hannah Arendt, Kötülüğün Sıradanlığı ve Sadakat Bırakın da bu işi tarih veya yukarıdaki Tanrı yapsın.’ cevabını vermişti. (Nitekim general Alfred Jodl Nürnberg’de asıldı). Pek zeki sayılmasa da, sözünü etmeye değecek bir eğitimi olmasa da Eichmann en azından herkesi suçlu hale getirenin bir emir değil de kanun olduğunu az çok sezmişti. Führer’in ağzından çıkan sözler, sıradan emirden farklıydı; bu sözlerin geçerliliği zaman ve mekânla sınırlı değilken, bu sınırlama, sıradan emrin en göze çarpan özelliği idi (Arendt, 2012b, s. 156). 492 Sadakat Bir Erdem... Böylece Eichmann, sadakat gösterdiği yasaların ruhunu, yasa koyucunun ancak “kendisi” iken yahut yasayı ancak kendisi onayladığında bir geçerliliğe sahip olacağını unutarak bir başkasını, Hitler’i yasa koyucu olarak tasarlayabilmiş ve kabullenebilmiştir. Arendt’e göre Kantçı kategorik imperatif, kötülükler karşısında bir tutum geliştirebilmeyi, nihai olarak kişilerin “iyiye sadakat” duymalarına tahvil eden, ne olduğu belirsiz bir 493 DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 Duva Kaya, Ö. 82) ve belki de bu bağ onun “kendi kimliğine” ve “bireysel haklarına” sadakatsizliğinden beslenmektedir. Bu durumda mutlak sadakatin sadakate ilişkin olarak “ne, neden, niçin?” gibi soruları yöneltemeyeceğimiz denli bir düşünce yoksunluğunun ürünü olduğunu söyleyebiliriz. Görülmektedir ki sadakatin otorite ile olan ilişkisi, modern kitle toplumu söz konusu olduğunda gerek Antik dönemde Platon’un tiranlıkla ilişkisinde ortaya koyduğu sadakat, gerekse modern çağda Machiavelli’nin birtakım çıkarlarla gelişen ya da korku uğruna hükümdara karşı duyulan bir bağlılık ve sadakat ile karşılaştırıldığında, bambaşka bir biçime ve içeriğe sahiptir. Burada artık söz konusu olan herhangi bir şeye değil, “sadakate sadakat”tir; zira kitle toplumu iyiyi kötüden, doğruyu yanlıştan ayıracak temel yetisini, yani “yargı yetisini” kaybetmiştir. Arendt’ten yola çıkarak diyebiliriz ki işte bu koşulsuz ve iyi ya da kötü herhangi bir şeyi kendisine nesne olarak almayan sadakat, radikal kötülükleri üretme kapasitesine sahip olmasıyla en tehlikelisidir. Duva Kaya, Ö. vicdan çerçevesine sığdırmaktaydı, oysa ki vicdanlar kendilerini bir şekilde meşrulaştırmanın yolunu kolaylıkla bulabilir ve bunu sadakate uygun davranmak gibi bir gerekçeyle ortaya koyabilirlerdi: “Nazi hiyerarşisinin vicdanla ilgili sorunları çözme konusunda en yetenekli üyesi Himmler’di. Hitler’in 1931’de SS’e yaptığı bir konuşmadan alınan şu meşhur ‘Sadakatim Onurumdur’ gibi sloganları-Eichmann’ın ‘kanatlı kelimeler’, hakimlerin ‘boş laflar’ dediği sloganları-buldu ve Eichmann’ın hatırladığı kadarıyla sene sonuna doğru muhtemelen Noel ikramiyesi ile birlikte dağıttı. Eichmann bunlardan bir tanesini hatırlıyor ve sürekli tekrar ediyordu: Bu savaşlar sayesinde gelecek nesiller tekrar savaşmak zorunda kalmayacak. Savaşlar derken kadınlara, çocuklara, yaşlılara ve diğer ‘faydasız boğazlara’ karşı savaşmaktan bahsediyordu. (…) Böylece katiller, ‘insanlara ne korkunç şeyler yaptım!’ demek yerine, ‘görevlerimi yerine getirirken ne korkunç şeyler görmek zorunda kaldım, bu görevin omuzlarıma yüklediği yük nasıl da ağır’ diyebiliyorlardı” (Arendt, 2012b, s. 114). Arendt, totaliter pratikler içerisinde karşımıza çıkan kitle toplumunun, bireyleri "fuzuli", “gereksiz”, “her zaman vazgeçilebilir” hale getirdiğini belirtir (Arendt, 2014, s. 260). Gereksizleşen insanın kendi kararlarını otonom bir biçimde alması mümkün olmadığı gibi, eylemleri üzerine düşünme ve sorgulama yetisi de kaybolmuştur; bu durumda kişi, kendisine karşı en büyük sorumluluğu olan “kendi üzerine düşünme”den vazgeçecektir. (Arendt, 2010, s. 262). Arendt’e göre “gereksizleşmiş” bir insan için bu kendinden vazgeçişin yarattığı boşluğun yerini, ideolojik aygıtların belirlediği bir “sadakat” ilkesi almakta ve böylece “sadık” bir bağlanma formu üzerinden otoriteye boyun eğen bireyler, her türlü kötülük karşısında sessiz kalarak, kendi vicdan yükünü hafifletmektedir. Göreve sadakat, üstlerine sadakat, partiye sadakat, yasaya sadakat vb. sadakat formları, radikal kötülüklerin ardına gizlendiği bir maskeden de fazlası, bizatihi o kötülükleri yaratan boyun eğme biçimleridir. Sadakate yaslanarak kendisini güçlendiren sistem içerisinde merkezi bürokrasinin, ulus-devletin ve yönetim aygıtlarının güç kazanması sayesinde gerçek bireysel kimliklerin yok olması, bunun sonucunda bireylerin bir yerlere ait olma arzularının giderek silikleşmesi ve mutlak itaat ve sadakat talep eden totaliter hareketlerin içerisinde eritilmesi, bireylerin pasif özneler haline getirilmesine değil, onların kötülüğün failleri haline gelmesine neden olur. Özetle “sadakat” yüzünden ve “sadakat” sayesinde birey, eylemleri üzerine düşünmekten vazgeçer. Berktay’ın ifadesiyle kitle insanının kendisini bir aidiyet içerisinde görmesini sağlayan aygıtlar bireyi "demirden bir bağ" ile kendilerine 494 Sadakat Bir Erdem... DEU Journal of GSSS, Vol: 24, Issue: 2 bağlarlar (Berktay, 2012, s. 82) ve belki de bu bağ onun “kendi kimliğine” ve “bireysel haklarına” sadakatsizliğinden beslenmektedir. Bu durumda mutlak sadakatin sadakate ilişkin olarak “ne, neden, niçin?” gibi soruları yöneltemeyeceğimiz denli bir düşünce yoksunluğunun ürünü olduğunu söyleyebiliriz. bağlarlar (Berktay, 2012, s. “Hakikate Sadakatin” Unutuluşu Arendt’in bu analizlerinden yola çıkarak sadakatin en açık ve saf halinin deneyimlendiği totalitarizmin, insanların iyiyi kötüden ayırmasına, yani yargı yetilerini kaybetmesine neden olması nedeniyle kanaatlerin oluşmasını, farklılaşmasını ve karşı karşıya gelmesini sağlayacak olan kamusal alanı yok ettiğini; felsefenin kadim arayışı olan “hakikate sadakat”in bile anlamını yitirmesine neden olduğunu söyleyebiliriz. Oysaki Arendt’in de belirttiği gibi “düşünme, bir hakikat deneyiminin insanı çarpmasıyla başlar” (Arendt, 1995, s. 25) ve hakikate olan bağlılık ve sadakat, dünyayla ilgili düşünmenin somut başlangıcıdır. Totaliter koşullar içerisinde bireylere ideolojik aygıtlar, propaganda, endoktrinasyon ve yalan yoluyla “hakikate sadakat” unutturulmuş, bunun yerini yargı kapasitelerini kullanamayan bireylerin mutlak sadakati ve koşulsuz itaati almıştır: “Yalandan bahsettiğimizde, özellikle de eyleyen insanların yalanı söz konusu olduğunda hatırlamalıyız ki yalan, insan günahkârlığının tesadüfi bir sonucu olarak siyasete sızmış değildir. Tam da bu nedenle, ahlaki öfkenin onu yok edebilmesi mümkün değil. Kasıtlı yalan, olumsal olgularla ilgilidir; yani, içkin bir doğruluk barındırmayan doğru olma zorunluluğu taşımayan meselelerle. Olgusal gerçeklerin doğruluğu, yalanlanamayacak nitelikte olmaz hiçbir zaman. Tarihçi, gündelik hayatımızı çevreleyen olguların ne denli narin bir dokusu olduğunu bilir. Bu doku her zaman tehdit altındadır, tekil yalanlarla delinme, grup, ulus veya sınıfların örgütlü yalanlarıyla paramparça edilme, ya da çoğu zaman olduğu gibi bir dolu yalanla üstü dikkatlice örtülerek inkâr edilip çarpıtılma veya kendi 495 Duva Kaya, Ö. DEÜ SBE Dergisi, Cilt: 24 halinde unutulmaya terk edilme tehlikesi barındırır” (Arendt, 2018, s. 14). Duva Kaya, Ö. DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 halinde unutulmaya terk edilme tehlikesi barındırır” (Arendt, 2018, s. 14). Yalanla ve ideolojik araçlarla sistematik bir biçimde gerçekleştirilen unutturma pratiğinin koşulsuz itaatle ve sadakatle olan ilişkisi bizi, sadakatin etik içeriğinin, aynı zamanda onun politik içeriği ile ilgili olduğu sonucuna götürür. Bununla birlikte Arendt’in düşüncelerine başvurarak diyebiliriz ki eğer sadakat kavramına etik bir içerik atfedilecekse, bu ancak geçmişle yüzleşme gibi, hakikati çağıran ve hakikatle ilişkisini kurabilen bir eylem motifi içerisinde mümkün olabilir. Zira hakikatin unutturulması aynı zamanda benliklerin, kimliklerin, “insan oluş” halinin de unutuluşudur; insanların yaşamlarından arda kalan herşeyin bir sayıyla, nicelikle yer değiştirdiği kitle toplumu ideolojisinin itaati sağlayabilmesinin yolu onları nesneleştiren ve niceliksel değerler haline getiren mekanizmalarında saklıdır. İnsanlığın yaşadığı en büyük çöküşün tam da böyle bir anda gerçekleştiğini söyleyen Arendt’e kulak vermek ve hakikatin unutturulması üzerine düşünmek belki de bugün “Post-truth çağ” söylemini nasıl kolayca benimsediğimizin de bir açıklamasını da verebilir. “Hakikate Sadakatin” Unutuluşu Oysaki Arendt bu durumdan çıkışın yolunu hakikati tamamen yadsıyarak değil, bizi başka türden bir hakikati çağırmaya davet ederek; sadakat kavramının başka türden bir kullanımına belli belirsiz de olsa yönelterek sunuyordu. Zira Arendt’e göre Eichmann’ın savunmasında ortaya koymuş olduğu ve insanları bir ceset haline getiren (Kadavergehorsam) itaate dönüşmüş bir sadakatten kurtarabilecek bir şey vardır: Tıpkı Eichmann davasında olduğu gibi geçmişin hatırlanması, ya da geçmişe ve onun hakikatine duyulan sadakat. Panzehir Olarak Sadakat Daha önce Arendt’in bakış açısına yerleşerek sadakatin hem zehir, hem de panzehir olabilen ikili bir anlamının olabileceğini belirtmiştik. Burada belki de panzehir, bellek ve hatırlama ile olan ilişkisinde “sadakat”tir. Ve Sponville’in “iyi sadakat” olarak belirlediği içeriğe bugün en uygun düşebilecek sadakat biçimi, geçmişin ve onun hakikatinin hatırlanmasıdır. Kaldı ki sadakatin kavram olarak doğası da aslında buna uygundur. Dikkat edilirse sadakat kavramından söz ederken aslında gizil bir zamandan bahsetmekteyiz ve bu gizil zaman genellikle geçmiştir. Sadakat, önceden verili yahut bilinen birşeye dair olacağından yapısı gereği geçmişten gelip şimdiki zamanda duyumsanan ve hissedilen bir süreci içerir. Bu anlamda onun bellek ve hatırlama ile ilişkisi vardır; fakat sadakati geçmişe duyulan bir hayranlık, bir fanatizm, saplantılı bir nostalji, otantik bir köklere dönüş arzusu olarak tanımlamak da onun anlamını daraltmak olacaktır. Sadakatin geçmişle ilişkisini düşünürken daha çok geçmişte yapılan bir iyiliği unutmamak, geçmiş zamanda verdiğimiz bir sözü tutmak, geçmişteki ilişkilerle aynı tarzda bütünleşilmese bile onlara vefa duymak söz konusudur; veyahut yapılan adaletsizliklere ses çıkarmak söz konusu olduğunda sadakatli ya da sadakatsiz oluruz; o halde sadakat, onu hatırlayacak bir eyleme, bir anımsamaya, yani bir belleğe ihtiyaç duyar. Bellek ile bir hatırlama işlemi gerçekleştiğinde geçmiş zamanı kapsayan bir düşünümün içine yerleşiriz. Hatırlama, geçmişte ne olduğumuzu, neye 496 DEU Journal of GSSS, Vol: 24, Issue: 2 Sadakat Bir Erdem... ve nereye ait olduğumuzu, şimdimizi ve gelecekte ne olacağımızı da kapsayan geniş bir düşünümün ifadesidir. Geçmişin hakikatine sadakatle olayları yargılamak ise, geçmişi tekrar etmek değildir; geleceği, geçmişteki hakikate sadakatin üzerinden yeniden kurmak demektir. Herşeyden önce mutlak ve düşünümsüz bir sadakatin aksine bu tür bir sadakat, “yargı vererek” kurulan bir sadakattir. O halde sadakat bir erdem olacaksa bile onun, belleğin, hatırlamanın erdemi olabileceğini iddia edebiliriz. Unutmanın değil, belleğin ve hatırlamanın erdemi olarak sadakat, aynı zamanda ortak bir dünyayı yeniden inşa etmenin yollarını açabilir. Zira geçmişin hakikatine duyulan sadakat, geleceği ve gelecekteki eylemleri belirleme gücüne sahiptir. Geçmiş ve Gelecek Arasında adlı eserinde Arendt, sadece geleceğin değil, geçmişin de bir güç olarak görülmesi gerektiğini ifade etmiştir (Arendt, 2010, ss. 23- 27). Totaliter deneyimlerden hareketle Arendt, geçmişin ve geçmişin hakikatine sadakatin bir güç olarak tanımlanmasının, o güne değin hiç görülmedik ölçekte bir “yurtsuzluk” ve “köksüzlük” fenomeniyle karşı karşıya kalınması ile ilintili olduğunu belirtir. “Yurtsuzluk” (homelessness) ile karşı karşıya kalan bireylerin aidiyet duyguları yok olmuştur; “ köksüzlük” (rootlessness) fenomeni de insanların yaşantısını belirsiz kılarak anlam dünyasını ortadan kaldırmaktadır; bu durumda insanlar, diğerleriyle bağ kurabilecekleri ortak referanslarını kaybetmektedirler ki bu da dünyanın parçalanışı anlamına gelmektedir. Panzehir Olarak Sadakat Bu nedenle Arendt, bütün bu sorunlar karşısında “geçmişin gücü”ne yaslanarak insanların yeni bir dünya kurabileceklerini belirtir. Modern zaman anlayışının aksine geçmiş ve hatırlama Arendt için insanların geride bırakmaları gereken bir fazlalık, bir yük değildir; Arendt geçmişteki hakikatlere sadakatin, geçmiş olaylara yönelik tanıklıkların, geçmişle yüzleşmenin, insanı başlangıç noktasına doğru, ileriye, geleceğe götürdüğüne inanır. Böylece geçmişe sadakat, Arendt’in düşüncesinde kamusal alan içerisinde kolektif bir hatırlama mekânı yaratarak öznelerarası bir iletişimin olanağını sağladığı gibi, insanların yitip giden anlam dünyalarının yeniden biçimlenmesine de katkıda bulunur. İnsanların yeniden kuracakları bu ortak dünyaya sadece güvenlik, adalet vb. gibi temel gereksinimlere ilişkin talepler nedeniyle değil; yeni bir anlam dizgesi bulmak için de ihtiyaçları vardır. Geçmişle bu tür bir ilişkilenme aynı zamanda bize, bu dünyada başkalarıyla birlikte yaşadığımız gerçeğini gösterir ve sadece geçmişteki nesillerle değil, gelecekteki nesillerle de ortak bir dünyayı paylaştığımızı hatırlatarak bir sorumluluk yükler. SONUÇ Sadakat erdemini düşünürken onun etik içeriğini politik sorunlarla ilişkilendirmemiz gerektiği ya da onun salt etik bir kavram olarak ele alınamayacağı görülmektedir. Özellikle totalitarizmin yol açtığı felaketler, insanlığa “sadakat için sadakat”in yıkıcı sonuçlarını bütün ağırlığı ile göstermiştir; buna karşın hakikate ve birarada yaşamanın koşullarına ilişkin değer ve hatırlama pratiklerini içeren bir sadakat ise, bizi dünyaya karşı açık olmaya ve politik sorumluluğa götürür. Arendt’in analizleri göstermektedir ki sadakatin yeniden bir erdem olarak tesisi, onun politik içeriğinin yeniden ele alınmasından ve geçmişle ve hakikatle yeniden kurulacak bir ilişkiden geçmektedir. Ancak hemen belirtmeliyiz ki Arendt, geçmişe sadakatle 497 DEÜ SBE Dergisi, Cilt: 24, Sayı: 2 Duva Kaya, Ö. yaklaşmayı asla bir kimliğe ya da kapalı bir topluluğa/kültüre bağlanma olarak anlamaz. O halde köklerimize sadakatin ölçütü kimlikler ve aidiyetler değil, ortak yarattığımız bir geçmişin, şimdiyi kurabilecek potansiyelleri olmalıdır. Bu potansiyeller, Arendt’in deyimiyle “anlayan bir yüreğin yetisi” olan tahayyül yetisi sayesinde geliştirebilir (Berktay, 2012, s. 30). Yeni ve çoğulluğu temel alan bir dünyayı tahayyül etmek, geçmişin “sadık” izleyicileri olabilmekten geçmektedir. Elbette ki buradaki sadakatle izlemek, olaylara sorgulayıcı bir biçimde ve propagandayla kirletilmiş sahte hakikatlere de şüpheci bir gözle bakmayı gerektirmektedir. Geçmişi unutmaya, ya da geçmişe sadakatsizliğe teslim olmuş bir toplumdan “şimdi”sini düşünmesi ve geleceğini kurması, ve daha temel olarak da “kim” olduğu üzerine düşünmesi beklenemez. Arendt’in politika felsefesinden çıkardığımız bu sonuçlar, bu noktalarda felsefî antropolojik bir bakış açısına bağlanmaktadır. Ancak bunu başka bir çalışmanın konusu olarak bir kenara bırakıp sadece şunu söylemekle yetinelim: Sadakat eğer bizim için değerli bir şey olacaksa, en temel olarak bize “ kim” olduğumuz sorusunu sordurabilmeli, yani öncelikle benliğimize, “nereden gelip nereye gittiğimize” ve geçmiş deneyimlerimize ve belleğimize ilişkin olmalıdır. KAYNAKÇA Arendt, H. (1976). The Origins of Totalitarianism. San Diego, New York, London: A Harvest Book. Arendt, H. (1976). The Origins of Totalitarianism. San Diego, New York, London: A Harvest Book. Arendt, H., McCarthy, M (1995). Between Friends: The Correspondence of Hannah Arendt and Mary McCarthy, 1949-1975 (Ed: Carol Brightman), New York: Harcourt Brace Arendt, H., McCarthy, M (1995). Between Friends: The Correspondence of Hannah Arendt and Mary McCarthy, 1949-1975 (Ed: Carol Brightman), New York: Harcourt Brace Arendt, H.(2010). Geçmişle Gelecek Arasında, (çev. Bahadır Sina Şener), İstanbul: İletişim Yayınları. Arendt, Hannah (2012a). Totalitarizmin Kaynakları /2 Emperyalizm, (çev. Bahadır Sina Şener), İstanbul: İletişim Yayıncılık. Arendt, H. (2012b). Kötülüğün Sıradanlığı: Eichmann Kudüs’te (çev. Özge Çevik). İstanbul: Metis Yayınları Arendt, Hannah (2014). Totalitarizmin Kaynakları /3 Totalitarizm, (çev. İsmail Serin), İstanbul: İletişim Yayıncılık Arendt, H. (2018). Siyasette Yalan, (çev. İmge Oranlı, Berfu Şeker), İstanbul: Sel Yayıncılık. Berktay, F. (2012). Dünyayı Bugünde Sevmek: Hannah Arendt’in Politika Anlayışı, İstanbul: Metis Yayınları Dürr, T. (2009). “Gereksizlik: Arendt’in Dışlanma Üzerine Düşünceleri”, Doğumunun 100. Yılında Hannah Arendt (Haz: Sanem Yazıcıoğlu), İstanbul: Yapı Kredi Yayınları. 498 Sadakat Bir Erdem... DEU Journal of GSSS, Vol: 24, Issue: 2 Machiavelli, N. (1999). Prens (çev. N. Güvenç). İstanbul: Anahtar Kitaplar. Sponville A.C. (2004). Büyük Erdemler Risalesi, (çev: Işık Ergüden), İstanbul: İstanbul Bilgi Üniversitesi Yayınları. TDK Online Sözlük, https://sozluk.gov.tr/ TDK Online Sözlük, https://sozluk.gov.tr/ TDK Online Sözlük, https://sozluk.gov.tr/ Stanford Felsefe Ansiklopedisi, https://plato.stanford.edu/entries/loyalty/ Stanford Felsefe Ansiklopedisi, https://plato.stanford.edu/entries/loyalty/ 499
https://openalex.org/W3211667181	https://research.tue.nl/files/199962073/Schulpen_2022_2D_Mater._9_025016.pdf	English	null	Controlling transition metal atomic ordering in two-dimensional Mo<sub>1−x </sub>W <sub>x</sub> S<sub>2</sub> alloys	2D materials	2,022	cc-by	8,912	Document status and date: Published: 01/04/2022 Document status and date: Published: 01/04/2022 Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Document Version: Publisher’s PDF, also known as Version of Record (includes final page, issue and volume numbers) Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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Oct. 2024 2D Materials You may also like A molecular dynamics study of heterogeneous nucleation in generic liquid/substrate systems with positive lattice misfit Z Fan and H Men - Enhancement of open-circuit voltage in InGaP solar cells grown by solid source molecular beam epitaxy Ryuji Oshima, Yuki Nagato, Yoshinobu Okano et al. - Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al. - You may also like A molecular dynamics study of heterogeneous nucleation in generic liquid/substrate systems with positive lattice misfit Z Fan and H Men - Enhancement of open-circuit voltage in InGaP solar cells grown by solid source molecular beam epitaxy Ryuji Oshima, Yuki Nagato, Yoshinobu Okano et al. - Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al. - PAPER • OPEN ACCESS This content was downloaded from IP address 131.155.94.127 on 04/04/2022 at 13:36 PAPER • OPEN ACCESS You may also like Abstract The unique optical and electronic properties of two-dimensional transition metal dichalcogenide (2D TMDs) make them promising materials for applications in (opto-)electronics, catalysis and more. Specifically, alloys of 2D TMDs have broad potential applications owing to their composition-controlled properties. Several important challenges remain regarding controllable and scalable fabrication of these alloys, such as achieving control over their atomic ordering (i.e. clustering or random mixing of the transition metal atoms within the 2D layers). In this work, atomic layer deposition is used to synthesize the TMD alloy Mo1−xWxS2 with excellent composition control along the complete composition range 0 ⩽x ⩽1. Importantly, this composition control allows us to control the atomic ordering of the alloy from well-mixed to clustered while keeping the alloy composition fixed, as is confirmed directly through atomic-resolution high-angle annular dark-field scanning transmission electron micrography imaging. The control over atomic ordering leads to tuning of the bandgap, as is demonstrated using optical transmission spectroscopy. The relation between this tuning of the electronic structure and the atomic ordering of the alloy was further confirmed through ab-initio calculations. Furthermore, as the atomic ordering modulates from clustered to well-mixed, the typical MoS2 and WS2 A1g vibrational modes converge. Our results demonstrate that atomic ordering is an important parameter that can be tuned experimentally to finely tune the fundamental properties of 2D TMD alloys for specific applications. Controlling transition metal atomic ordering in two-dimensional Mo1−xWxS2 alloys eff J P M Schulpen1, Marcel A Verheijen1,2, Wilhelmus M M (Erwin) Kessels1, Vincent Vandalon1 1 Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands 2 Eurofins Materials Science BV, High Tech Campus, Eindhoven, The Netherlands ∗Author to whom any correspondence should be addressed. Supplementary material for this article is available online Controlling transition metal atomic ordering in two- dimensional Mo1−xWxS2 alloys Polariton condensate and Landau-Zener- Stückelberg interferometry transition in multilayer transition metal dichalcogenides C M Ekengoue, C Kenfack-Sadem, J E Danga et al - View the article online for updates and enhancements. https://doi.org/10.1088/2053-1583/ac54ef 2D Mater. 9 (2022) 025016 OPEN ACCESS RECEIVED 10 September 2021 REVISED 9 February 2022 ACCEPTED FOR PUBLICATION 14 February 2022 PUBLISHED 3 March 2022 Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 2.1.1. In-situ growth characterization g The growth of the Mo1−xWxS2 films by ALD was characterized in-situ by spectroscopic ellipsometry (SE). Figure 2(a) shows the deposited film thickness per ALD cycle (GPC) as a function of the cycle frac- tion n/(n + m). The GPC of the pure WS2 and MoS2 and films are determined to be 0.08 and 0.13 nm respectively, in line with our previously reported characterization of these ALD processes [31, 33]. The GPC of the alloy increases linearly with the molyb- denum content, which is in line with the rule of mix- tures for alloys [35]: GPCalloy = n n + m × GPCMoS2 + m n + m × GPCWS2, (1) (1) demonstrating well-behaved alloying behavior free from the non-idealities commonly observed when depositing alloys by ALD [35]. 2.1. Control over alloy composition y p A series of six Mo1−xWxS2 samples was prepared using supercycle fractions of 0.0 (pure WS2), 0.2, 0.4, 0.6, 0.8 and 1.0 (pure MoS2), each for a total of 50 ALD cycles. We first investigate the growth and chem- ical composition of the alloys, after which we charac- terize their composition-dependent vibrational spec- trum and electronic structure. In this work, we introduce a plasma-enhanced ALD (PE-ALD) process for one-step deposition of Mo1−xWxS2 alloys at a low substrate temperature of 350 ◦C without the need for post-deposition sul- furization. Using this process, we demonstrate fine control over the alloy composition x by manipulat- ing the relative number of MoS2 and WS2 deposition cycles in a supercycle scheme, which is an established method of growing alloys of e.g. oxides by ALD [9]. Furthermore, we demonstrate for the first time con- trol over the atomic ordering within the Mo1−xWxS2 layers, which is confirmed directly through high- angle annular dark-field scanning transmission elec- tron micrography (HAADF-STEM). The control over the atomic ordering of the alloys is achieved by manipulating the supercycle period of the ALD pro- cess (i.e. the total number of ALD cycles per super- cycle). Beyond HAADF-STEM imaging, we report the effects of atomic ordering on the electronic struc- ture and phonon spectrum of the alloy, which are probed through optical transmission spectroscopy and Raman spectroscopy respectively. Finally, dens- ity functional theory (DFT) calculations are carried out to provide fundamental insight into how the elec- tronic structure of the alloys is modulated by their atomic ordering. 1. Introduction The cycle fraction n/(n + m) determines the composition x of the alloy, whereas the number of cycles per supercycle n + m (i.e. the supercycle length) is exploited to control the atomic ordering of the alloy. The total number of cycles N × (n + m) determines the film thickness. Atomic layer deposition (ALD) is a thin film deposition technique that exploits self-limiting sur- face reactions of vapor-phase precursors to achieve sub-nanometer growth control at low deposition temperatures (<450 ◦C). While direct ALD growth of pure TMDs has been reported [31–33], efforts on ALD of TMD alloys have focused on high- temperature sulfurization of metal (oxide) parent films, with the parent film synthesized by ALD [34]. While this approach allows a graded compos- ition profile of the alloy [34], the high-temperature sulfurization step nevertheless diminishes the low- temperature benefit of ALD, such that there remains a need for a fully low-temperature ALD synthesis process for better control over the composition and atomic ordering of the TMD alloys. ALD cycles followed by m WS2 ALD cycles are repeated, as illustrated in figure 1. The supercycle fraction n / (n + m) determines the composition of the alloy (the Mo/W ratio), whereas the supercycle length n + m determines the atomic ordering of the alloy. The total number of ALD cycles N × (n + m) determines the thickness of the deposited film. 1. Introduction nanoelectronics [5], versatile nanophotonics [6], and highly efficient photovoltaics [7]. Two-dimensional (2D) materials consist of molecu- lar layers whose mutual interaction is weak compared to the bonding within the layers [1], such that these materials can be thinned down to a single molecu- lar layer while maintaining predictable properties [2] which often include interesting physical phenom- ena induced by quantum confinement [3, 4]. Trans- ition metal dichalcogenides (TMDs) are a particularly important group of 2D materials since many of them, including the well-studied materials MoS2 and WS2, are semiconductors. This makes them interesting for a wide array of applications such as ultimately-scaled Alloying of semiconducting 2D TMDs further expands the possible applications of these materi- als [8]. For example, their electronic bandgap can be tuned continuously over a wide energy range both through chalcogen substitution [9–17] and through metal substitution [18–20]. Furthermore, alloying can improve the quality of TMD films, such as by reducing the density of chalcogen vacancy defects [21]. Important challenges remain in achieving con- trollable and scalable synthesis of TMD alloys. Tech- niques such as chemical vapor deposition (CVD) and © 2022 The Author(s). Published by IOP Publishing Ltd 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 1. The ALD supercycle scheme used for growth of Mo1−xWxS2 alloys by alternating ALD cycles of MoS2 and WS2. The cycle fraction n/(n + m) determines the composition x of the alloy, whereas the number of cycles per supercycle n + m (i.e. the supercycle length) is exploited to control the atomic ordering of the alloy. The total number of cycles N × (n + m) determines the film thickness. chemical vapor transport (CVT), which are known to produce high-quality samples of pure TMDs [22–24], are being used to synthesize such alloys [25–27]. However, control over the growth is usually limited when using these techniques. For instance, it has not been possible to control the atomic ordering of the alloys (i.e. well-mixed or clustered) using these tech- niques [8]. Instead, the alloys grown by CVD and CVT invariably exhibit random mixing of the con- stituents, likely due to strong surface diffusion of atoms during synthesis owing to the inherently high processing temperatures of these techniques [28–30]. Figure 1. The ALD supercycle scheme used for growth of Mo1−xWxS2 alloys by alternating ALD cycles of MoS2 and WS2. 2.1.2. Chemical composition On the same set of samples, the alloy composition x and stoichiometry (defined as the S/(Mo + W) ratio) as a function of the supercycle fraction n/(n + m) Mo1−xWxS2 alloys were grown by ALD using a scheme whereby supercycles consisting of n MoS2 2 2D Mater. 9 (2022) 025016 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 2. Impact of the composition x (controlled through the ALD cycle fraction n/(n + m)) of the Mo1−xWxS2 films on their (a) growth per cycle measured by in-situ SE, (b) elemental composition measured by XPS, (c) vibrational spectrum measured by Raman spectroscopy, (d) electronic structure measured by optical extinction spectroscopy. The insets in (d) show the positions of the absorption peaks as a function of alloy composition, as well as the difference between the alloy spectrum and a superposed MoS2 + WS2 spectrum. Spectra in (c) and (d) have been offset vertically for legibility. Figure 2. Impact of the composition x (controlled through the ALD cycle fraction n/(n + m)) of the Mo1−xWxS2 films on their (a) growth per cycle measured by in-situ SE, (b) elemental composition measured by XPS, (c) vibrational spectrum measured by Raman spectroscopy, (d) electronic structure measured by optical extinction spectroscopy. The insets in (d) show the positions of the absorption peaks as a function of alloy composition, as well as the difference between the alloy spectrum and a superposed MoS2 + WS2 spectrum. Spectra in (c) and (d) have been offset vertically for legibility. salloy = n n + m × sMoS2 + m n + m × sWS2, were measured using x-ray photoelectron spectro- scopy (XPS) and are shown in figure 2(b). The trend in alloy composition x closely follows the rule of mix- tures given by (3) where sMoS2 and sWS2 are the stoichiometries of the pure MoS2 and WS2 films respectively. The stoi- chiometry of the alloys as a function of the cycle fraction is well described by equation (3), again con- firming the well-behaved growth of the Mo1−xWxS2 alloys over the complete composition range 0 ⩽x ⩽1. x = [Mo] [Mo] + [W] = n n+m × GPCat,Mo n n+m × GPCat,Mo + m n+m × GPCat,W , (2) (2) where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. 2.1.2. Chemical composition By fitting equation (2) to the data, the atomic GPC ratio between Mo and W is found to be 1.67 ± 0.03. This value is within experimental error of the GPC ratio found from the ellipsometry data, indicating that the trend in film thickness as a function of the cycle fraction can be attributed to the difference between the number of Mo and W atoms deposited per ALD cycle. As also shown in figure 2(b), the deposited alloys are approxim- ately stoichiometric (sulfur to metal ratio ≈2), with a trend towards over-stoichiometry (excess sul- fur) for tungsten-rich films. Analogously to equation (1), a rule of mixtures can be formulated for the stoichiometry: where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. where the GPCat,Mo is the number of Mo atoms deposited per MoS2 ALD cycle, and GPCat,W the number of W atoms deposited per WS2 ALD cycle. By fitting equation (2) to the data, the atomic GPC ratio between Mo and W is found to be 1.67 ± 0.03. This value is within experimental error of the GPC ratio found from the ellipsometry data, indicating that the trend in film thickness as a function of the cycle fraction can be attributed to the difference between the number of Mo and W atoms deposited per ALD cycle. As also shown in figure 2(b), the deposited alloys are approxim- ately stoichiometric (sulfur to metal ratio ≈2), with a trend towards over-stoichiometry (excess sul- fur) for tungsten-rich films. Analogously to equation (1), a rule of mixtures can be formulated for the stoichiometry: 2.1.3. Crystallinity and vibrational spectrum 2.1.3. Crystallinity and vibrational spectrum The impact of the alloy composition on the crystallin- ity and vibrational spectrum of Mo1−xWxS2 alloys was studied by Raman spectroscopy: the spectra are shown in figure 2(c). In the spectrum of the pure MoS2 film, two dominant peaks at 384 and 409 cm−1 are observed which can be identified as the in-plane E12g mode and out-of-plane A1g mode respectively [36]. Peaks corresponding to the same vibrational modes are observed in the spectrum of the pure WS2 films at 356 and 420 cm−1 [37]. It should be noted that the E12g peak of WS2 overlaps with its 2LA(M) mode, which dominates when using 514 nm excita- tion light on pure WS2 [38, 39]. For simplicity, we nevertheless refer to the peak complex at 356 cm−1 as the E12g peak. The E12g and A1g peaks of MoS2 and 3 3 2D Mater. 9 (2022) 025016 2D Mater. 9 (2022) 025016 J J P M Schulpen et al WS2 persist at all alloy fractions 0 < x < 1, indicat- ing a polycrystalline structure of the alloys along the complete composition range. are not independent. Since our deposition process for Mo1−xWxS2 shows no such non-ideal behavior, it is a very interesting case study into the isolated effects of supercycle length at a fixed alloy composition. Fur- thermore, since it takes approximately 15 ALD cycles to grow a closed monolayer film with our process, a short supercycle (<10 cycles) process could be used to tune the atomic ordering within the individual molecular layers of the alloy without changing their composition. As a function of the alloy fraction x, the MoS2- like and WS2-like E12g peaks remain spectrally sep- arated and their relative intensities scale with the alloy fraction, i.e. they exhibit two-mode beha- vior [40]. Similarly, though the spectral proxim- ity of the A1g modes makes their deconvolution more challenging, the asymmetric lineshape of the A1g complex at intermediate alloy fractions sug- gests two-mode behavior for this mode as well. The same behavior is observed from alloys deposited by CVT [27]. To confirm that the supercycle length only influ- ences the atomic ordering of the Mo1−xWxS2 alloys and not their composition, XPS and Rutherford backscattering spectroscopy (RBS) measurements were carried out. From these measurements, it was confirmed that the relative change in alloy fraction ∆x/x was less than 3% for supercycle lengths ranging from 2 to 48 cycles. 2.1.4. Electronic structure To study the evolution of the electronic structure of the alloys as a function of their composition, optical transmission measurements were carried out: the spectra are shown figure 2(d). In the spectrum of pure MoS2, two extinction peaks are seen around 1.86 and 2.02 eV, which can be identified as the fundamental electronic transitions of the A exciton and B exciton [41–43]. Analogous peaks are observed in the spec- trum of pure WS2 around 2.01 and 2.38 eV, which is in line with literature values for the A and B exciton energies of this material [41–43]. 2.2.1. HAADF-STEM imaging To directly study the atomic ordering of the ALD- grown Mo1−xWxS2 films, HAADF-STEM imaging was performed on two sub-monolayer samples grown using different supercycle lengths. The first was made through a single supercycle of ten ALD cycles, i.e. five MoS2 cycles followed by five WS2 cycles. The second was made through five supercycles of two cycles each, i.e. 5 × (1 MoS2 cycle + 1 WS2 cycle), again for a total of ten ALD cycles. In the atomic-resolution STEM micrographs (figures 3(a) and (b)), Mo and W atoms can be distinguished by their Z-contrast: the heavier W atoms appear brighter than the lighter Mo atoms, while S atoms are not visible in HAADF-STEM mode. The spectra of intermediate alloys each exhibit two distinct extinction peaks in the spectral region between 1.8 and 2.5 eV, analogous to the A and B excitonic peaks of pure MoS2 and WS2. Further- more, the positions of these peaks vary smoothly with the alloy composition. This behavior signi- fies good mixing of the alloy’s constituents, since any significant phase separation in the alloy would result in a spectrum that resembles a superposi- tion of the spectra of the constituents, which is not observed. Additionally, the shifts of the excitonic peaks as a function of alloy composition follow a quadratic bowing trend, in line with theoretical calculations [20]. The hexagonal crystal structure of MoS2 and WS2 is clearly visible in the STEM micrographs, dir- ectly confirming the polycrystallinity of the depos- ited alloys: the crystal (grain) sizes are approxim- ately 5–10 nm. Comparing the STEM images of the long-supercycle sample (figure 3(a)) and the short- supercycle sample (figure 3(b)) shows a clear dif- ference in atomic ordering of the alloys. The long- supercycle sample exhibits clusters of Mo atoms which are bordered by W atoms. The short-supercycle sample does not exhibit such clustering of Mo and W atoms: instead the Mo and W atoms appear to be ran- domly distributed in the alloy. 2.2. Control over atomic ordering The longer supercycle length results in clustering of Mo and W while the shorter supercycle length produces a well-mixed alloy, as also illustrated by the TEM brightness profiles (c) and (d). Results of stochastic simulations of the film growth (e) and (f) reproduce the observed differences in atomic ordering, indicating that the supercycle length together with the preferential edge growth of the material play a large role in determining the atomic ordering. in the supplementary information. The result for long supercycles is the formation of 2D core/shell-like nan- oflakes with MoS2 cores and WS2 shells. Conversely, for the short-supercycle sample, the number of con- secutive MoS2 and WS2 cycles is too low for sig- nificant clusters of MoS2 or WS2 to form. To sup- port this proposed mechanism, simulations of the initial film growth were conducted using a simpli- fied 2D growth model based on sequential precursor adsorption with preferential adsorption at grain edge sites. The results of these simulations are shown in figures 3(e) and (f), and are in qualitative agreement with the observed differences in atomic ordering as a function of supercycle length. In summary, the atomic ordering within the individual Mo1−xWxS2 molecular layers can be controlled through the super- cycle length n + m of the ALD process. Further- more, the lateral grain sizes of the deposited films can be estimated from the HAADF-STEM micro- graphs (figures 3(a), (b), S1 and S2 available online at stacks.iop.org/TDM/9/025016/mmedia) to be typ- ically 5–10 nm. supercycle length of 24 cycles, four peaks are seen at frequencies corresponding to the E12g and A1g modes of bulk MoS2 and WS2 [37, 45], indicating that the sample resembles a heterostructure as is expected for long supercycles [46]. As the supercycle period decreases, the MoS2-like E12g vibration at 383 cm−1 softens by 3.0 ± 0.5 cm−1 along with a broadening of the peak. The WS2-like E12g peak does not shift as much as the MoS2-like E12g peak, though some softening is still observed. The A1g peaks are spectrally separated at a super- cycle period of 24 cycles, while for shorter super- cycle periods the two individual peaks cannot be resolved. This merging of the two A1g phonon peaks cannot be caused purely by a broadening of these peaks, since that would broaden the total two-peak complex, which is not observed. 2.2. Control over atomic ordering The ideal rule-of-mixtures alloying behavior of the described deposition process opens up interesting new possibilities of fine-tuning the growth of the alloy. As demonstrated in the previous section, the cycle fraction of the supercycle process controls the composition of the alloy. On the other hand, it is known that the supercycle period can be used to con- trol the ordering of an alloy: short supercycle periods produce a well-mixed film while long supercycle periods produce a nanolaminate film [35]. Usually [35], non-ideal growth characteristics such as het- eronucleation delays lead to changes in composition when the supercycle length is changed, such that the effects of the cycle fraction and the supercycle length Since the long-supercycle sample was grown by performing five MoS2 cycles followed by five WS2 cycles, the distribution of Mo and W atoms in this sample is consistent with MoS2 island growth dur- ing the first five cycles followed by edge-on growth of WS2 at the edges of the MoS2 islands in the last five ALD cycles. This growth mode is consistent with earlier reports of 2D TMD growth by ALD, where nucleation is followed by preferential edge-on growth [31, 33, 44]. This growth mechanism is also discussed 4 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 Figure 3. HAADF-STEM imaging of sub-monolayer samples of ALD-grown Mo1−xWxS2 with supercycle lengths of ten cycles (a) and two cycles (b), both for a total of ten ALD cycles. The scalebars indicate a size of 2 nm. The insets show local Fourier transforms, demonstrating the hexagonal crystal structure of both samples. The longer supercycle length results in clustering of Mo and W while the shorter supercycle length produces a well-mixed alloy, as also illustrated by the TEM brightness profiles (c) and (d). Results of stochastic simulations of the film growth (e) and (f) reproduce the observed differences in atomic ordering, indicating that the supercycle length together with the preferential edge growth of the material play a large role in determining the atomic ordering. Figure 3. HAADF-STEM imaging of sub-monolayer samples of ALD-grown Mo1−xWxS2 with supercycle lengths of ten cycles (a) and two cycles (b), both for a total of ten ALD cycles. The scalebars indicate a size of 2 nm. The insets show local Fourier transforms, demonstrating the hexagonal crystal structure of both samples. 2.2. Control over atomic ordering Hence, a frequency shift of one or both of the A1g peaks must be involved. Spectral deconvolution indicates the MoS2- like A1g mode stiffens by 3.5 ± 0.5 cm−1 while the WS2-like A1g mode softens by 2.0 ± 0.3 cm−1 as the supercycle length is shortened from 24 to 2 cycles. 2.2.2. Impact of atomic ordering on the vibrational structure The broadening of the two E12g peaks for short supercycle lengths can be understood as a consequence of the enhanced mixing of the alloy constituents, which reduces the crystalline order and relaxes the momentum selection rule on the Raman scattering process [47]. On the other hand, the soften- ing of the E12g modes as a function of the supercycle period cannot easily be explained on the basis of known effects. Firstly, strain [48–50] of the alloy film is not expected to be the cause of these shifts The influence of the atomic ordering of the Mo1−xWxS2 alloys on their vibrational structure was investigated by Raman spectroscopy. Few-layer (6–8 L) samples were grown using supercycle lengths of 2, 6, 10, 16 and 24 cycles at a constant MoS2 cycle fraction n/(n + m) of 0.5 and a total number of 48 ALD cycles for each sample. The Raman spectra are shown in figure 4. For the sample with the longest 5 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 4. Effect of atomic ordering (i.e. supercycle length) on (a) the vibrational spectrum (Raman spectroscopy) and (b) the electronic structure (optical extinction spectroscopy) of the Mo1−xWxS2 alloys. Insets in (a) show the peak positions of the Raman peaks as a function of the supercycle length. Insets in (b) show the spectral positions of the extinction peaks A and B, and an overlay of the spectrum of the longest-supercycle sample with a superposition of the pure MoS2 and WS2 spectra, indicating the heterostructure-like nature of samples grown using long supercycle lengths. Spectra are offset vertically for legibility. Figure 4. Effect of atomic ordering (i.e. supercycle length) on (a) the vibrational spectrum (Raman spectroscopy) and (b) the electronic structure (optical extinction spectroscopy) of the Mo1−xWxS2 alloys. Insets in (a) show the peak positions of the Raman peaks as a function of the supercycle length. Insets in (b) show the spectral positions of the extinction peaks A and B, and an overlay of the spectrum of the longest-supercycle sample with a superposition of the pure MoS2 and WS2 spectra, indicating the heterostructure-like nature of samples grown using long supercycle lengths. Spectra are offset vertically for legibility. indistinguishable from a superposition of the spectra of pure MoS2 and WS2 (see inset figure 4(a)). 2.2.2. Impact of atomic ordering on the vibrational structure This is expected since for very long supercycles, the depos- ited film resembles a heterostructure of MoS2 and WS2, such that the extinction spectrum contains four peaks (A and B of MoS2 and WS2). Due to their spectral broadness, not all of these four peaks can be resolved, and in the following we deconvolve the extinction spectra using a two-peak model. We will focus our attention on the interpretation of the alloy- like samples made using short supercycles (i.e. up to ten cycles) and not on the heterostructure-like samples made using longer supercycles, for which the interpretation of a two-peak deconvolution is not straightforward. since MoS2 and WS2 have identical crystal structures with in-plane lattice constants differing by only 0.2% [51]. Furthermore, opposite strain on the constitu- ents MoS2 and WS2 would lead to opposite shifts in the two E12g peaks, but parallel shifts are observed. Secondly, an increase in sulfur vacancy concentration [52] can be ruled out as the cause of the observed E12g and A1g shifts since the stoichiometry was found to remain constant at 2.0 ± 0.1 between supercycle periods of 2 and 12 cycles from RBS analysis. Addi- tionally, an increase in sulfur vacancy concentration would shift the E12g frequencies more than the A1g frequencies [52], which we do not observe. Having ruled out these alternative causes, we conclude that the E12g and A1g Raman peaks of Mo1−xWxS2 are sensitive to changes in atomic order- ing of the alloy, making Raman spectroscopy an accessible tool for probing the atomic ordering of 2D alloys. Additionally, these findings indicate that the control over atomic ordering which was demon- strated for sub-monolayer films (figure 3) extends to the few layer (6–8 L) regime. The B-peak is seen to shift to higher energy as the supercycle length is shortened (i.e. towards ran- dom atomic ordering), while the A-peak shows no significant shift. Between supercycle lengths of two and ten cycles, the B-peak has shifted by approxim- ately 20 meV. Such behavior may be related to small changes in the alloy composition (Mo/W ratio) as a function of the supercycle length [20], or instead the atomic ordering could have a direct effect on the electronic structure of the alloy. To explore the lat- ter option, DFT electronic structure calculations were carried out. For these calculations, the atomic order- ing is quantified using the order parameter J [29]: 2.2.3. 2.2.2. Impact of atomic ordering on the vibrational structure Impact of atomic ordering on electronic structure The effect of atomic ordering on the electronic structure of the Mo1−xWxS2 films was investigated by optical transmission spectroscopy. Samples were grown using supercycle lengths of 2, 6, 10, 16, 24 and 48 cycles on transparent quartz substrates. The cycle fraction n/(n + m) was again kept fixed at 0.5 (result- ing in alloy composition x = 0.6), and the total num- ber of ALD cycles was 48 for each sample. The extinc- tion spectra (figure 4(b)) show two peaks around 1.85 and 2.1 eV, labeled A and B. The spectrum of the alloy grown with the longest supercycle (48 cycles) is J = Psample Prandom = Psample x2 + (1 −x)2 , (4) (4) where Psample is the fraction of neighboring identical metal atoms (Mo–Mo or W–W) of the sample and Prandom is the fraction of neighboring identical metal atoms when the alloy is randomly mixed. The value 6 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 J J P M Schulpen et al Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. A peak than of the B peak, which was not observed experimentally. Hence, our calculations indicate that atomic ordering indeed could impact the electronic structure of Mo1−xWxS2, though further study is required to experimentally distinguish the effects of atomic ordering on the electronic structure from the effects of small changes in alloy composition. 3. Conclusions We have shown that the 2D TMD alloy Mo1−xWxS2 can be grown with excellent composition control using a supercycle-based ALD process. This well- behaved alloy growth allows for control over the ordering of the transition metal atoms within the individual molecular layers by tuning the super- cycle length of the deposition process. This control over atomic ordering was confirmed directly through HAADF-STEM imaging, and the atomic ordering was shown to tune the vibrational spectrum and elec- tronic structure of the alloys. Our results indicate that atomic ordering of 2D semiconductors alloys can be experimentally manipulated during synthesis to finely tune the opto-electronic properties of these materials for specific applications. Furthermore, our results indicate that Raman spectroscopy is sensit- ive to changes in atomic ordering of 2D TMD alloys, making it an accessible method of probing the atomic ordering of these materials. Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. Figure 5. Calculated fundamental electronic transition energies corresponding to the A bottom and B top excitons of Mo1−xWxS2 as a function of atomic ordering. Plotted datapoints are averaged values over five unique configurations per order parameter, and the error bars denote their standard deviation. of J describes the atomic ordering of the alloy: J = 1 describes random mixing, J > 1 describes clustering of Mo and W (where larger J means larger clusters) and J < 1 describes ‘anti-clustering’, i.e. the preferential neighboring of non-identical metal atoms (Mo–W), tending towards a checkerboard pattern. 4. Methods Typical values of J for Mo1−xWxS2 alloys synthes- ized at high temperatures are around 1.0, i.e. these alloys exhibit random mixing of the transition metal atoms [29]. On the other hand, the value of J for our ALD-grown Mo1−xWxS2 using a supercycle length of ten cycles can be estimated from STEM (figure 3) to be approximately 1.8. However, since the unit cell size needed to perform DFT calculations for such large clusters would lead to prohibitively high computa- tional cost, we limit our calculations to a supercell of 5 × 5 × 1 unit cells, such that a maximum value of J of 1.41 can be simulated. The supercell configura- tions are shown in the supplementary information. The calculated electronic transition energies A and B as a function of the order parameter J are shown in figure 5. 4.1. PE-ALD process 4.1. PE-ALD process PE-ALD of Mo1−xWxS2 films was performed using an Oxford Instruments FlexAL-2 reactor equipped with a remote inductively coupled plasma source. Silicon wafers with 450 nm thermally grown oxide were used as substrates. The substrate table heater was kept at 450 ◦C during processing, resulting in a substrate temperature of 350 ◦C; a 20 min wait step was performed prior to deposition to ensure thermal equilibration. The ALD processes used for the MoS2 and WS2 cycles are described in detail in previous work [31, 33]. As precursors, Mo(NtBu)2(NMe2)2 (98%, Sigma Aldrich) and W(NtBu)2(NMe2)2 (99%, Strem Chemicals) were used for MoS2 and WS2 respectively. The precursors were kept in stainless steel canisters which were heated to 50 ◦C, and precursor deliv- ery into the reaction chamber was facilitated with a 50 sccm argon bubbling flow. Precursors were dosed for 6 s, followed by a 10 s purge of the reactor cham- ber with 100 sccm argon. A plasma of 10 sccm H2S and 40 sccm Ar was used as the coreactant in both the MoS2 and the WS2 processes. Plasma exposure was performed at a power of 100 W for 30 s at a pressure of approximately 6 mTorr. A subsequent 10 s purge of the reactor chamber with 100 sccm argon completes the ALD cycle. Since anti-clustering is not observed experiment- ally, the results for J ⩾1 are most relevant for com- parison to experiment. Between J = 1 (random mix- ing) and J = 1.41 (largest clustering), the calculated A transition energy redshifts by 11 meV, whereas the calculated B transition energy redshifts by 5 meV. Linear extrapolation to the appropriate J-value of 1.8 for Mo1−xWxS2 alloys deposited using a supercycle length of ten cycles yields redshifts of 22 and 10 meV for the A and B transitions respectively. These val- ues are of the same magnitude as the experiment- ally observed shift in the optical extinction spectrum, although the calculations predict a larger shift of the 7 2D Mater. 9 (2022) 025016 J J P M Schulpen et al 2D Mater. 9 (2022) 025016 to a residual force tolerance of 10−4 eV Å−1 using a converged plane-wave cut-off energy (400 eV) and k- space sampling (3 × 3 × 1 Monkhorst–Pack mesh). Effective electronic band structures were obtained through the unfolding procedure [59]. 4.5. Atomic ordering stochastic simulation The data that support the findings of this study are available upon reasonable request from the authors. The simulation starts with an empty hexagonal grid. In a loop, a random grid point is chosen. If this grid point is empty, there is a chance of 0.001 that a ‘MoS2 unit cell’ is placed there (simulating nucleation). If the empty grid point is next to a grid point that is already filled, this chance is 1 (simulating preferential edge growth). This loop is repeated N times (simulat- ing a full ALD cycle). By alternating such virtual ALD cycles of MoS2 and WS2, the full supercycle process of Mo1−xWxS2 alloy deposition is simulated. 4.3. Elemental composition measurement 4.3. Elemental composition measurement The relative elemental composition of the Mo1−xWxS2 films was determined through XPS using a Thermo Scientific K-alpha spectrometer with an aluminum K-α (1486.6 eV) radiation source. In the experiments on atomic ordering, the obtained atomic abundances were corrected for the exponential atten- uation of the emitted photoelectrons with depth. RBS was used to determine the absolute elemental com- position of selected samples as additional validation. RBS measurements were performed with a 2 MeV 4He beam and a detector at 170◦scattering angle. 4.4. Raman and PL measurements Raman scattering spectroscopy and photolumines- cence (PL) spectroscopy measurements were per- formed with a Renishaw InVia Raman microscope equipped with a 514.5 nm laser and a CCD detector. Raman peak positions corresponding to the A1g modes were extracted by deconvolution of the spec- tra using a five-peak Voigt model, which is a strategy similar to that used in the literature [32]. Acknowledgments This work was partially funded by the Dutch Research Council (NWO) through the Gravitation program ‘Research Centre for Integrated Nanophotonics’ (No. 024.002.033). This work has also been supported by the European Research Council (Grant Agreement No. 648787-ALD of 2DTMDs). The authors acknow- ledge C O van Bommel, M G Dijstelbloem, C A A van Helvoirt, B Krishnamoorthy and J J A Zeebregts for their technical assistance, and thank S ter Huurne for performing UV-vis absorption measurements. Sol- liance and the Dutch province of Noord-Brabant are acknowledged for funding the TEM facility. 4.6. Ab-initio electronic structure calculations DFT calculations were carried out with the projector- augmented-wave [55] framework as implemented in the VASP software [56] with the exchange and correl- ation contributions to the electronic energy described semi-locally by the PBE functionals [57]. Van der Waals interactions were modeled using the DFT-D3 method of Grimme [58]. Supercells of 5 × 5 × 1 primitive cells were used with J-values between 0.64 and 1.41, which were geometrically optimized down 4.1. PE-ALD process The elec- tronic band-to-band transition energies were derived from the calculated conduction band minimum and valence band maximum at the K-point of the Bril- louin zone (where the A and B excitons are loc- ated). For each degree of atomic ordering, five unique supercells were generated and the obtained electronic transition energies were averaged over these struc- tures. The band structures were calculated at the PBE level and exciton binding was not included, such that a systematic offset is expected between the DFT-calculated band-to-band transition energies and the experimentally measured absorption peak positions. 4.2. Film thickness measurement Film growth was monitored by SE using a J.A. Wool- lam M-2000 ellipsometer in the spectral range from 1.25–4 eV. The film thickness and optical constants were determined by parametrizing the SE data using a B-spline [53]. 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https://openalex.org/W4315783725	https://ieeexplore.ieee.org/ielx7/6287639/10005208/10015756.pdf	English	null	Personal Learning Environments: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform	IEEE access	2,023	cc-by	13,975	SARAH ALSERHAN 1, TURKI MESFER ALQAHTANI 2, NORAFFANDY YAHAYA 1, WALEED MUGAHED AL-RAHMI1, AND HASSAN ABUHASSNA 1 1Faculty of Social Sciences and Humanities, School of Education, Universiti Teknologi Malaysia (UTM), Skudai, Johor 81310, Malaysia 2Department of Instructional Technology, Jazan University, Jazan 45142, Saudi Arabia Corresponding author: Sarah Alserhan (sarah.alsarhan@hotmail.com) SARAH ALSERHAN 1, TURKI MESFER ALQAHTANI 2, NORAFFANDY YAHAYA 1, WALEED MUGAHED AL-RAHMI1, AND HASSAN ABUHASSNA 1 1Faculty of Social Sciences and Humanities, School of Education, Universiti Teknologi Malaysia (UTM), Skudai, Johor 81310, Malaysia 2Department of Instructional Technology, Jazan University, Jazan 45142, Saudi Arabia Corresponding author: Sarah Alserhan (sarah.alsarhan@hotmail.com) ABSTRACT A personal learning environment (PLE) is known as a crucial support for educators who lead learners through the process of collection, creation, and organization of personalized learning tools. In this manner, the learner can interpret a variety of new tools in their own interest, which makes the learning process easier. The PLE approach represents a considerable movement away from traditional learning, where learners are considered consumers of information through isolated channels, particularly learning management systems (LMSs), to a model where learners draw significant connections from numerous resources that they choose. Thus, educational settings have implemented LMSs fully into their respective learning contexts. In this sense, LMS is identified as a learning platform that helps learners and educators submit assignments, share ideas, and communicate through web-based systems with numerous benefits. Under these circumstances, self-regulation is addressed as a significant component that explains how learners build and manage PLEs and come up with more choices; they take ownership of their own learning and enhance self-regulated learning (SRL) practices. On this occasion, there is a belief that teachers can utilize LMSs to shift from passive to active learning and to improve self-reflection (SR). Therefore, considering all the above issues, the current study examines integrating a third-generation LMS to enhance learners’ SR. This study considered PLEs by utilizing Zimmerman’s SRL model to investigate the integration of the third- generation LMS. SR is applied in this study in the form of a pretest and posttest following the involvement of the PLE course, which was designed and applied during the COVID-19 pandemic. Finally, the experimental findings of the current study formulated a model of SR factors in PLEs through the LMS platform with partial least squares structural equation modeling (SEM) before and after the intervention. INDEX TERMS Personal learning environments, learning management systems, self-regulated learning, partial least squares structural equation modeling. INDEX TERMS Personal learning environments, learning management systems, self-regulated learning, partial least squares structural equation modeling. Personal Learning Environments: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform Personal Learning Environments: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 Received 9 December 2022, accepted 8 January 2023, date of publication 12 January 2023, date of current version 19 January 2023. Digital Object Identifier 10.1109/ACCESS.2023.3236504 II. LITERATURE REVIEW II. LITERATURE REVIEW The development and utilization of technologies in the infor- mation and communication sectors in educational contexts have led to various changes in the employed tools for teach- ing and learning progress [1]. In this context, LMSs play a crucial role by focusing on higher institutions. Accord- ing to Wesch, learners should not learn only in informal environments, in particular in thematic courses with tools that are not prepared and controlled by the institutions [12]. Therefore, to recognize these methods of learning, including the personalization features that are unique to our new digital age, PLEs have been announced. PLEs are defined as ways to make people learn for the rest of their lives. However, what occurs in them must be utilized with a consideration of the institution [13]. Conde et al. believe that PLEs facilitate learners’ learning progress by guiding learners to employ the significant tools that they require and not collecting them for a specific institutional context as an LMS does [2]. As a result, interactions in the eventual environment have a great effect on learning outcomes and student academic performance. Apart from the considered components, although an LMS balances the learner’s ability to acquire both formal and informal learning contexts [6], [7], educators face difficul- ties in tracking, adapting, and evaluating LMS effectiveness, along with adapting to various new personalized tools. In the same vein, Shaikh and Khoja consider that a lack of sup- port for PLEs makes learning environments ineffective [8]. Therefore, driven by the significant role of PLEs and LMSs, educators must be the most knowledgeable party, which is crucial for learners to develop a strong and multifunctional association between themselves and PLEs. From the perspective of Dabbagh and Kitsantas, the devel- opment of PLEs in the e-learning context through addressing learning control and personalization problems is regularly ignored in institutional LMSs [4]. An LMS is known as the most crucial method and has significant impacts on differ- ent purposes, such as the organization’s objectives, online training strategy, and desired outcomes [14]. As stated by Martindale and Dowdy, the LMS is considerably presented as a vital element to emphasize that learning has an indi- vidual aspect, as they believe that every learner is unique and has different capabilities, interests, and limitations [15]. II. LITERATURE REVIEW Therefore, it is obvious that learners need their own process of acquiring knowledge, which leads to the development of PLEs and the enhancement of SRL. Although LMSs were initially framed to offer a flexible framework for advanced learning pedagogies [15], it has been considered that LMSs focus on institutional broadcasting tools over learner learning tools. Under these circumstances, Dabbagh and Kitsantas state that SRL is recognized as a skill in which learners must comprehend how to set goals, determine what is important to reach the goals, and how to utilize the reached goals [4]. PLEs play a crucial role in assisting educators in guiding learners by gathering, making, and organizing personalized learning tools. Similarly, Moreillon believes that learners can understand and interpret fresh tools in their own interest to thus make the learning process easier [9]. However, learners may face several difficulties in the PLE context, such as managing the information presented to them due to a lack of customization of learning materials [10]. In this man- ner, a study conducted by Hartley shows that learners face issues when articulating or discussing complex issues, which PLEs may use to attract learners as a significant learning component [10]. Fueled by the crucial role of PLEs, sev- eral researchers have employed Zimmerman’s SRL in tech- nologically assisted learning, such as Web.2, social media applications, and LMSs, to transform institution-centered environments into learner-centered environments [3], [4]. In line with the significant role of PLEs, Dabbagh and Kitsan- tas hold that PLEs are a highly promising pedagogical theory for learning through using social media and learners’ self- regulating themselves [4]. In this context, several educators lack in one or both of these areas. A study by Schaffert and I. INTRODUCTION of technologies and learning management systems (LMSs), it is gradually moving toward personalized-based learning that seeks to improve self-regulated learning (SRL). This clearly shows that personal learning (PL) is a crucial approach to enhancing a learner’s capability to benefit from SRL. In this decade, the role of technology, particularly LMSs, has changed. An LMS serves various purposes, such as the organization of organizations, online learning and teaching techniques, and desired results. Moreover, it is critical to mention that an LMS is an individual aspect, as every learner The enhancement of modern learning techniques has been developed by adopting information and communication tech- nologies in the education system [1]. In this manner, the adop- tion of information has distorted the limitations of formal, informal, and nonformal teaching techniques and face-to-face and online education systems. Through the swift development The associate editor coordinating the review of this manuscript and approving it for publication was Chin-Feng Lai . 5464 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform is unique and has different capabilities, interests, and limi- tations. Therefore, it is obvious that learners need their own processes to seek knowledge. This process leads to the devel- opment of personal learning environments (PLEs) and SRL enhancement, which is the major purpose of this paper. Hilzensauer reveals that many teachers are not comfortable working with technology-driven equipment [11]. Therefore, this is a problematic issue because if educators are not privy to the skills needed to facilitate PLEs, then their students will not receive the maximum number of benefits. Thus, considering all the above issues, the current study examines integrating a third-generation LMS into PLEs to enhance learners’ self-regulation. A. PROBLEM STATEMENT The general concept of a learning management system (LMS) is recognized as an institutional platform that focuses only on learners [2]. From the perspective of [3], an LMS helps to extend the diversity of functionalities in a user-oriented con- text. On this occasion, several researchers claim that an LMS includes the combination of numerous services from a variety of sources to develop a customized learning experience in which the integration of a more personalized LMS tool is necessary [2], [4], [5]. Fueled by the significance of an LMS to offer personalized tools, Conde et al. believed that there are several issues with LMSs that provide an open environment in personalized learning, such as learners’ information tech- niques, idea and experience sharing networks, information sharing through management systems and identity techniques integrated via organizations, which are utilized in learning settings [2]. B. PLES AND LMSS This explanation, in the perspective of Panagiotidis, is iden- tified as a particularly designed system that covers many external tools and resources for developing a customized learning experience that can be accessed individually [23], which is consistent with the perspective of several researchers who believe that PLEs enable learners to engage with their peers, resources, and services in a broad context [24]. Apart from the significant role of PLEs in learning and engaging learners, PLEs have been challenging the traditional (LMSs. According to Ullrich et al., an LMS should be recognized as a suitable solution for institutions [24]. In this sense, PLEs should ideally aim to enhance learners’ cognitive abil- ities, redefine the pedagogical process, and integrate third- generation LMSs to design technology-enhanced practices and opportunities [11]. Therefore, Hicks and Sinkinson indi- cate that it is crucial to enhance PLEs, particularly because digital information is being developed. Thus, learners will be able to develop their own self-reflective and learning envi- ronments [25]; they will then need to develop and manage their PLEs through the required tools [26], [27]. According to Schwartz and Al-Rahmi et al., since components such as learning progress, technology, and resources are provided to help all learners and premiums, the outcome will be even Learning LMSs originated in the 1960s. During this time. A learning system named PLATO was developed at the University of Illinois. According to Mott, the system offered computer-based learning instruction [33]. Tradition- ally, LMSs were created to provide, manage, cover, and examine learning tasks in a formal learning context. Accord- ing to Mott [33], a new wave of systems is evolving to promote teaching and learning through new modes of col- laboration, information sharing, and social networking ser- vices. All of the developments in the educational and training ecosystem have exposed the conventional LMS to an increas- ing range of challenges. From the perspective of Westphal, the learning environment has been a transforming outcome of technology development and resource accessibility [34]. An LMS desires to cover itself to make the environment transformation and developmental necessities of learners and teachers more open, individualized, social, updated, analyt- ical, and accessible. Thus, in this sense, Broderick argues that to enable learners to complete prepared tasks, one must employ the instructional design factor to create a conducive instructional environment for learners [35]. B. PLES AND LMSS PLEs help the educator guide learners via the progress of gathering, making, and organizing personalized learning tools [9]. In this context, Hicks and Sinkinson claim that PLEs enable learners to utilize and customize their reason- able individual objectives to cover their learning through the management of the tools that they integrate, and they enable communication among learners [25]. The combination of all of these aspects creates a very successful learning process. However, learners face some difficulties managing the infor- mation presented to them, which may be due to a lack of customization of the learning materials [10]. Yilmaz suggests that PLEs play a significant role as an efficient approach because they easily manage the learning process [29]. In addi- tion, PLEs enable learners to hold their own personal, edu- cational records, in which the tool will grant mobility of educational records by empowering learners to maintain their learning space [25]. From the perspective of Ragupathi, PLEs make it possible to promote peer and independent learning [30]. Although education is driven by the role of PLEs, there are still many challenges and obstacles that educators must overcome when implementing PLEs in the classroom setting. Not all of the obstacles are equally shared among educa- tors, but some trends persist throughout the educational envi- ronment. A study conducted by Schaffert and Hilzensauer reveals that many teachers are not comfortable working with technology-driven equipment [11]. Burns claims that teachers can develop a positive, inspiring, and imaginative influence on learners by personalizing their teaching skills [31]. Counted as the major point of PLE research, Buchem et al. believe that the creation of customized platforms is not the purpose of the research conducted by focusing on obtaining the learners’ activities in terms of their employment of tech- nology for supporting their learning process [21]. In addition, Van Harmelen considers that PLE plays a significant role as a system that assists learners in managing and taking control over the method of learning by setting their targets and com- municating with other learners to fulfil their objectives [22]. A. PERSONAL LEARNING ENVIRONMENTSi PL is identified as one of the means of lifelong learning, which addresses the fact that students obtain information in different ways [16]. According to Schwartz, learners should be provided with the flexibility to enhance their various skills 5465 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform more interesting to students, which prompts deeper respon- sibility and improves the results [16], [28]. to critically analyze information [16]. In this manner, the idea of PLEs was initially examined by Olivier and Liber, who address PLEs as a crucial resource for e-learning and interactive learning environments (ILEs) [14]. In the same vein, Kulathuramaiyer and Maurer portray PLEs as a great help in managing information and the cognitive overload that comes with it [17]. Lifelong learners have been defined as having a persistent user interface to meet their needs of with the purpose of managing their learning career [14]. According to Taraghi and Ebner and Alalwan et al., in an envi- ronment where learners can integrate distributed resources, applications, and tools into a single platform, this can pro- vide individual learners with an acceptable circumstance to develop their specific needs in a study environment that allows people to interact within collaborative and distributed environments [18], [19]. In this situation, PLEs are known as an instructive approach that allows students to employ social media to obtain an experience of enhancing self- motivated learning in both formal and informal pedagogi- cal contexts [4]. Many researchers have conducted different studies and found that the PLE concept has implications for open-access online learning, learner-based guidance, self- direction, and self-direction issues [8], [20]. B. PLES AND LMSS Thus, according to Ellis, LMSs cover the following six key objectives that are in line with the objectives of this paper: i) centralize 5466 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform parts: (1) the main prediction module, which is the alpha-beta filter algorithm, and (2) the learning module, which is a feed- forward artificial neural network (FF-ANN). Additionally, a prediction method is employed in the model to forecast actual sensor values from noisy sensor readings. The model takes two inputs, temperature sensor and humidity sensor data. By incorporating the feed-forward backpropagation neural network into the innovative technique, prediction accuracy is greatly increased, and the root mean square error (RMSE) and mean absolute error are also decreased (MAE). The standard alpha-beta filter method and other algorithms, such as the Kalman filter, were used to assess the performance of the sug- gested model. The RMSE was 38.2%, while the MAE ranged from 35.1% to better forecast accuracy. When compared to conventional methods, the final proposed model’s perfor- mance results demonstrated improved performance [40]. and automate administration; ii) utilize self-service tools; iii) sharply set and transform learning content; iv) develop teaching techniques in a scalable web-based context; v) guide and lead portability; and vi) personalize content and accessi- ble knowledge sharing [36]. Exploring ever-larger amounts of data from educational contexts, such as LMSs, and creating computational tech- niques to better comprehend students’ actions and learning environments are the main goals of educational data mining. Numerous studies have been conducted on the learning pro- cess of students, and as a result, many widely used frame- works and theories that describe students’ learning behaviors have been developed. However, the most recent focus of study states that it is necessary to create effective models with ideas that can be put into practice to understand student learning practices and to influence students’ academic performance. By analyzing fine-grained data samples gathered by LMSs at the student level, existing studies examine various individ- ual, emotional, and social factors related to student learning behaviors, but they do not take into account the dynamics of learning behaviors and do not look at the full picture of students’ learning lives. B. PLES AND LMSS For instance, the majority of stud- ies on student learning behavior concentrate on particular courses and associated academic achievement but do not consider the fact that students usually attend many courses at once. Therefore, it may be difficult to develop effective teaching methods for students by using the implications and knowledge from a static understanding of students’ learning behavior in a single isolated course [37]. Furthermore, Khan et al. introduced a novel Multiple learn- ing to prediction algorithm model that employed three dis- tinct combinations of machine-learning techniques to raise the accuracy of the - filter algorithm. Instead of static settings, the parameters of and were optimized in dynamic situations. The deep extreme learning machine (DELM), the deep belief network (DBN), and the support vector machine (SVM) were chosen as the three different learning algorithms to be used in the suggested system. The final anticipated outcomes were then provided by these learned parameters after being taught using machine learning algorithms that were customized to the - filter method as a prediction module. The proposed approach produced results that were more accurate when compared to the standard alpha-beta filter algorithm [41]. A useful approach for discovering the links buried in edu- cational data and forecasting students’ academic success is educational data mining (EDM). EDM is the use of conven- tional DM techniques to address the issues that pertain to education. EDM refers to the application of DM techniques to educational data, such as student data, academic records, exam results, participation in class, and the frequency of questions raised by students. EDM has developed into a useful tool in recent years for predicting academic success, finding hidden patterns in educational data, and enhancing the learning and teaching environment [38]. III. RESEARCH MODEL AND HYPOTHESES DEVELOPMENT This research used Zimmerman’s model [49]. The model was used as a self-regulatory learning process system in three stages, namely, forethought, performance, and self-reflection (SR) [50]. Zimmerman considered that the forethought stage process is used in PL for learning efforts (LEs) and is meant to boost learning. He also identified the performance phase process that is used to help in the self-monitoring (SM) of one’s performance during LEs [49]. Finally, in Zimmer- man, the SR phase process occurs after making an attempt to learn and is supposed to enhance a person’s responses to his or her results [49]. In particular, these SRs shape forethought mechanisms and assumptions about future LEs. Finally, a self-regulatory cycle was completed. Prior studies provide different evidence of the relationship among the SRL factors. However, this suggests that a theoretical explanation of this relationship has been provided. Several studies provide many examples of evidence for the relationship between these factors in the learning environment [50], [51]. Given the complexity of SRL, such an explanation would necessitate the inclusion of multiple variables. In this manner, structural equation modeling (SEM) is an appropriate statistical analy- sis procedure for confirming indirect and direct relationships among many variables while also taking into account errors in measurement [52]. Following the utilization of SEM, path analysis models were developed in this study, including indi- rect and direct relationships among the observed variables. This method was utilized by Kormos and Csizér to examine a proposed model of the relationship among SRL factors [53]. See Figure 1. Hypothesis 1 (H1): A significant relationship exists between SE and EF. C. LMSS IN PLES AND THE TREND TOWARDS SELF-REGULATED LEARNING SRL is a philosophical paradigm to understand the cognitive, motivational, and emotional dimensions of learning [42]. According to Zimmerman [43], SRL has had a considerable influence on educational psychology since the relationship between SRL and metacognition began to be recognized. Since then, from the perspective of Sitzmann and Ely, vari- ous models of SRL have been addressed [42]. For instance, Puustinen and Pulkkinen published a theoretical analysis in 2001 that depicts the most important models developed by Boekaerts, Borkowski, Pintrich, Winne, and Zimmerman [44]. The fact that there are currently three meta-analyses of SRL results is a first indicator of the evolution of SRL mod- els [37]. The second predictor is that in the area of educational psychology, there are already modern SRL models that did not exist in 2001 [45]. Finally, there is a recent handbook by Zimmerman [43] that illustrates a number of well-established approaches to analyze SRL. According to Ernesto Panadero, PLEs should ideally aim to improve learners’ cognitive and metacognitive abilities, redefine the pedagogical process, and integrate third-generation LMSs [46] to design technology- enhanced practices and opportunities [11]. Therefore, from the perspective of Hicks and Sinkinson [25], it is crucial to enhance PLEs, particularly because digital information In order to improve the performance of the prediction algorithms in dynamic conditions, Khan et al. added a new learning to the prediction model. They have put forth a novel technique called ’’learning to alpha-beta filter’’ that is based on the alpha-beta filter and the deep extreme learn- ing machine (DELM) algorithm. The prediction unit and the learning unit are the two key parts of the suggested methodology. In the prediction unit, we used an alpha-beta filter, and a DELM is used in the learning unit. The primary issue with the traditional alpha-beta filter is that the values are often chosen through the method of trial and error. The results demonstrated that the proposed model outperforms the traditional alpha-beta filter in terms of results [39]. In addition, Khan et al. suggested a new prediction learning model to boost the alpha-beta filter algorithm’s dynamic performance. The suggested model consists of two main 5467 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform well-known motivational construct in educational psychol- ogy [55], and it is also a central motivational feature in most self-regulation models, such as [49]. C. LMSS IN PLES AND THE TREND TOWARDS SELF-REGULATED LEARNING SE is one of the motivat- ing elements of the forethought process in Zimmerman’s [49] self-regulation model [56]. As considered by Zimmerman, SE refers to learners’ views about their abilities to fulfil a task, which has a key effect on personal processes [57]. In addition, Zimmerman considers SE as a central variable that influences SRL, which is also according to social cog- nitive theorists [57]. In a study conducted by Kurtz and Borkowski, the key aspects of students’ SM were attributed to SE expectations [58]. Kurtz and Borkowski believed that high SE students demonstrated higher quality learning methods and more SM of their academic achievement than low SE students [58]. From the perspective of Bandaura, one of the motivating factors in the forethought process is SE [54]. However, notably, SE is not a stand-alone factor but rather one of the multiple factors considered during the self-regulation cycle [59]. Studies emphasize the beneficial impact of SE on self-regulation activities during forethought, performance, and SR and even on academic achievement [60], [61], [62]. It is indeed essential to note that SE is not only a condition for self-regulation but also a function of it. Diseth stated that the effects of SE can either directly affect learning strategies or indirectly affect learning strategies by its influence on other variables of motivation [63]. Self-motivation derives from student assumptions about learning, including SE per- ceptions about possessing a PL capacity and performance perceptions about the personal effects of learning [54]. As a result, Zimmerman suggests that students who feel self- efficient in learning to divide fractions and plan to use this ability to pass a college entrance exam are more driven to practice in a self-regulatory way [64]. A study by Moos indicates that significant monitoring and learning success are affected by SE [65]. In addition, the study shows that the association between SE and learning success is mediated by the monitoring factor. The findings are additionally con- firmed by Moos and Azevedo, where the SE of learners is estimated as positive under these circumstances [65]. Under these circumstances, Moos and Azevedo believe that learners who have had little exposure to hypermedia learning can approximate the demands of the learning domain but not the demands of the learning community [65]. As a result, learners first overrate their knowledge and capacity. Self-efficiency also impacts self-assessment processes [66]. C. LMSS IN PLES AND THE TREND TOWARDS SELF-REGULATED LEARNING According to several researchers, SE is a strong indicator of academic performance [37], [64]. In addition, researchers consider that SE has beneficial effects on self-regulation processes during forethought, efficiency, and SR, along with learning achievement [37], [64]. Therefore, considering all the above explanations, this study proposes the following hypotheses to d t i th l ti is being developed. Thus, learners will be able to develop their own self-reflective and learning environments and then need to develop and manage their PLEs through the required tools [26]. Several studies have evaluated the association between PLEs and SRL experimentally. For instance, in a study conducted by Cho et al., PLE-based learning is pre- sented as the self-regulating setting of learners who can pre- dict their social presence [47]. Similarly, Türker and Zingel conducted a study and organized learning resources accessi- ble to PLEs into comprehensible learning tasks to reach set- ting objectives to be stated as a performance of instructional form to the forethought phase considered by Zimmerman’s SRL model [48]. This explanation was also considered by Mott [33]. In this sense, Dabbagh and Kitsantas developed a three-phase system for using social media to promote SRL in PLEs, including personal knowledge management, social networking and communication, and information aggregation and management [4]. As Dabbagh and Kitsantas point out, engaging learners in personal information management prac- tices through blogs and wikis can enable them to participate in a self-regulated forethought learning process [4]. A. SELF EFFICACY (SE) SE is described as faith in one’s ability to prepare and carry out the actions necessary to achieve clear goals [54]. It is a Hypothesis 2 (H2): A significant relationship exists between SE and EV. 5468 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 1. Research model. Hypothesis 3 (H3): A significant relationship exists between SE and GS. to tackle educational exercises with a schedule as a feasible way of providing self-regulation and learning [69]. As stated by Hardin et al., the setting of goals progress develops pro- ductivity, fulfilment, inherent motivation, commitment in the face of challenges and employee behavior examination [68]. Therefore, this study proposes the following hypotheses. Hypothesis 4 (H4): A significant relationship exists between SE and SM. Hypothesis 5 (H5): A significant relationship exists between SE and RE. Hypothesis 6 (H6): A significant relationship exists between GS and EF. B. GOAL SETTING AND PLANNING (GS AND PL) Hypothesis 7 (H7): A significant relationship exists between GS and SM. The forethought phase occurs before learning occurs, and it covers two main classes that include task analysis and self-motivation [50]. From Zimmerman’s perspective, at this stage, learners analyze the learning activity and create par- ticular objectives into covering this activity [57]. As con- sidered by Pajares and Schunk [67], the PL phase helps learners self-regulate their learning prior to encouraging learning activities. The current study argues that GS and PL are complementary components because PL helps learn- ers address well-thought-out objectives and techniques to reach the success step. According to Pajares and Schunk, objectives are significantly identified as desired end states to be obtained through a particular timeline [67]. Specifi- cally, setting goals enhances the identification with the team’s decision-making process, which impacts downstream adher- ence to the goals [68]. Hardin et al. find that teams are more dedicated to challenging, concrete goals than rela- tively difficult or idiosyncratically defined ’’do-your-best’’ goals [68]. That is, the accomplishment of difficult, partic- ipatory targets helps improve dedication to these goals [68]. At this stage, Pajares and Schunk indicate that PL takes place in three phases, namely, setting a learning task objective, determining strategies to achieve the goal, and defining how much time and what resources will be required to reach the goal [67]. Zumbrunn et al. consider teaching students A. SAMPLE SIZE The designed survey in this study was conducted on total. Previous literature has provided recommendations for the minimum sample size required to perform certain analy- ses. Thus, this study followed [79]. It is recommended that PLSSEM users who are not methodological researchers use the inverse square root method for the minimum sample size estimation in the early stages of their research design. These researchers will generate estimates that are both reasonably precise and safe by using both normal and nonnormal data. According to [80]’s analyses, their estimates will always be slightly larger than the true minimum sample sizes needed but not by much, which puts light demands on data collection beyond what is needed. As a result, in accordance with [80], the sample size of this study was kept small to be simpler (by relying on a simple equation), is also fairly precise, which results in small overestimations and is ’’safe’’ in its slight IV. RESEARCH METHODOLOGY A pre-experimental design (one-group pretest-posttest design) is utilized in this study. A one-group pretest-posttest design is known as a type of research method that is most often utilized by behavioral researchers to examine the effect of a treatment or intervention on a given sample [78]. In this study, one experimental group was involved as the pretested (O1) group, which was exposed to the PLE-based course model as the treatment (X1). After conducting the pretest, the same group was selected for utilizing the posttest (O2) to examine the proposed model. This study was conducted in a particular time period (eight weeks) as follows. In the first week, the PLE course model was introduced to the teacher, followed in the second week by the introduction of the PLE course model to the students. This was followed by the third week when the pretest was conducted on the students. Afterwards, the applied PLE course-based model was conducted during week four. The PLE course model was continued until week seven. Finally, the posttest was performed during week eight to evaluate the students’ self- regulation. Hypothesis 9 (H9): A significant relationship exists between SM and EV. C. SELF EVALUATION (SE) Learners who can evaluate their own learning independently of teacher-centered summative assessments are more likely to become self-regulated students [69]. According to Zimmer- man [57], teachers may empower students to assess them- selves in the classroom by aiding them in monitoring their learning objectives and strategies and then making improve- ments to certain priorities and strategies based on learning performance. During the reflecting on results process, stu- dents measure their performance on the learning challenge in terms of the usefulness of the strategies that they choose [69]. Zumbrunn believes that throughout this process, students must monitor their feelings about the learning experience’s outcomes. Students’ future PL and aspirations are influenced by these SRs, which clearly shows the relation between SE with GS and PL [69]. Therefore, it can be concluded that self-assessment refers to measures of self-assessment results against some standard, such as previous performance, the per- formance of another person, or a total performance standard. Therefore, this study proposes another hypothesis as follows.i Hypothesis 8 (H8): A significant relationship exists between EV and RE. 5469 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform i.e., the higher-order processing aspect of reflective learning that relates to comprehending course material [76]. RE is also recognized as a move beyond material understanding to more involved participation in learning that elicits prior information and experience, includes challenging what has been learned, and can require a search for alternate meanings. In addition, RE from the perspective of Peltier et al. is cov- ered by two main conditions, namely, student-to-student and instructor-to-student interactions. According to Peltier et al., the combination of these two conditions addresses the ulti- mate success of the educational process [77]. Under this con- dition, all members of the learning community are inspired by and in harmony with their teachers and fellow students and encourage them to experiment with new ideas. This model is selected by the current study, as shown in the relationships in Figure 1. E. EFFORT (EF) Several self-regulation experiments have presented the EF variable as one of the essential aspects of motivation or as a separate variable [73]. Self-regulated learners, according to Zimmerman, not only report SE and intrinsic motiva- tion but also bring forward exceptional EF and persever- ance while learning [59]. This explanation clearly addresses the correlation between EF and SE. Zimmerman also illus- trates the relationship between initiative and SE in which SE beliefs influence motivation by defining how individuals set goals and strategies for themselves, how much work they put forward, and how long they persevere in the face of hardship [73]. Their goals are often proposed as a means to encourage human action and inspire people to produce desired results [65]. Therefore, it clearly considers the sig- nificant relationship between EF and goals, as it influences performance through several mechanisms as stated by Ban- dura [75]. First, goals inspire people to perform more both cognitively and behaviorally. Goals also ensure commitment in reaching end states, which is especially important when goals are difficult to attain. Finally, targets implicitly boost job efficiency by encouraging individuals to check and apply applicable information and techniques. D. SELF MONITORING (SM) In the monitoring phase, students utilize multiple techniques to make progress on the learning activity and SM the efficacy of such strategies and their incentive to keep progressing toward the goals defined for the task [70], [71]. Furthermore, according to Kistner et al. if students need to be strategic, then they must take responsibility for their learning and perfor- mance outcomes, which self-regulated learners accomplish by measuring their progress against learning objectives [72]. Many of the above techniques are used in the SM phase [58]. A learner must set their own learning objectives, schedule accordingly, individually encourage themselves to achieve their goals, concentrate their energy on the task at hand, and use learning techniques to promote their comprehen- sion of knowledge to self-monitor their success [58], which clearly addresses the relation between SM and both SGs and PL. Similarly, monitoring covers metacognitive understand- ing, and monitoring cognition is a central feature of self- regulation information retrieval models [73]. Thus, Butler and Winne believe that internal feedback is provided by successful self-regulated learners as they track their inter- action with learning experiences and assignments and mea- sure their progress against goals [74]. Students evaluate their self-monitored results to an absolute baseline or previous performance during this SE [57]. Therefore, the following hypothesis is proposed. F. SELF-REFLECTION (SR)l Reflection (RE) is recognized as the reflective thought that involves critiquing deeply held beliefs of what is learned, 5470 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 2. Goal setting tasks. FIGURE 3. Selecting the main library. for the following phase [85]. ADDIE provides a strategy to create and design instructional courses in education. In the process, each phase is informed by rapid prototyping to obtain the instructors’, students’, and any targeted users’ feedback needed for the next phase [86]. First, in the analysis phase, th fi t d t i ifi t h d l i h ld imprecision; thus, the sample size of this study was seventeen students. FIGURE 2. Goal setting tasks. B. THE PARTICIPANTS AND DATA COLLECTION The experiment was conducted on convenience samples because of their availability and easy accessibility. A non- purposing sample was utilized to select 17 participants out of the population of 8th-grade students in government sec- ondary schools in Saudi Arabia in 2019. However, the progress of blended learning and face-to-face learning has faced some limitations because of COVID-19. For the pur- pose of this study, 17 pretests were distributed, all of which were answered under the teacher’s supervision. In addition, all 150 students attended the PLE course-based program. After school was postponed due to the COVID-19 pandemic, only 17 students completed the posttest. Therefore, a man- ual analysis of the total of 17 pretests and posttests was completed, which is an acceptable sample size in PLSSEM according to Marcoulides and Saunders and Privitera and Delzell [81], [82]. FIGURE 3. Selecting the main library. V. THE PLE-BASED COURSE A. THE CONTEXT OF THE TREATMENT for the following phase [85]. ADDIE provides a strategy to create and design instructional courses in education. In the process, each phase is informed by rapid prototyping to obtain the instructors’, students’, and any targeted users’ feedback needed for the next phase [86]. First, in the analysis phase, as the first and most significant phase, a needs analysis should be implemented at the start of any development effort to decide whether the course is required to fill a gap in the audi- ence knowledge skills and to determine whether the course design is the best way to deliver the course. In this regard, the researcher collected the data from the first phase of the study (assessment phase), topic content, learning outcomes, and student background. A target audience analysis is considered to be a crucial step. C. INSTRUMENTATION The self-regulation of learning self-report scale (SRL-SRS) was utilized by this study for different components, includ- ing PL, SM, assessment, meditation, EF, and SE [83]. This instrument was utilized to investigate the self-regulating pro- cesses of the learners. The current study investigated PLEs as a measure of self-regulation. The SRL-SRS was used in this research for PL, SM, assessment, RE, EF, and SE, all of which, according to Toering et al., are essential aspects of learning. The SRL-SRS helps to evaluate learners’ self- regulating processes. In a study conducted by Toering et al., which utilized the SRL-SRS, the adoption of the instrument was guided by the components related to the six subscales of PL, SM, EV, RE, EF, and SE [83]. The instrument was first translated into Arabic and then reverse-translated [84]. The items were translated by two bilingual translators from English into Arabic. Two independent translators who were fluent in both languages translated these interpretations from Arabic to English without using the original scale. FIGURE 3. Selecting the main library. FIGURE 3. Selecting the main library. B. THE COURSE DESIGN AND DELIVERY OF THE PLE The course design and delivery of the PLE course are influ- enced by the outcomes of the students’ characteristics and their learning context and access to technology regarding the platform that was implemented in the study, which is Future Gate (FG). The second is the implementation phase, where the developed course is put into action, and the final product is presented to the target audience. The implementation phase consisted of two main steps according to Watson: installation and the distribution and management learning activities [87]. This study took place during the COVID-19 pandemic. Thus, all classes were conducted online. The instructor met with the learner online by using ZOOM software a few times before conducting the experiment. Before and during the actual online course, online meetings were put into action to intro- duce the learning system to ensure that the learners were well prepared and familiar with the learning system. Moreover, the learners were asked to create and sign up for the FG platform during these online meetings with an assistant from the lab technician to ensure that all learners had an accepted account to participate in the FG platform. Third, in the design phase, the researcher prepared the course outlines, user interface, menus, and guidelines by referring to the Curriculum book of the Arabic course according to the topics mentioned in the book. Thus, in this section, the researcher elaborated on the interface and all menus needed for both students and teachers who used the FG platform. Additionally, a proper framework was chosen to design the PLE-based course. The main menu of the Future Gate platform is shown in Figure 5. FIGURE 5. Main menu of the future gate platform. transformed all products from both the analysis and design phases into the learning system. In this stage, the content was produced. The content varied considerably, depending on the available resources. For example, some of the content consisted of only simpler materials (i.e., those with little or no interactivity or multimedia, such as structured PDF documents), which can be combined with other materials (e.g., audio or video files), assignments and tests. In this situation, storyboard development and the development of media and electronic interactions would not be conducted. Finally, in the evaluation (EV) phase, the online courses could be evaluated for specific EV purposes. A. MEASUREMENT MODEL (BEFORE INTERVENTION)i A. MEASUREMENT MODEL (BEFORE INTERVENTION)i V. THE PLE BASED COURSE A. THE CONTEXT OF THE TREATMENT The intervention of this study was based on the PLE-based course. First, self-efficacy in goal-setting tasks was applied by encouraging the students to set the goals and strategy instruction and the contents for the first lesson for each unit (see Figures 2 and 3). In this research, the context of the PLE-based course involved the experimental distance group, where the students in the experimental group were introduced to the PLE-based course during the 2nd semester of 2020. The researcher applied a systematic instructional system design approach called ADDIE. ADDIE is a sequence of five stages that must be followed logically for each phase to provide input 5471 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 4. Browsing the public library. FIGURE 4. Browsing the public library. FIGURE 5. Main menu of the future gate platform. B. THE COURSE DESIGN AND DELIVERY OF THE PLE B. THE COURSE DESIGN AND DELIVERY OF THE PLE These evaluations might be to evaluate the learners’ reactions, the achievement of learning objectives, the transfer of job-related knowledge and skills, and the impact of the project on the organization. According to Watson, EV is a constant process that starts from the beginning of the project until the end. Accordingly, feedback from the learners through post questionnaires was gathered in this phase [87]. C. THE THEORETICAL BASE OF THE COURSE However, based on [91], to increase the CR, indicators with outer loadings between 0.40 and 0.70 were removed from the scale. according to Table 1. In addition, the measurement model is portrayed in Figure 2. C. THE THEORETICAL BASE OF THE COURSE The Cronbach’s alpha (CA) reliability coefficient was utilized in this study to evaluate the reliability of the factors that influenced SRL without the intervention of the PLE course based on three conditions. As stated by Hair et al., the variable indices must be less than 0.80, and the validity outcome in this study was found to be 0.7 [89], [90]. In addition, as claimed by Fornell and Larcker, each construct’s average variance extracted (AVE) must be equal to or greater than 0.5 [91]. They also claimed that each construct’s AVE square root must be greater than the interconstruct correlations (IC) for a factor. Therefore, a report by Fornell and Larcker stated that the con- struct must be greater than the standard of 0.5 [91]. Regarding the explanation, the factor loadings in this study are defined The theoretical base of the course focused on enhancing self- regulated learning through PLEs. The theoretical framework of the study was based on the following three theories: i) the theory of constructivism originated by Savery and Duffy [88]; ii) the theory of PLE originated by Schaffert and Hilzen- sauer [11]; and iii) the theory of self-regulation originated by Zimmerman [54]. Central to the theory was the responsibility of the learner to determine the contents, goals, and evaluation decisions of the learning program. Fourth, the development phase was the actual production and assembly of the materials of the planned system from the design phase. This phase began once the design phase was completed. The researcher 5472 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform TABLE 1. Outer loadings. TABLE 2. Construct reliability. according to Table 1. In addition, the measurement model is portrayed in Figure 2. 1) CONSTRUCT RELIABILITY As suggested by Fornell and Larcker regarding factor check AVE), as illustrated in Table 2. However, based on [91], to increase the CR, indicators with outer loadings between 0.40 and 0.70 were removed from the scale. 2) DISCRIMINANT VALIDITY S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform TABLE 1. Outer loadings. AVE), as illustrated in Table 2. However, based on [91], to increase the CR, indicators with outer loadings between 0.40 and 0.70 were removed from the scale. AVE), as illustrated in Table 2. 2) DISCRIMINANT VALIDITY As suggested by Fornell and Larcker regarding factor check- ing, this study examined the composite reliability (CR) [91]. The findings of this study presented values that were consid- ered greater than the standard value (0.7 for CR and 0.5 for Discriminant validity (DV) is explained to present the differ- ences among sets of concepts and their indicators. From the perspective of [87], the entire constructs’ DV was considered 5473 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform TABLE 3. Latent variable correlations. TABLE 4. Hypothesis testing. TABLE 3. Latent variable correlations. The proposed seventh hypothesis presented a direct rela- tionship between GS and SM as the findings considered β = 0.877, t = 27.51, p < 0.001.i to have values greater than 0.50 and to be significant at p = 0.001. Furthermore, [88] stated that the AVE square root as presented by a single construct’s items must be less than the correlations between the items in the two constructs. Accord- ing to this explanation, the outcomes of DV are presented in Table 3. The eighth hypothesis (H8) showed a significant and posi- tive relationship with SM (β = 0.241, t = 13.876, p < 0.001). Furthermore, the ninth hypothesis showed a positive and significant relationship between EV and RE according to the findings (β = 1.12, t = 25.171, p < 0.001), which supported the hypothesis. 3) STRUCTURAL MODEL (BEFORE INTERVENTION)i After conducting the first phase, this study utilized the boot- strapping method with the aim of examining the normality of the data in the second phase. In this process, a large number of subsamples (n = 5,000) were taken from the original sample with the purpose of checking for errors. As illustrated in Figure 3, the findings offer the path coefficient values and the T values to present the importance of the measurement model, as illustrated in Figure 4. B. AFTER INTERVENTION MODEL ANALYSIS The reliability of the factors that affect SRL with the PLE course-based intervention was examined via the same tech- niques as before the intervention. The construct was above the standard of 0.5. The factor loadings are shown in Table 5. Figure 5 illustrates the measurement model. According to the presented information in Table 4, the findings of this study addressed the first hypothesis (H1) through a positive and significant relationship between SE and EF (β = 1.12, t = 25.17, p < 0.001). Therefore, the findings show that students’ SE significantly affects their EF. To address the second hypothesis (H2) in this study, the findings portrayed the relationship between SE and EV (β = 0.75, t = 48.38, p < 0.001).i According to the presented information in Table 4, the findings of this study addressed the first hypothesis (H1) through a positive and significant relationship between SE and EF (β = 1.12, t = 25.17, p < 0.001). Therefore, the findings show that students’ SE significantly affects their EF. 1) CONSTRUCT RELIABILITY The research then went on to the next phase, which looked at the CR via the same model that was utilized before the intervention. According to the results, Table 6 shows that the values were higher than the normal value (0.7 for CR and 0.5 for AVE). To address the second hypothesis (H2) in this study, the findings portrayed the relationship between SE and EV (β = 0.75, t = 48.38, p < 0.001). To present the findings according to the third hypothe- sis, a positive and significant relationship between SE and GS was presented. The analysis findings were β = 0.69, t = 22.144, and p < 0.001. 2) DISCRIMINANT VALIDITY After examining the CR and AVE, this study evaluated the DV. The DV in this study portrayed the square root of the AVE with each hidden variable in the addressed model. In addition, as shown in Table 7, the latent variable correlations are pre- sented to verify the normality of the DV. The fourth hypothesis proposed in this study was rejected based on the results of β = −0.03 t = 1.01, and p > 0.001. The fifth hypothesis was predicted to show a significant and direct effect between SE and RE. The findings showed β = −0.34, t = 6.84, and p < 0.001, which supports hypothesis (H5) and states that SE influenced students’ RE. and EF (β = 0.588, t = 16.503, p < 0.001). Therefore, the students’ SE significantly affects their EF. 3) STRUCTURAL MODEL (AFTER INTERVENTION) This study utilized bootstrapping to evaluate the normality of the data. Thus, a large number of subsamples (n = 5,000) were taken from the original sample for error checking. The findings addressed the path coefficients in Figure 6, and the T values are defined in Figure 7. The sixth hypothesis (H6) proposed in this study pre- sented a direct and significant relationship between GS and EF through the results that supported this relationship (β = 0.349, t = 9.633, p < 0.001). 5474 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform han et al.: PLEs: Modeling Students Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 6. Before intervention measurement model. FIGURE 7. Path coefficient results. e findings based on the proposed first hypothesis (H1) dered a positive and significant relationship between SE and EF (β = 0.588, t = 16.503, p < 0.001). Therefore students’ SE significantly affects their EF. FIGURE 6. Before intervention measurement model. FIGURE 6. Before intervention measurement model. FIGURE 7. Path coefficient results. he findings based on the proposed first hypothesis (H1) idered a positive and significant relationship between SE and EF (β = 0.588, t = 16.503, p < 0.001). Therefore, the students’ SE significantly affects their EF. FIGURE 7. Path coefficient results. The findings based on the proposed first hypothesis (H1) considered a positive and significant relationship between SE and EF (β = 0.588, t = 16.503, p < 0.001). Therefore, the students’ SE significantly affects their EF. 5475 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 8. Path coefficient T values. FIGURE 9. After intervention measurement model. e findings to address the second proposed hypothesis supported the relationship between SE and EV (β = t = 10.94, p < 0.001). The third hypothesis proposed that SE and GS hav and significant relationship, as the results showed 0.87, t = 92.68, and p < 0.001. FIGURE 8. Path coefficient T values. FIGURE 8. Path coefficient T values. FIGURE 9. After intervention measurement model. FIGURE 9. After intervention measurement model. The findings to address the second proposed hypothesis (H2) supported the relationship between SE and EV (β = 0.44, t = 10.94, p < 0.001). 3) STRUCTURAL MODEL (AFTER INTERVENTION) Finally, the ninth hypothesis showed a positive and significant relationship between EV and RE according to the addressed results (β = 0.39, t = 9.63, p < 0.001). TABLE 5. Outer loadings. TABLE 6 Construct reliability TABLE 6. Construct reliability. TABLE 6. Construct reliability. The sixth proposed hypothesis showed a direct and signif- icant relationship between GS and EF (β = 0.33, t = 7.9, p < 0.001).i because the results showed that β = 0.506, t = 12.70, and p < 0.001). Finally, the ninth hypothesis showed a positive and significant relationship between EV and RE according to the addressed results (β = 0.39, t = 9.63, p < 0.001). The findings of this study to address the seventh hypothesis proposed a direct relationship between GS and SM, as the results showed (β = 0.22, t = 5.71, p < 0.001). 3) STRUCTURAL MODEL (AFTER INTERVENTION) The third hypothesis proposed that SE and GS have a direct and significant relationship, as the results showed that β = 0.87, t = 92.68, and p < 0.001. The third hypothesis proposed that SE and GS have a direct and significant relationship, as the results showed that β = 0.87, t = 92.68, and p < 0.001. 5476 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform FIGURE 10. Path coefficients results. FIGURE 11. Path coefficients T values. ourth proposed hypothesis addressed a direct and nt relationship between SE and SM, as the results that β = 0.7 t = 19.24, and p > 0.001. The fifth hypothesis addressed a direct effect be and RE because the results showed that β = −0 13.766, and p < 0.001. FIGURE 10. Path coefficients results. FIGURE 10. Path coefficients results. FIGURE 10. Path coefficients results. FIGURE 10. Path coefficients results. FIGURE 10. Path coefficients results. FIGURE 11. Path coefficients T values. urth proposed hypothesis addressed a direct and t relationship between SE and SM, as the results at β = 0.7 t = 19.24, and p > 0.001. The fifth hypothesis addressed a direct effect between SE and RE because the results showed that β = −0.540, t = 13.766, and p < 0.001. FIGURE 11. Path coefficients T values. The fifth hypothesis addressed a direct effect between SE and RE because the results showed that β = −0.540, t = 13.766, and p < 0.001. The fourth proposed hypothesis addressed a direct and significant relationship between SE and SM, as the results showed that β = 0.7 t = 19.24, and p > 0.001. 5477 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform TABLE 5. Outer loadings. TABLE 6. Construct reliability. The sixth proposed hypothesis showed a direct and signif- icant relationship between GS and EF (β = 0.33, t = 7.9, p < 0.001). The findings of this study to address the seventh hypothesis because the results showed that β = 0.506, t = 12.70, and p < 0.001). C. FINDINGS AND DISCUSSIONi (β p ) The relationship between SM and EV according to the eighth hypothesis was proposed to be significant and direct This study provided two significant contributions to PLEs through the LMS platform and modeled the relations between 5478 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform TABLE 7. Latent variable correlations. TABLE 8. Hypothesis testing. TABLE 7. Latent variable correlations. the SRL factors after a PLE intervention. The findings of this study confirmed that PLEs have a positive effect on students’ SRL skills. The findings were in line with the perspective of Mott [33], who suggested that enhancing PLEs as learner- created and administered resource matrices can help learn- ers develop their metacognition and self-regulation, which results in more positive learning experiences for learners. In addition, the findings of this study are consistent with the perspectives of several researchers that have addressed a positive and significant impact among the SRL factors [43], [47], [57], [60], [61], [64].i preparation are related processes, with PL aiding learners in creating well-thought-out goals and plans for progress. This explanation has been supported by several researchers [63], [64]. More specifically, the findings of this study proved that goals to stimulate human activity and inspire people to attain successful results are recommended, which is in line with the perspective of Hardin et al. [68]. Under these circumstances, this study showed a positive impact of GS on performance phase factors, which are covered by SM and EF.i According to the findings of several studies, successful learning is based on self-regulation balancing with educa- tional PL [59], [67], [68]. Furthermore, when they monitor their interactions with learning experiences and assignments and evaluate progress toward goals, successful self-regulated learners produce internal feedback [69]. Students’ self- monitored performance was compared to an absolute baseline or previous performance during this SE [60]. Similarly, in the current study, the findings indicate that SM has a positive effect on SE. In addition, the findings portrayed the posi- tive impact of SE on students’ RE, which is similar to the perspective of Hadwin and Winne who argued that learners are eager to be self-regulated when they can assess their own learning [71]. However, some of the relationships were confirmed after the intervention of PLEs. C. FINDINGS AND DISCUSSIONi Accordingly, this study indicates that applying the 3rd generation LMS as a platform in PLEs develops students’ SRL skills. According to Zimmer- man [44], SE is not an isolated factor in the forethought phase; rather, it impacts a wide variety of factors in the self- regulation phases. In the same vein, the findings of this study portrayed SE as a key variable that affects SRL. In addition, several studies outline the beneficial impact of SE on self- regulation, including forethought (GS), performance (SM, ring SE), ion), and SR (reflection). In line with the perspec- tive of Zimmerman [43], the findings of this research also indicated that SE shows a positive and significant impact on goal setting, EF, SE, and RE. This is in contrast with the findings of this study that demonstrated the effect of SE on SM to be insignificant, although after the intervention PLEs, the findings improved and showed a positive impact, which is also in line with a study conducted by Kurtz and Borkowski who found that students’ perceptions of self-efficiency have proven to be associated with students’ SM [58]. In addition, according to Kurtz and Borkowski, students with high SE demonstrate a greater SM of their academic achievement than students with low SE [58]. Furthermore, the findings of this study are consistent with the findings of a study conducted by Moos and Azevedo, who showed that the rela- tionship between SE and learning performance is mediated by monitoring [65]. The findings further suggest that GS and VII. CONCLUSION AND RECOMMENDATIONS FOR FURTHER STUDIESi Therefore, according to the findings and discussion, this study claimed that enhancing SRL skills by using PLEs would be crucial in the education system. Furthermore, regarding the findings of this study, it can be considered that all the stated hypotheses were accepted after the intervention of PLEs. Therefore, the contributions of this study are as follows. • The impact of SRL factors before and after PLEs inter- vention should be modeled. • The impact of SRL factors before and after PLEs inter- vention should be modeled. • The use of PLEs in a learning context should enhance stu- dents’ SRL skills. In addition, this can improve components such as goal-setting skills, EF, self-monitoring, SE, and RE VOLUME 11, 2023 5479 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platfo and can emphasize the effect on SE improvement and effect, among other factors. [14] B. Olivier and O. Liber, ‘‘Lifelong learning: The need for portable personal learning environments and supporting interoperability standards,’’ JISC Centre Educ. Technol. Interoperability Standards, Bolton Inst., Bristol, U.K., Tech. Rep., 2001, pp. 1–8. [Online]. Available: Retrieved from http://wiki.cetis.ac.uk/uploads/6/67/Olivierandliber2001.doc • Educational institutions are advised to integrate the 3rd generation as a PLE platform. [15] T. Martindale and M. Dowdy, ‘‘Personal learning environments,’’ in Emerging Technologies in Distance Education. Edmonton, AB, Canada: Athabasca Univ. Press, Jul. 2010, pp. 177–193. [Online]. Available: http://www.aupress.ca/books/120177/ebook/09_Veletsianos_2010- Emerging_Technologies_in_Distance_Education.pdf Although this study offered several significant points, it also has its own limitations, which may offer some signif- icant recommendations for further studies to be conducted. The first limitation refers to the sample size, which was lim- ited due to the COVID-19 pandemic. This study recommends that further studies work on a larger sample to obtain gener- alizable findings. Second, the current research was restricted to a single country. It is recommended that further studies be conducted on several learners from different regions and societies to overcome the limitations and expand the reach of new findings. Finally, the findings of this study suggest that further studies could work in the vast context of PLEs. For instance, further studies may be conducted on the LMS as a PLE platform. Further studies could also focus on the implementation of PLE platforms to enhance learners’ self- regulation skills to obtain more generalizable findings. Emerging_Technologies_in_Distance_Education.pdf [16] S. H. REFERENCES [1] F. J. García-Peñalvo and M. Á. 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He had eight years of teaching experience at the Department of Computer Science, Hodeidah Uni- versity, and a Teaching Assistant with the Faculty of Computing, UTM, for a period of two and half years. Moreover, he was a Postdoctoral Researcher with the Faculty of Information and Communica- tion Technology, International Islamic University, Malaysia; and the Faculty of Science, UTM. Currently, he is a Postdoctoral Researcher with the Faculty of Education, UTM. REFERENCES Bandura, ‘‘The anatomy of stages of change,’’ Amer. J. Health Promo- tion, vol. 12, no. 1, pp. 8–10, 1997. [55] P. K. Murphy and P. A. Alexander, ‘‘A motivated exploration of motivation terminology,’’ Contemp. Educ. Psychol., vol. 25, no. 1, pp. 3–53, Jan. 2000.l [76] D. Kember, D. Y. P. Leung, A. Jones, A. Y. Loke, J. McKay, K. Sinclair, H. Tse, C. Webb, F. K. Yuet Wong, M. Wong, and E. Yeung, ‘‘Devel- opment of a questionnaire to measure the level of reflective thinking,’’ Assessment Eval. Higher Educ., vol. 25, no. 4, pp. 381–395, Dec. 2000, doi: 10.1080/713611442. [56] B. J. Zimmerman and A. Bandura, ‘‘Impact of self-regulatory influ- ences on writing course attainment,’’ Amer. Educ. Res. J., vol. 31, no. 4, pp. 845–862, Dec. 1994. 5481 VOLUME 11, 2023 VOLUME 11, 2023 S. Alserhan et al.: PLEs: Modeling Students’ Self-Regulation Enhancement Through a Learning Management System Platform [77] T. C. Peltier, P. S. Barboza, and J. E. Blake, ‘‘Seasonal hyperphagia does not reduce digestive efficiency in an Arctic grazer,’’ Physiol. Biochem. Zool., vol. 76, no. 4, pp. 471–483, Jul. 2003, doi: 10.1086/377744. NORAFFANDY YAHAYA received the Ph.D. degree in computer-based learning from the Uni- versity of Leeds, U.K. [78] W. W. Chin and P. R. Newsted, ‘‘Structural equation modeling analysis with small samples using partial least squares,’’ Stat. Strategies Small Sample Res., vol. 1, no. 1, pp. 307–341, 1999. y He has been an Associate Professor with the Faculty of Education, Universiti Teknologi Malaysia, since 2013. He was the Head of the Department of Educational Science, Mathematics, and Creative Multimedia, for nine years. He con- ducted studies on students’ interaction in online learning environment, learning analytics, and mas- sive open online courses (MOOCs). He has published more than 70 papers in journals and conferences proceedings in the research area of online learning, ICT in education, and the use of technology in teaching and learning. He has been a supervisor to more than 25 completed master’s degree students and seven completed Ph.D. students in the area of educational technology, online learning, and ICT in education. He had also been appointed as an external examiner for universities in Malaysia and Australia for doctoral theses and had been an assessor for master’s dissertation for university in New Zealand. His research interests include multimedia in education, online learning, and ICT in education. [79] E. Whitley and J. TURKI MESFER ALQAHTANI re TURKI MESFER ALQAHTANI received the B.S. degree in computer science from King Khalid University, Abha, Saudi Arabia, and the master’s degree in information technology from Griffith University, Australia. HASSAN ABUHASSNA received the master’s degree in instructional technology and the Ph.D. degree (Hons.) in education technology. He worked at Universiti Teknologi Malaysia (UTM), as a Postdoctoral Fellow for six months, a part-time Lecturer for two months, and a Research Assistant for three years. He is currently an Assistant Professor with UTM. He has many recent publications in WOS and Scopus indexed. He has more than six years of experience working in the field of education and multimedia training. His current research interests include MOOC, multimedia in education, online learning, and e-learning. REFERENCES His research interests include information sys- tem management, information technology management, human–computer interaction, implementation process, technology acceptance model (TAM), communication and constructivism theories, impact of social media net- works, collaborative learning, e-learning, knowledge management, massive open online course (MOOCs), and statistical data analysis (IBM SPSS, AMOS, NVIVO, and SmartPLS). He received the Best Student Award, the Doctor of Philosophy (Faculty of Computing-Information System), the Excellent Academic Achievement in Conjunction with the 56th Convocation Ceremony, UTM, in 2016. [88] J. R. Savery and T. M. Duffy, ‘‘Problem based learning: An instructional model and its constructivist framework,’’ Educ. Technol., vol. 35, no. 5, pp. 31–38, 1995. [89] J. F. Hair Jr., G. T. M. Hult, C. M. Ringle, and M. Sarstedt, A Primer on Partial Least Squares Structural Equation Modeling (PLS-SEM), 2nd ed. Thousand Oaks, CA, USA: Sage, 2016. [90] J. F. Hair, G. T. M. Hult, C. M. Ringle, and M. Sarstedt, A Primer on Partial Least Squares Structural Equation Modeling. New York, NY, USA: Sage, 2014. [91] C. Fornell and D. F. Larcker, ‘‘Evaluating structural equation models with unobservable variables and measurement error,’’ J. Marketing Res., vol. 18, no. 1, pp. 39–50, Feb. 1981, doi: 10.1177/002224378101800104. SARAH ALSERHAN received the master’s degree in educational technology from King Saud University, Saudi Arabia. She is currently pursuing the Ph.D. degree with the College of Education, Universiti Teknologi Malaysia, Johor. Her research interests include educational technology, instructional design, personalize learning, and virtual environment. TURKI MESFER ALQAHTANI received the B.S. degree in computer science from King Khalid University, Abha, Saudi Arabia, and the master’s degree in information technology from Griffith University, Australia. HASSAN ABUHASSNA HASSAN ABUHASSNA received the master’s degree in instructional technology and the Ph.D. degree (Hons.) in education technology. He is currently a Lecturer with the Depart- ment of Instructional Technology, Jazan Univer- sity, Saudi Arabia. He is also an expert in data analysis using IBM SPSS and SmartPLS. He had experience around nine years of teaching instruc- tional design principles course and technology-based learning tools and applications. He also focuses on the flipped classroom and online anxi- ety. His research interests include flipped classroom, blend learning, and blockchain. He was a Committee Member of the Jury in the Final Selection of the National Olympiad for Scientific Creativity ‘‘Ibdaa,’’ under the super- vision of King Abdulaziz and his Companions Foundation for Giftedness and Creativity (Mawhiba) for several years in Riyadh, Saudi Arabia. VOLUME 11, 2023 5482
https://openalex.org/W2611665327	https://iris.unimore.it/bitstream/11380/1256449/1/Cappuccilli%202017.pdf	English	null	Identification of expanded T-cell clones by spectratyping in nonfunctioning kidney transplants	Journal of inflammation research	2,017	cc-by	6,056	Dovepress open access to scientific and medical research Dovepress open access to scientific and medical research Journal of Inflammation Research Journal of Inflammation Research O R I G I N A L R E S E A R C H Identification of expanded T-cell clones by spectratyping in nonfunctioning kidney transplants This article was published in the following Dove Press journal: Journal of Inflammation Research 3 May 2017 y Number of times this article has been viewed Number of times this article has been viewed Maria Cappuccilli1 Gabriele Donati1 Giorgia Comai1 Olga Baraldi1 Diletta Conte1 Irene Capelli1 Valeria Aiello1 Andrea Pession2 Gaetano La Manna1 Background: The aim of this study was the application of complementarity-determining region-3 spectratyping analysis to determine T-cell-repertoire complexity and to detect T-cell- clone expansion, as a measure of immune response in nonfunctioning kidney transplants (group hemodialysis-transplant [HD-Tx]), nontransplanted dialysis patients (group hemodialysis [HD]), and normal subjects as controls (group C). Patients and methods: Analysis of T-cell receptor (TCR) diversity by spectratyping was applied to peripheral blood samples collected from 21 subjects: eight in group HD-Tx, seven in group HD, and six in group C. Results: Considering the extent of the skew in TCR variable region repertoires as a measure of clonal T cells, we found that the number of altered spectra showed a progressive increase from normal subjects to dialysis patients and to nonfunctioning kidney transplants, respectively. Healthy subjects had the lowest number of altered spectra, and patients with nonfunctioning kid- ney transplants the highest. Differences were significant for group HD-Tx vs group C (P=0.017) and group HD vs group C (P=0.015), but not between nonfunctioning kidney-transplant recipients and dialysis patients (group HD-Tx vs group HD). 1Department of Experimental Diagnostic and Specialty Medicine (DIMES), Nephrology, Dialysis, and Renal Transplant Unit, 2Molecular Laboratory of Pediatrics, Hematology–Oncology Unit, St Orsola Hospital, University of Bologna, Bologna, Italy Conclusion: Although dialysis appears to be a weaker trigger for clonal expansion of T cells, our data suggest that the utilization of complementarity-determining region-3 spectratyping analysis of the TCR repertoire might be useful to monitor specific immunoactivation in patients before and after kidney transplantation. Keywords: dialysis, nonfunctioning kidney transplant, T-cell repertoire, TCR spectratyping Correspondence: Gaetano La Manna Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Nephrology, Dialysis and Renal Transplant Unit, St Orsola Hospital, University of Bologna, 9 Via Giuseppe Massarenti – Pad 15, Bologna 40138, Italy Tel +39 051 214 4577 Fax +39 051 344 439 Email gaetano.lamanna@unibo.it © 2017 Cappuccilli et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Journal of Inflammation Research 2017:10 41–47 41 Introduction Kidney transplantation is considered the treatment of choice for patients with end- stage renal disease, since it restores patients’ quality of life and reduces morbidity and mortality rates associated with dialysis.1,2 The introduction of new immunosuppres- sant drugs has greatly decreased the number and impact of acute rejections, which however is still a critical complication.3,4 Unfortunately, several pathological changes due to immunological and nonimmunological components for organ loss lead to a progressive loss of graft function, and this condition does not seem to have received consistent benefits from the new pharmacological treatments.5,6 The main consequence is the necessity to retransplant patients who are often sensitized or highly sensitized by a previous transplant, with a resulting higher percentage of patients in transplant- waiting lists needing a second transplantation, longer average waiting times, and a further increase in the gap between organ demand and availability.7–9 There is much evidence suggesting how immunosuppressive therapy remains too aspecific a strategy 41 submit your manuscript \| www.dovepress.com Dovepress http://dx.doi.org/10.2147/JIR.S124944 y p \| p Dovepress http://dx.doi.org/10.2147/JIR.S124944 Dovepress Cappuccilli et al to improve graft survival, and additionally it is not practicable to stress systemic immunosuppression, owing to the related possible complications, mainly viral infections, malignancy, nephrotoxicity, and cardiovascular disease.10–14 order to investigate the immune response in patients with a nonfunctioning graft compared to those under chronic dialysis treatment with no previous kidney transplant, we evaluated the differences in the number of altered spectra in peripheral blood samples in three groups of patients: non- functioning kidney-transplant recipients vs nontransplanted dialysis patients vs healthy control subjects. Growing attention is currently focused on the develop- ment and protective mechanisms of graft tolerance, in which T-cell responses in both innate and adaptive immunity play a major role.15 Accumulating evidence suggests that innate immunity interacts with the adaptive immune system to identify antigens and eliminate them from the host.16 Both components are implicated not only in acute rejection but also in chronic allograft nephropathy, which in turn is related to immune and nonimmune mechanisms, including the activation of CD4+ T cells.5,6,10,17 T-cell recognition of the antigen is mediated by the T-cell receptor (TCR), composed of α- and β-chains, each composed of a variable (V) gene segment, a joining (J) segment, and a constant (C) region. The β-chains have a diversity (D) gene segment between the V and J segments. Introduction During T-cell development, one V, one D, and one J gene segment are randomly rearranged, resulting in more than 1,000 possible junctional combinations of α- and β-chains.18 The specificity of antigen recognition is mainly due to the presence of the complementarity-determining region-3 (CDR3), a junctional region between the V, D, and J gene segments.19,20 The possibility to characterize the specific T-cell repertoires may have considerable repercussions on many areas of interest, including autoimmunity, response to superantigens, tumor immunity, and immunoreconstitution after bone marrow and solid-organ transplantation. Since the hypervariable TCR region is related to the clonal expansion of T cells against specific antigens, the early identification of these clones via TCR-diversity analysis might provide a valid tool to monitor their progression and to delineate specific treatments.21–26 submit your manuscript \| www.dovepress.com Dovepress Patients and methods Patients A total of 21 subjects were enrolled in the study and assigned to three groups: the first composed of eight nonfunctioning kidney-transplant recipients under dialysis treatment, nega- tive for the presence of preformed human leukocyte antigen (HLA) antibodies (group hemodialysis-transplant [HD-Tx]); the second group consisted of seven dialysis patients with no previous renal transplant (group hemodialysis [HD]); and the third included six healthy subjects with normal renal func- tion, used as the control group (group C). Patients in groups HD-Tx and HD were on regular bicarbonate hemodialysis therapy three times a week at our dialysis center. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204 For personal use only. Inclusion criteria were age above 18 years, and for dialysis patients in groups HD-Tx and HD: 1) clinical stabil- ity of at least 3 months before study entry; 2) functioning arteriovenous fistula as vascular access; and 3) absence of active gastrointestinal bleeding, diabetes, amyloidosis, focal segmental glomerulosclerosis, autoimmune diseases, vasculitis, and malignancy. As shown in Table 1, the subjects included in each group were matched for general parameters (sex distribution and age). Groups HD-Tx and HD were comparable for dialysis vintage, chronic kidney-disease duration, and serum creatinine. Causes of transplant failure in the HD-Tx group were biopsy-proven chronic allograft nonimmune injury (interstitial fibrosis and tubular atrophy) in five patients, recurrent primary disease in two patients, and de novo disease in one patient. Spectratyping is a method able to evaluate T-cell-reper- toire complexity through CDR3-size heterogeneity of the TCR β-chain, and represents a more powerful approach than lymphocyte-subset analysis through flow cytometry, for the clinical immunomonitoring of solid-organ recipients.25–27 The CDR3 sequence defines a unique clonotype that behaves as a fingerprint for the T-cell line bearing it. The study of TCR repertoires using length analysis of CDR3 hypervariable regions could concur to follow the immunological status of each patient and to identify early expansion of T-cell clones involved in immune response, thus providing the necessary steps for clone-specific therapeutic strategies.22,23 To exclude interference due to anti-HLA antibody pro- duction in patients with nonfunctioning kidney transplants, all subjects in group HD-Tx were assayed for posttransplant HLA-specific IgG antibodies through enzyme-linked immu- nosorbent assay panel-reactive antibodies), as previously described by Costa et al.28 Therefore, patients in group HD-Tx were selected only if they tested negative for specific HLA antibodies in three consecutive controls. All patients in HD-Tx group had been tapered from immunosuppression to low-dose monotherapy steroids (5 mg/day prednisone). Dialysis patients with no previous transplant (group HD) were always negative for panel-reactive antibodies and HLA specificity. In this study on a limited number of subjects, we inves- tigated the application of spectratyping for the identifica- tion of T-cell-clone expansion after renal transplantation. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.99.250 on 20-May-2017 For personal use only. Notes: Categorical variables presented as percentages, and continuous variables presented as mean ± standard deviation. Differences between the groups were computed using Kruskal–Wallis analysis of variance followed by Mann–Whitney U tests for post hoc comparison. Abbreviations: CKD, chronic kidney disease; NS, not significant; HD-Tx, hemodialysis-transplant; HD, hemodialysis. 30 seconds 95°C, annealing 30 seconds 58°C, extension 45 seconds 72°C, 29 cycles; final extension 5 minutes 72°C. An aliquot of 5 μL of the final PCR product was analyzed by electrophoresis in a 2% agarose gel (FMC, Rockland, ME, USA) with 0.5 μg/mL of ethidium bromide in 0.5× Tris–borate–ethylenediaminetetraacetic acid buffer (Thermo Fisher Scientific), and then visualized under an ultraviolet transilluminator. The study was carried out in conformity with the Declaration of Helsinki. Written informed consent was obtained before inclusion from all subjects, and the proto- col was approved by the ethical committee of St Orsola– Malpighi University Hospital, Bologna, Italy (protocol BO-SO_NEPH2011_07). T-cell spectratyping Peripheral blood mononuclear cells were isolated from hep- arinized blood by Isopaque Ficoll (Lymphoprep; Nycomed, Zurich, Switzerland) gradient centrifugation, then total RNA was extracted with an RNeasy minikit (Qiagen NV, Venlo, the Netherlands) according to the manufacturer’s instructions. For each sample, 1 μg of RNA was reverse- transcribed to complementary DNA using random hexa- nucleotide primers and reverse transcriptase (Superscript; Thermo Fisher Scientific, Waltham, MA, USA), with a thermal profile of 10 minutes 25°C, 60 minutes 42°C, and 5 minutes 99°C. p yp g PCR product (1 μL) was denatured in 15 μL formamide and electrophoresed through POP-4® on an ABI 310 auto- mated sequencer in the presence of Tamra 400HD ROX size standard (all Thermo Fisher Scientific). As a result of TCR β-chain rearrangements, each amplification shows a certain pattern of bands related to the complexity of the T-cell rep- ertoire and the concentration of one or few expanded clones species within the size class. Spectratyping analysis was performed in duplicate for the Vβ families Vβ2, Vβ4, Vβ5.1, Vβ5.3, Vβ7, Vβ9, Vβ12, Vβ13, Vβ14, Vβ15, Vβ16, Vβ17, Vβ19, Vβ21,Vβ23, and Vβ24. GeneScan and Genotyper 2.1 softwares (Thermo Fisher Scientific) were used to classify each Vβ histogram peak by its PCR-size length and compute the area under curve (AUC) of each peak. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204 For personal use only. In Journal of Inflammation Research 2017:10 42 Dovepress Spectratyping in renal transplants Dovepress Table 1 Demographic, clinical, and biochemical parameters of the study groups Parameter A HD-Tx (n=8) B HD (n=7) C Healthy (n=6) P (Kruskal–Wallis) Post hoc comparisons Sex (male/female) 4/4 4/3 3/3 NS – Age (years) 47.9±8.6 48.3±6.3 47.1±7.9 NS – Dialysis vintage (months) 50.1±22.3 51.1±20 – NS – CKD duration (months) 64.5±65.1 75.6±83 – NS – Serum creatinine (mg/dL) 9.1±3.6 9.8±3 0.9±0.2 <0.001 A, B < C Primary disease Glomerulonephritis Polycystic kidney disease Interstitial nephritis Vascular nephropathy Hereditary nephropathy Not diagnosed 1 2 1 3 0 1 2 1 1 2 0 1 – – – – – – – Notes: Categorical variables presented as percentages, and continuous variables presented as mean ± standard deviation. Differences between the groups were computed using Kruskal–Wallis analysis of variance followed by Mann–Whitney U tests for post hoc comparison. Abbreviations: CKD, chronic kidney disease; NS, not significant; HD-Tx, hemodialysis-transplant; HD, hemodialysis. Table 1 Demographic, clinical, and biochemical parameters of the study groups Results All the subjects participating in the study presented skewed spectra, including those in the control group. The technique showed high specificity and repeatability. The number of altered spectra revealed no correlation with sex, age, or dialysis vintage. Qualitative evaluation of skewed spectra revealed high variability in the three groups, although the number of patients was too small to provide statistically significant correlations of specific altered Vβ-chain CDR3 regions with clinical outcomes. Figure 1 shows the complete spectra patterns for patients in each group who presented an intermediate number of alterations. In our study, we selected a limited number of renal transplant recipients with a nonfunctioning graft negative for donor-specific antibodies to avoid the introduction of a further confounding variable. The eight-patient HD-Tx group had lost graft function, due to nonimmune injury (interstitial fibrosis/tubular atrophy) previously diagnosed by renal biopsy in five, recurrent primary disease in two, and de novo disease in one. The group of renal transplant recipients with a nonfunctioning graft showed the maximum number of skewed spectra, although the difference from dialysis patients did not reach statistical significance. It is feasible that previ- ous cell-mediated acute and/or chronic reactions might have elicited persistent specific HLA-sensitization responsiveness, leading to allograft intolerance. On the other hand, the limited number of patients prevented us from applying other statisti- cal analyses or to draw any firm conclusion about the real influence of dialysis on T-cell-clone expansion. The higher extent of skewing in patients with a failed graft seems to indicate a link between altered Vβ-chain CDR3 regions and immunological response against HLAs. To determine the number of skewed spectra for each patient in the three groups, we observed electropherograms of TCR Vβ amplicons and expressed the “area under the peak” as the quantity of labeled Vβ-fragment PCR product of a defined length measured by the detector. From the evaluation of the amount of altered spectra in each group, we applied Krus- kal–Wallis analyses of variance, followed by Mann–Whitney U tests to compare the AUC for each peak of all Vβ families across the three groups. Table S1 shows the AUC of each peak for all Vβ families analyzed for each subject in the three groups: HD-Tx (n=8) vs HD (n=7) vs C (n=6). As Table 2 shows, there was a progressive increase in the number of skewed spectra from healthy subjects to dialysis patients and to nonfunctioning kidney transplants, respec- tively. Discussion The possibility of identifying oligoclonal T-cell expansions of TCR Vβ families in kidney transplants can represent a fascinating research field in kidney-transplant tolerance. The analysis of TCR diversity through spectratyping in our patients suggested that renal transplant recipients who experienced graft failure were characterized by more persistent expanded T-cell clones, with a potential specific role in the acceptance of transplanted solid organs.27,29 Previous evidence has indicated that the extent of skewing of TCR repertoires and levels of clonal T-cell expansion are significantly correlated with renal transplant outcomes. It has been reported that a decrease in the number of donor-reactive T-cell clones is more directly associated with tolerance induction, whereas allograft rejec- tion can trigger clonal expansion of T cells.30,31 Polymerase chain reaction The TCR Vβ gene subfamilies were amplified by a con- stant primer labeled at the 5’ terminus with the dye FAM (6-carboxyfluorescein), coupled with other specific primers, first designed by Gorski et al.21 Briefly, 20 μL aliquots of working solution containing GeneAmp® polymerase chain- reaction (PCR; Thermo Fisher Scientific), 2 mM MgCl2, 0.2 mM of each deoxyribonucleotide triphosphate, 0.1 mM of each primer, and 1 μL of complementary DNA were amplified with the following PCR conditions: denaturation Normally, in healthy subjects, the CDR3-length distri- butions of each TCR Vβ family contain at least six peaks in a Gaussian distribution, suggestive of a polyclonal T-cell expansion. Any perturbation in TCR Vβ CDR3 repertoire, referred to as “skewing”, results in a markedly non-Gaussian CDR3-length distribution, suggesting a clonal or oligoclonal T-cell population. Specifically, we considered as skewed any Journal of Inflammation Research 2017:10 submit your manuscript \| www.dovepress.com Dovepress 43 Dovepress Dovepress Cappuccilli et al TCR Vβ family exhibiting a predominant peak, defined by an AUC within a given CDR3 spectratype of greater than 40% of the sum of the total AUC. TCR Vβ family exhibiting a predominant peak, defined by an AUC within a given CDR3 spectratype of greater than 40% of the sum of the total AUC. submit your manuscript \| www.dovepress.com Dovepress Statistical analysis Continuous variables are presented as mean ± standard deviation and categorical variables as number and percentage. Differences between groups were computed using Kruskal– Wallis analyses of variance followed by Mann–Whitney U tests. P-values below 0.05 were considered significant, and all statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) for Windows version 16.0. Journal of Inflammation Research 2017:10 Results When each Vβ family was considered separately, no significant differences were found among the three groups, but the differences were significant for the sum of total skewed spectra (P<0.001). In spite of the small sample size and differences in the patient population, these findings show a certain consistency with a recent report by Morris et al, who described a drop in donor-reactive T-cell clones in tolerant combined kidney and bone marrow-transplant recipients. The same reductions were not observed in intolerant patients or standard renal transplant patients treated with standard immunosuppressive regimens. T-cell-repertoire turnover due to lymphocyte-depleting condi- tioning only partially accounted for the observed reductions in tolerant patients, supporting the hypothesis that one key mechanism of allograft-tolerance induction may be T-cell- clonal deletion.31 Healthy subjects had the fewest altered spectra and patients with a nonfunctioning kidney transplant the highest. Applying post hoc Mann–Whitney nonparametric tests, we found that differences were significant for group HD-Tx vs group C (P=0.017) and group HD vs group C (P=0.015), but not for nonfunctioning kidney-transplant recipients vs dialysis patients (group HD-Tx vs group HD). The actual difficulty is related to the apparent variabil- ity of the spectra-alteration pattern detected in each group Journal of Inflammation Research 2017:10 44 Dovepress Spectratyping in renal transplants Group C Vβ1 Vβ2 Vβ2 Vβ4 Vβ6 Vβ7 Vβ8 Vβ9 Vβ10 Vβ11 Vβ12 Vβ13 Vβ14 Vβ15 Vβ16 Vβ17 Vβ18 Vβ19 Vβ20 Vβ21 Vβ5.1 Vβ5.3 Patient 12 Group HD-Tx Group HD Group C Vβ1 Vβ2 Vβ2 Vβ4 Vβ6 Vβ7 Vβ8 Vβ9 Vβ10 Vβ11 Vβ12 Vβ13 Vβ14 Vβ15 Vβ16 Vβ17 Vβ18 Vβ19 Vβ20 Vβ21 Vβ22 Vβ24 Vβ23 Vβ5.1 Vβ5.3 Vβ1 Vβ2 Vβ2 Vβ4 Vβ6 Vβ7 Vβ8 Vβ9 Vβ10 Vβ11 Vβ12 Vβ13 Vβ14 Vβ15 Vβ16 Vβ17 Vβ18 Vβ19 Vβ20 Vβ21 Vβ22 Vβ24 Vβ23 Vβ5.1 Vβ5.3 Vβ1 Vβ2 Vβ2 Vβ4 Vβ6 Vβ7 Vβ8 Vβ9 Vβ10 Vβ11 Vβ12 Vβ13 Vβ14 Vβ15 Vβ16 Vβ17 Vβ18 Vβ19 Vβ20 Vβ21 Vβ22 Vβ24 Vβ23 Vβ5.1 Vβ5.3 Patient 2 Patient 7 Patient 12 re 1 Complete spectra patterns for patients 2, 7, and 12. es: Patients 2, 7, and 12 were those with the intermediate number of skewed spectra in group HD-Tx, group HD, and group C, respectively. reviations: C, control; HD-Tx, hemodialysis-transplant; HD, hemodialysis. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.99.250 on 20-May-2017 For personal use only. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.9 For personal use only. 2. Sá H, Leal R, Rosa MS. Renal transplant immunology in the last 20 years: a revolution towards graft and patient survival improvement. Int Rev Immunol. Epub 2016 Sep 28. 2. Sá H, Leal R, Rosa MS. Renal transplant immunology in the last 20 years: a revolution towards graft and patient survival improvement. Int Rev Immunol. Epub 2016 Sep 28. 3. Tanriover B, Jaikaransingh V, MacConmara MP, et al. Acute rejec- tion rates and graft outcomes according to induction regimen among recipients of kidneys from deceased donors treated with tacrolimus and mycophenolate. Clin J Am Soc Nephrol. 2016;11(9):1650–1961. 3. Tanriover B, Jaikaransingh V, MacConmara MP, et al. Acute rejec- tion rates and graft outcomes according to induction regimen among recipients of kidneys from deceased donors treated with tacrolimus and mycophenolate. Clin J Am Soc Nephrol. 2016;11(9):1650–1961. 4. Ville S, Poirier N, Branchereau J, et al. Anti-CD28 antibody and belata- cept exert differential effects on mechanisms of renal allograft rejection. J Am Soc Nephrol. 2016;27(12):3577–3588. 5. Solez K, Colvin RB, Racusen LC, et al. Banff ‘05 meeting report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (‘CAN’). Am J Transplant. 2007;7(3): 518–526. 6. Scolari MP, Cappuccilli ML, Lanci N, L et al. Predictive factors in chronic allograft nephropathy. Transplant Proc. 2005;37(6):2482–2484. 7. Heaphy EL, Poggio ED, Flechner SM, et al. Risk factors for retrans- plant kidney recipients: relisting and outcomes from patients’ primary transplant. Am J Transplant. 2014;14(6):1356–1367. 8. Panchal H, Muskovich J, Patterson J, Schroder PM, Ortiz J. Expanded criteria donor kidneys for retransplantation: United Network for Organ Sharing update – proceed with caution. Transpl Int. 2015;28(8): 990–999. 9. Gandolfini I, Buzio C, Zanelli P, et al. The Kidney Donor Profile Index (KDPI) of marginal donors allocated by standardized pretransplant donor biopsy assessment: distribution and association with graft out- comes. Am J Transplant. 2014;14(11):2515–2525. 10. Martin-Gandul C, Mueller NJ, Pascual M, Manuel O. The impact of infection on chronic allograft dysfunction and allograft survival after solid organ transplantation. Am J Transplant. 2015;15(12):3024–3040. 11. Fischereder M. Cancer in patients on dialysis and after renal transplanta- tion. Nephrol Dial Transplant. 2008;23(8):2457–2460. 12. Gaston RS. Chronic calcineurin inhibitor nephrotoxicity: reflections on an evolving paradigm. Clin J Am Soc Nephrol. 2009;4(12):2029–2034. 13. Ghanta M, Kozicky M, Jim B. Pathophysiologic and treatment strate- gies for cardiovascular disease in end-stage renal disease and kidney transplantations. Cardiol Rev. 2015;23(3):109–118. 14. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.9 For personal use only. Shirali AC, Bia MJ, Denton MD, Magee CC, Sayegh MH. Management of cardiovascular disease in renal transplant recipients. Clin J Am Soc Nephrol. 2008;3(2):491–504. 15. Nakanishi K. Innate and acquired activation pathways in T-cells. Nat Immunol. 2001;2(2):140–142. 16. Pratt JR, Basheer SA, Sacks SH. Local synthesis of complement C3 regulates acute renal transplant rejection. Nat Med. 2002;8(6):582–587. Disclosure The authors report no conflicts of interest in this work. Results Individual comparisons through post hoc Mann–Whitney nonparametric tests revealed that healthy subjects had the fewest altered spectra (group HD-Tx vs group C, P=0.017; group HD vs group C, P=0.015), while differences between nonfunctioning kidney- transplant recipients and dialysis patients were not statistically significant (group HD-Tx vs group HD). Abbreviations: C, control; NS, not significant; HD-Tx, hemodialysis-transplant; HD, hemodialysis. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.99.250 on 20-May-2017 For personal use only. Acknowledgment The authors would like to acknowledge Dr Vania Cuna for her essential research assistance. Results Vβ7 Vβ7 Vβ15 Vβ17 Vβ17 Vβ16 Vβ17 Vβ18 Vβ19 Vβ19 Vβ19 Vβ18 Vβ20 Vβ21 Vβ20 Vβ20 Vβ21 Vβ21 Vβ24 Vβ24 Figure 1 Complete spectra patterns for patients 2, 7, and 12. Notes: Patients 2, 7, and 12 were those with the intermediate number of skewed spectra in group HD-Tx, group HD, and group C, respectively. Abbreviations: C, control; HD-Tx, hemodialysis-transplant; HD, hemodialysis. gu e Co p ete spect a patte s o pat e ts , 7, a . Notes: Patients 2, 7, and 12 were those with the intermediate number of skewed spectra in group HD-Tx, group HD, and group C, respectively. Abbreviations: C, control; HD-Tx, hemodialysis-transplant; HD, hemodialysis. approach for renal transplant recipients, considering the changes over time in T-cell CDR3 regions as a reflection of a specific immune status after kidney transplant. and not characterizing the particular status of the subject (posttransplant, dialysis or healthy), in line with previous data by Barth et al, suggesting that T-cell Vβ-repertoire is restricted and individual in both acute and ongoing chronic allograft rejection before getting to transplant failure.26 Thus far, these preliminary data might suggest a possible follow-up Delayed T-cell recovery and restricted TCR diversity after both hematopoietic stem cell transplantation and solid-organ transplantation appear to increase the risk of opportunistic Journal of Inflammation Research 2017:10 submit your manuscript \| www.dovepress.com 45 Dovepress Dovepress Cappuccilli et al Cappuccilli et al infections and cancer, with a significant impact on trans- plant-associated morbidity and mortality.32 van Heijst et al investigated clinical parameters potentially able to affect T-cell-repertoire recovery, and found cytomegalovirus or Epstein–Barr virus infections to be associated with lower TCR diversity.33 However, our study had the main limitation of considering T-cell-repertoire features in nonfunctioning kidney-transplant recipients vs dialysis patients vs healthy subjects, without examining the outcomes specifically. Although the limited sample size prevents us from drawing firm conclusions about a possible correlation between a given pattern of spectra and a specific clinical condition, the main preliminary finding emerging from our study is the elevated number of skewed spectra in nonfunctioning kidney-transplant recipients under dialysis treatment. Perhaps dialysis treatment alone represents a weaker trigger for clonal expansion of T cells, even if the difference between nonfunctioning kidney- transplant recipients vs dialysis patients did not meet statistical significance, maybe owing to the small number of subjects. Results Abbreviations: C, control; NS, not significant; HD-Tx, hemodialysis-transplant; HD, hemodialysis. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.99.250 on 20-May-2017 For personal use only. infections and cancer, with a significant impact on trans- plant-associated morbidity and mortality.32 van Heijst et al investigated clinical parameters potentially able to affect T-cell-repertoire recovery, and found cytomegalovirus or Epstein–Barr virus infections to be associated with lower TCR diversity.33 However, our study had the main limitation of considering T-cell-repertoire features in nonfunctioning kidney-transplant recipients vs dialysis patients vs healthy subjects, without examining the outcomes specifically. Although the limited sample size prevents us from drawing firm conclusions about a possible correlation between a given pattern of spectra and a specific clinical condition, the main preliminary finding emerging from our study is the elevated number of skewed spectra in nonfunctioning kidney-transplant recipients under dialysis treatment. Perhaps dialysis treatment alone represents a weaker trigger for clonal expansion of T cells, even if the difference between nonfunctioning kidney- transplant recipients vs dialysis patients did not meet statistical significance, maybe owing to the small number of subjects. peak (in brackets) for each Vβ family analyzed in the three groups. HD-Tx (n=8) HD (n=7) C (n=6) P Vb2 3/8 (4,928) 0/7 (9,316) 0/6 (6,861) NS Vb4 3/8 (7,182) 2/7 (10,367) 0/6 (11,598) NS Vb5.1 2/8 (4,940) 3/7 (4,885) 0/6 (5,429) NS Vb5.3 3/8 (3,493) 3/7 (1,465) 0/6 (2,594) NS Vb7 4/8 (8,501) 2/7 (9,850) 0/6 (17,732) NS Vb9 2/8 (4,284) 2/7 (2,260) 0/6 (11,509) NS Vb12 1/8 (2,096) 0/7 (3,145) 0/6 (719) NS Vb13 1/8 (12,303) 2/7 (12,693) 0/6 (23,654) NS Vb14 2/8 (1,935) 1/7 (25,177) 0/6 (1,876) NS Vb15 2/8 (5,846) 3/7 (7,575) 0/6 (2,802) NS Vb16 3/8 (3,094) 2/7 (7,457) 0/6 (6,777) NS Vb17 3/8 (1,586) 1/7 (3,963) 0/6 (6,169) NS Vb19 1/8 (11,208) 1/7 (25,098) 0/6 (15,592) NS Vb21 1/8 (4,354) 3/7 (2,018) 0/6 (13,481) NS Vb23 3/8 (10,385) 1/7 (15,294) 0/6 (27,751) NS Vb24 1/8 (1,614) 3/7 (1,410) 4/6 (2,520) NS Total 35/128 (27.3%) 29/112 (25.9%) 4/96 (4.2%) <0.001 Notes: Although no differences across the groups emerged when each Vβ family was considered separately (Kruskal–Wallis analysis of variance), significant differences were found in the total number of skewed spectra (χ2 test). Results Conclusion Our data indicate that early identification of T-cell clonality renal transplantation might predict a state of immunoactiva- tion against HLAs thus providing a diagnostic tool not only Table 2 Number of skewed bands with median area under the peak (in brackets) for each Vβ family analyzed in the three groups. HD-Tx (n=8) HD (n=7) C (n=6) P Vb2 3/8 (4,928) 0/7 (9,316) 0/6 (6,861) NS Vb4 3/8 (7,182) 2/7 (10,367) 0/6 (11,598) NS Vb5.1 2/8 (4,940) 3/7 (4,885) 0/6 (5,429) NS Vb5.3 3/8 (3,493) 3/7 (1,465) 0/6 (2,594) NS Vb7 4/8 (8,501) 2/7 (9,850) 0/6 (17,732) NS Vb9 2/8 (4,284) 2/7 (2,260) 0/6 (11,509) NS Vb12 1/8 (2,096) 0/7 (3,145) 0/6 (719) NS Vb13 1/8 (12,303) 2/7 (12,693) 0/6 (23,654) NS Vb14 2/8 (1,935) 1/7 (25,177) 0/6 (1,876) NS Vb15 2/8 (5,846) 3/7 (7,575) 0/6 (2,802) NS Vb16 3/8 (3,094) 2/7 (7,457) 0/6 (6,777) NS Vb17 3/8 (1,586) 1/7 (3,963) 0/6 (6,169) NS Vb19 1/8 (11,208) 1/7 (25,098) 0/6 (15,592) NS Vb21 1/8 (4,354) 3/7 (2,018) 0/6 (13,481) NS Vb23 3/8 (10,385) 1/7 (15,294) 0/6 (27,751) NS Vb24 1/8 (1,614) 3/7 (1,410) 4/6 (2,520) NS Total 35/128 (27.3%) 29/112 (25.9%) 4/96 (4.2%) <0.001 Notes: Although no differences across the groups emerged when each Vβ family was considered separately (Kruskal–Wallis analysis of variance), significant differences were found in the total number of skewed spectra (χ2 test). Individual comparisons through post hoc Mann–Whitney nonparametric tests revealed that healthy subjects had the fewest altered spectra (group HD-Tx vs group C, P=0.017; group HD vs group C, P=0.015), while differences between nonfunctioning kidney- transplant recipients and dialysis patients were not statistically significant (group HD-Tx vs group HD). Abbreviations: C, control; NS, not significant; HD-Tx, hemodialysis-transplant; HD, hemodialysis. Journal of Inflammation Research downloaded from https://www.dovepress.com/ by 137.204.99.250 on 20-May-2017 For personal use only. Table 2 Number of skewed bands with median area under the peak (in brackets) for each Vβ family analyzed in the three groups. clones involved in chronic allograft failure, the leading cause of renal graft loss. Results infections and cancer, with a significant impact on trans- plant-associated morbidity and mortality.32 van Heijst et al investigated clinical parameters potentially able to affect T-cell-repertoire recovery, and found cytomegalovirus or Epstein–Barr virus infections to be associated with lower TCR diversity.33 However, our study had the main limitation of considering T-cell-repertoire features in nonfunctioning kidney-transplant recipients vs dialysis patients vs healthy subjects, without examining the outcomes specifically. Although the limited sample size prevents us from drawing firm conclusions about a possible correlation between a given pattern of spectra and a specific clinical condition, the main preliminary finding emerging from our study is the elevated number of skewed spectra in nonfunctioning kidney-transplant recipients under dialysis treatment. Perhaps dialysis treatment alone represents a weaker trigger for clonal expansion of T cells, even if the difference between nonfunctioning kidney- transplant recipients vs dialysis patients did not meet statistical significance, maybe owing to the small number of subjects. Table 2 Number of skewed bands with median area under the peak (in brackets) for each Vβ family analyzed in the three groups. HD-Tx (n=8) HD (n=7) C (n=6) P Vb2 3/8 (4,928) 0/7 (9,316) 0/6 (6,861) NS Vb4 3/8 (7,182) 2/7 (10,367) 0/6 (11,598) NS Vb5.1 2/8 (4,940) 3/7 (4,885) 0/6 (5,429) NS Vb5.3 3/8 (3,493) 3/7 (1,465) 0/6 (2,594) NS Vb7 4/8 (8,501) 2/7 (9,850) 0/6 (17,732) NS Vb9 2/8 (4,284) 2/7 (2,260) 0/6 (11,509) NS Vb12 1/8 (2,096) 0/7 (3,145) 0/6 (719) NS Vb13 1/8 (12,303) 2/7 (12,693) 0/6 (23,654) NS Vb14 2/8 (1,935) 1/7 (25,177) 0/6 (1,876) NS Vb15 2/8 (5,846) 3/7 (7,575) 0/6 (2,802) NS Vb16 3/8 (3,094) 2/7 (7,457) 0/6 (6,777) NS Vb17 3/8 (1,586) 1/7 (3,963) 0/6 (6,169) NS Vb19 1/8 (11,208) 1/7 (25,098) 0/6 (15,592) NS Vb21 1/8 (4,354) 3/7 (2,018) 0/6 (13,481) NS Vb23 3/8 (10,385) 1/7 (15,294) 0/6 (27,751) NS Vb24 1/8 (1,614) 3/7 (1,410) 4/6 (2,520) NS Total 35/128 (27.3%) 29/112 (25.9%) 4/96 (4.2%) <0.001 Notes: Although no differences across the groups emerged when each Vβ family was considered separately (Kruskal–Wallis analysis of variance), significant differences were found in the total number of skewed spectra (χ2 test). Individual comparisons through post hoc Mann–Whitney nonparametric tests revealed that healthy subjects had the fewest altered spectra (group HD-Tx vs group C, P=0.017; group HD vs group C, P=0.015), while differences between nonfunctioning kidney- transplant recipients and dialysis patients were not statistically significant (group HD-Tx vs group HD). References 1. Ghanta M, Jim B. Renal transplantation in advanced chronic kidney disease patients. Med Clin North Am. 2016;100(3):465–476. 1. Ghanta M, Jim B. Renal transplantation in advanced chronic kidney disease patients. Med Clin North Am. 2016;100(3):465–476. 1. Ghanta M, Jim B. Renal transplantation in advanced chronic kidney disease patients. Med Clin North Am. 2016;100(3):465–476. Conclusion 17. Alvarez CM, Opelz G, Garcia LF, Süsal C. Expression of regulatory T-cell-related molecule genes and clinical outcome in kidney transplant recipients. Transplantation. 2009;87(6):857–863. Our data indicate that early identification of T-cell clonality renal transplantation might predict a state of immunoactiva- tion against HLAs, thus providing a diagnostic tool not only for acute rejection but also for the identification of cellular 18. Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition. Nature. 1988;334(6181):395–402. 19. Fields BA, Ober B, Malchiodi EL, et al. Crystal structure of the Vα domain of a T-cell antigen receptor. Science. 1995;270(5243):1821–1824. Journal of Inflammation Research 2017:10 submit your manuscript \| www.dovepress.com Dovepress submit your manuscript \| www.dovepress.com 46 Dovepress Dovepress Spectratyping in renal transplants 27. Haarer J. TCR spectratyping in transplantation. Transplantation. 2015; 99(12):2438–2439. 20. Bentley GA, Boulot G, Karjalainen K, Mariuzza RA. Crystal structure of the β chain of a T-cell antigen receptor. Science. 1995;267(5206):1984–1987. 27. Haarer J. TCR spectratyping in transplantation. Transplantation. 2015; 99(12):2438–2439. 28. Costa AN, Scolari MP, Iannelli S, et al. The presence of posttransplant HLA-specific IgG antibodies detected by enzyme-linked immunosor- bent assay correlates with specific rejection pathologies. Transplanta- tion. 1997;63(1):167–169. 21. Gorski J, Yassai M, Zhu X et al. Circulating T-cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. J Immunol. 1994;152(10):5109–5119. 22. Vignes C, Chiffoleau E, Douillard P, et al. Anti-TCR-specific DNA vac- cination demonstrates a role for a CD8+ T-cell clone in the induction of allograft tolerance by donor-specific blood transfusion. J Immunol. 2000;165(1):96–101. 29. Ducloux D, Bamoulid J, Courivaud C, et al. Thymic function, anti- thymocytes globulins, and cancer after renal transplantation. Transpl Immunol. 2011;25(1):56–60. 23. Jiang H, Kashleva H, Xu LX, et al. T-cell vaccination induces T-cell receptor Vβ-specific Qa-1-restricted regulatory CD8+ T-cells. Proc Natl Acad Sci U S A. 1998;95(8):4533–4537. 30. Matsutani T, Ohashi Y, Yoshioka T, et al. Skew in T-cell receptor usage and clonal T-cell expansion in patients with chronic rejection of trans- planted kidneys. Transplantation. 2003;75(3):398–407. 24. Plasilova M, Risitano A, Maciejewski JP. Application of the molecular analysis of the T-cell receptor repertoire in the study of immune- mediated hematologic diseases. Hematology. 2003;8(3):173–181. 31. Morris H, DeWolf S, Robins H, et al. Tracking donor-reactive T-cells: evidence for clonal deletion in tolerant kidney transplant patients. Sci Transl Med. 2015;7(272):272ra10. 32. Lai L, Wang L, Chen H, et al. Conclusion T cell repertoire following kidney trans- plantation revealed by high-throughput sequencing. Transpl Immunol. 2016;39:34–45. 25. Baron C, McMorrow I, Sachs DH, LeGuern C. Persistence of domi- nant T-cell clones in accepted solid organ transplants. J Immunol. 2001;167(8):4154–4160. 26. Barth C, Von Menges A, Zanker B et al. Restricted T-cell Vβ repertoire in renal allografts during acute and chronic rejection. Kidney Int. 1996; 50(6):2020–2026. 33. van Heijst JW, Ceberio I, Lipuma LB, et al. Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation. Nat Med. 2013;19(3):372–377. 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The Journal of Inflammation Research is an international, peer-reviewed open access journal that welcomes laboratory and clinical findings on the molecular basis, cell biology and pharmacology of inflammation including original research, reviews, symposium reports, hypothesis for- mation and commentaries on: acute/chronic inflammation; mediators of Submit your manuscript here: https://www.dovepress.com/journal-of-inflammation-research-journal Journal of Inflammation Research 2017:10 submit your manuscript \| www.dovepress.com 47 submit your manuscript \| www.dovepress.com Dovepress Dovepress Dovepress
https://openalex.org/W3020062430	https://strathprints.strath.ac.uk/72917/1/van_Teijlingen_etal_NA_2020_Size_dependent_melting_point_depression_of_nickel.pdf	English	null	Size-dependent melting point depression of nickel nanoparticles	Nanoscale advances	2,020	cc-by	3,636	Introduction Metal and metal oxide nanoparticles exhibit uniquely size- dependent properties which normally follow an inverse surface area to volume proportionality.1 This has a depressive eﬀect on the melting point, and has been shown experimentally for many metals.2–9 This eﬀect was rst predicted by P. Pawlow in 190910 and demonstrated experimentally in 1954 by M. Takagi.11 Buﬀat et al. investigated the melting point depression of spherical gold nanoparticles using the same ED method as Takagi et al., and noted that this method could only determine the range of melting points for an ensemble of particles.4 This has long been a standard technology in determining nano- particle melting points.12 It has been purported, however, that measurements of melting points via ED becomes increasingly inaccurate as nanoparticle size decreases due to line-broad- ening.8 Determination of melting point depression has also been undertaken through the use of nano-DSC, Lai et al. investigated this property of tin nanoparticles, determining the melting point to be when there was an abrupt increase (“jump”) in heat ow required to sustain the increasing temperature.8 We apply a similar method as Zhang et al. in order to measure individual nanoparticles by direct observation of nanoparticles via TEM recognising record melting as an abrupt increase in observed diameter.13 In a typical synthesis (synthesis 1) a 5% (w/v) nickel nitrate solution was prepared by dissolving nickel nitrate hexahydrate (11.25 mg, 38.67 mmol) in water (0.225 mL). A 5% (w/v) alkaline NaBH4 solution was prepared in a similar fashion. Two reverse microemulsions (RME-1 and RME-2) were made up of cyclo- hexane (22.744 mL), 1-hexanol (0.3 mL) and TX-100 (1.731 mL, 2.50 mmol). To RME-1 under stirring 5% (w/v) nickel nitrate solution (0.225 mL) was added so that W0 was equal to 5 or 1 respectively. To RME-2 under stirring 5% (w/v) alkaline NaBH4 (0.225 mL) solution was added. Both reverse microemulsions were leto stir for 30 min to achieve homogeneity aer which RME-2 was added dropwise to RME-1 with continuous stirring under an inert (argon) atmosphere. The resulting reverse micro- emulsion was leto stir for 3 hours to allow for nanoparticle growth via Ostwald ripening. The nickel nanoparticles were separated via centrifugation and washed with ethanol and separately with toluene then dispersed in water (Fig. 2). aDepartment of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, UK bSchool of Chemistry, University of Bristol, Bristol, BS8 1TS, UK. E-mail: s.a.davis@ bristol.ac.uk; Simon.Hall@bristol.ac.uk † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0na00153h Synthesis Nickel nitrate hexahydrate (Extra Pure SLR), cyclohexane ($99.5%) and sodium hydroxide ($97% NaOH) were purchased from Fisher Scientic. TX-100 (laboratory grade), 1-hexanol Open Access Article. Published on 21 April 2020. Downloaded on 6/25/2020 11:21:50 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Cite this: Nanoscale Adv., 2020, 2, 2347 Cite this: Nanoscale Adv., 2020, 2, 2347 Alexander van Teijlingen, a Sean A. Davis b and Simon R. Hall b We investigate the phase-transition behaviour of nickel nanoparticles (3–6 nm) via dynamic TEM. The nanoparticles were synthesized within a reverse microemulsion and then monitored via dynamic TEM simultaneously while undergoing controlled heating. The size-dependent melting point depression experimentally observed is compared with, and is in good agreement with existing thermodynamic and molecular dynamic predictions. Received 24th February 2020 Accepted 21st April 2020 DOI: 10.1039/d0na00153h rsc.li/nanoscale-advances Received 24th February 2020 Accepted 21st April 2020 (98%) and NaBH4 ($96%) were purchased from Sigma-Aldrich. The reaction procedure was scaled down from the work of Kumar et al.14 Four syntheses were conducted with diﬀerent reactant concentrations (ESI Table 1†). The size of the nickel nanoparticles was controlled by varying the molar ratio of water to surfactant (W0). Each reaction does however produce a range of sizes (ESI Table 1 & ESI Fig. 1–4†), which we nd itself useful in this study. Introduction These reverse microemulsions continually collide and coalesce, mix- ing their internal constituents – this allows the sodium boro- hydride to reduce the nickel ions which then grow into a nanoparticle within the microemulsions. The synthesis has been visualized in Fig. 1. Nanoscale Advances View Article Online View Journal \| View Issue Melting point characterization Phase-pure and oxide free nickel nanoparticles on carbon coated copper TEM grids were prepared by drop-casting of a colloidal suspension of nickel nanoparticles in toluene. Using a Gatan heating stage (model number 628) with applied water This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 2347–2351 \| 2347 View Article Online View Article Online Paper Nanoscale Advances Fig. 2 TEM images of (A) nickel nanoparticles encapsulated a reverse microemulsions of TX-100/1-hexanol (d ¼ 100 nm) i l i k l ti l (d 4 5 ) EM images of (A) nickel nanoparticles encapsulate i l i f TX 100/1 h l (d 100 Fig. 1 Nickel nanoparticle synthesis procedure consisting of prepa- ration of two reverse microemulsions (TX-100 with cosurfactant 1- hexanol stabilizing the spherical structure), one with nickel(II) ions within and another with sodium borohydride within. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 2 TEM images of (A) nickel nanoparticles encapsulated within a reverse microemulsions of TX-100/1-hexanol (d ¼ 100 nm) and (B) a single nickel nanoparticle (d ¼ 4.5 nm). measurements. Recording the expansion of nanoparticles around the melting point allows the determination of this physical property unambiguously. Nanoparticles were deter- mined to have melted when three requirements had been met: 1. The average diameter of the spherical nanoparticle increased by at least 4.3% (this percentage was chosen as it is the diﬀerence in diameter for two equimolar spherical nickel nanoparticles, one a solid and one a liquid with perfect wetting on a substrate, densities 8.908 g cm3 and 7.81 g cm3 respectively). 1. The average diameter of the spherical nanoparticle increased by at least 4.3% (this percentage was chosen as it is the diﬀerence in diameter for two equimolar spherical nickel nanoparticles, one a solid and one a liquid with perfect wetting on a substrate, densities 8.908 g cm3 and 7.81 g cm3 respectively). Fig. 1 Nickel nanoparticle synthesis procedure consisting of prepa- ration of two reverse microemulsions (TX-100 with cosurfactant 1- hexanol stabilizing the spherical structure), one with nickel(II) ions within and another with sodium borohydride within. Fig. 1 Nickel nanoparticle synthesis procedure consisting of prepa- ration of two reverse microemulsions (TX-100 with cosurfactant 1- hexanol stabilizing the spherical structure), one with nickel(II) ions within and another with sodium borohydride within. 2. This journal is © The Royal Society of Chemistry 2020 Melting point characterization 3) and the diameter calculated according to (1). d ¼ 2 ﬃﬃﬃﬃ A p r (1) (1) Fig. 3 shows the change in mean diameter as a function of projected nanoparticle surface area, for a single nickel nano- particle over increasing temperatures. There is an abrupt increase at the melting point (925 C 25 C). The melting point was taken as the temperature halfway between the upper and lower points of the increase. This method is similar to the nano-DSC method described by Lai et al.8 This process was repeated for seven diﬀerent nickel nanoparticles and the determined melting points plotted in Fig. 4. This experimental data is in good agreement with the MD studies and thermody- namic models also shown in Fig. 4. nanoparticles,2 it is generally believed that it is an incomplete model of solid–liquid phase transition in nanoparticles. More sophisticated models, such as the liquid nucleation and growth model (eqn (4) and (5)), which supposes that the liquid phase is initiated at a surface (a discontinuity) and propagates inwards towards the centre. At a critical radius (r) an unstable equi- librium is formed, where r ¼ r. The temperature at which this is formed marks the upper boundary of the melting range (green area, Fig. 4), while the lower boundary is given by eqn (5).17 Tm ¼ T0 1 1 d (2) (2) The empirical relationship between diameter and melting point can be described by eqn (2) where T0 denotes the bulk melting point, d the diameter, Tm the melting point of the nanoparticle and the melting point is inversely related to the diameter. r* ¼ 2gsvnmðsÞT0 DmHðT T0Þ (4) TmðdÞ ¼ T0 1 3 DmH gsvnmðsÞ r glvnðlÞ r (5) (4) (5) TmðdÞ ¼ T0 1 4gsl DmHrsd (3) (3) Thermodynamically, for the solid-to-liquid phase trans- formation to be energetically favourable, the sum of the ener- gies of the newly formed solid–liquid and liquid–vapor interfaces must be less than or equal to the energy of the solid– vapor interface (eqn (6)). There exist several proposed mechanisms in which melting initialization and propagation in nanomaterials is described as a function of interface energies and the enthalpy of fusion (DmH). Melting point characterization The green area shows the temperatures between the upper and lower bounds of the liquid nucleation and growth model (eqn (4) and (5)) – this is in very close agreement with the experimental data between 3.5 nm and 5.3 nm, where most of the experimental data points where collected. The orange trendline and markers display the experimental data collected in this study, the vertical error bars show a 50 C uncertainty in temperature and the horizontal error bars show a 0.1 nm uncertainty in diameter. Open Access Article. Published on 21 April 2020. Downloaded on 6/25/2020 11:21:50 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 3 A line graph showing the diameter of a 4.3 nm nickel nano- sphere over a temperature range of 700–1050 C, at intervals of 50 C. The solid nanoparticle diameter was taken as an average of 5 measurements between 700 C and 900 C. The red X in the centre marks the melting point at 925 25 C, which was determined from the abrupt, sustained and signiﬁcant increase in nanoparticle diameter. Fig. 4 A graph showing the results of evaluating the melting point depression of nickel nanoparticles by four diﬀerent methods. The red line denotes the melting point of nickel nanoparticles according to the empirical model (eqn (2)) from d ¼ 1 nm to d ¼ 6 nm with 1 nm increments. The blue trendline and markers plot the melting point results of the molecular dynamics study using the quantum-Sutton- Chen (QSC) force ﬁeld reported by Qi et al.3 The purple line denotes values predict by the liquid-drop model24 with a b value given by Hanszen et al.25 The black line and markers represent values for the melting point as a function of size predicted by the Gibbs–Thompson (eqn (3)). The green area shows the temperatures between the upper and lower bounds of the liquid nucleation and growth model (eqn (4) and (5)) – this is in very close agreement with the experimental data between 3.5 nm and 5.3 nm, where most of the experimental data points where collected. The orange trendline and markers display the experimental data collected in this study, the vertical error bars show a 50 C uncertainty in temperature and the horizontal error bars show a 0.1 nm uncertainty in diameter. projected TEM image surface area of individual nanoparticles (Fig. Melting point characterization The melting point is clearly identied by a rapid and sustained increase in size as opposed to gradual expansion due to heating of a solid. cooling coupled to a JEOL-2100F TEM, we recorded the melting point of eight diﬀerent nickel nanoparticles over a temperature range of 700–1100 C at a rate of 5 C min1. Nanoparticle diameters were measured only when the video capture showed clearly de-ned edges. The heating stage independently measured the temperature of the sample and therefore heating eﬀects from the electron beam are accounted for in our 3. The diameter of the liquid phase nanoparticle used to deter-mine the melting point does not have overlapping error bars with the diameter measurements of the solid nanoparticle. 3. The diameter of the liquid phase nanoparticle used to deter-mine the melting point does not have overlapping error bars with the diameter measurements of the solid nanoparticle. Images at diﬀerent temperatures were analysed using the so-ware package ImageJ2, Fiji distribution15,16 by recording the 2348 \| Nanoscale Adv., 2020, 2, 2347–2351 This journal is © The Royal Society of Chemistry 2020 View Article Online Paper Fig. 3 A line graph showing the diameter of a 4.3 nm nickel nano- sphere over a temperature range of 700–1050 C, at intervals of 50 C. The solid nanoparticle diameter was taken as an average of 5 measurements between 700 C and 900 C. The red X in the centre marks the melting point at 925 25 C, which was determined from the abrupt, sustained and signiﬁcant increase in nanoparticle diameter. Paper Nanoscale Advances Fig. 4 A graph showing the results of evaluating the melting point depression of nickel nanoparticles by four diﬀerent methods. The red line denotes the melting point of nickel nanoparticles according to the empirical model (eqn (2)) from d ¼ 1 nm to d ¼ 6 nm with 1 nm increments. The blue trendline and markers plot the melting point results of the molecular dynamics study using the quantum-Sutton- Chen (QSC) force ﬁeld reported by Qi et al.3 The purple line denotes values predict by the liquid-drop model24 with a b value given by Hanszen et al.25 The black line and markers represent values for the melting point as a function of size predicted by the Gibbs–Thompson (eqn (3)). This journal is © The Royal Society of Chemistry 2020 Acknowledgements where: (8) (9) The authors would like to acknowledge the Bristol Centre for Functional NanoMaterials funded by the Engineering and Physical Sciences Research Council (EPRSC) UK (grantsEP/ G036780/1 and EP/L015544). TEM studies were carried out in the Chemistry Imaging Facility with equipment funded by UoB and EPSRC (EP/K035746/1 and EP/M028216/1). (8) (9) The authors would like to acknowledge the Bristol Centre for Functional NanoMaterials funded by the Engineering and Physical Sciences Research Council (EPRSC) UK (grantsEP/ G036780/1 and EP/L015544). TEM studies were carried out in the Chemistry Imaging Facility with equipment funded by UoB and EPSRC (EP/K035746/1 and EP/M028216/1). (8) (9) The authors would like to acknowledge the Bristol Centre for Functional NanoMaterials funded by the Engineering and Physical Sciences Research Council (EPRSC) UK (grantsEP/ G036780/1 and EP/L015544). TEM studies were carried out in the Chemistry Imaging Facility with equipment funded by UoB and EPSRC (EP/K035746/1 and EP/M028216/1). and: (9) Conﬂicts of interest There are no conicts to declare. Utot ¼ X i¼1 3 "X isj V rij C ﬃﬃﬃﬃri p # (7) VðrÞ ¼ a rij n (8) ri ¼ X a rij m (9) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p (10) Utot ¼ X i¼1 3 "X isj V rij C ﬃﬃﬃﬃri p # (7) VðrÞ ¼ a rij n (8) ri ¼ X a rij m (9) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p (10) (7) Nanoscale Advances Paper A key limitation of thermodynamic models is that melting is equivalent to stability such that changes in individual atoms in a system are not considered and therefore cannot reveal the mechanism of the melting process alone. In 2001 Qi et al. published a molecular dynamics study calculating the degree of melting point depression in nickel nanoparticles.3 By super- heating and super-cooling nickel nanoparticles under the QSC potential (eqn (7)), they found the equilibrium melting temperature according to eqn (10). The data presented in Fig. 4 sheds light onto the accuracy of some thermodynamic and molecular dynamic models that have been used to predict the melting point depression as a function of size in nickel, showing that the liquid nucleation and growth model (green area) to be the model that t best with the experimental data (orange line). experiment and modelling to be tested over a larger size range. Future experiments may also include both heating/cooling cycles where the properties of diﬀerent nanoparticles can be analysed at the 2nd, 3rd, 4th etc. cycle of heating as well as their behaviour upon cooling.23 With suﬃcient distribution on the grid, we speculate that this method would be appropriate to measure the properties of completely diﬀerent nanoparticles simultaneously. Just as we have used it to study diﬀerent sizes of nickel nanoparticles simultaneously, it could be used to deter- mine melting point depression in magnesium oxide and aluminium oxide simultaneously or the sintering of any two or more diﬀerent nanoparticles. Melting point characterization One of these models is the Gibbs–Thompson eqn (3), which assumes an isolated spherical nanoparticle situated within it's a liquid of its own type.7 While this model has some success in predicting melting point depression in our gsv $ gsl + glv (6) (6) Nanoscale Adv., 2020, 2, 2347–2351 \| 2349 This journal is © The Royal Society of Chemistry 2020 Paper View Article Online View Article Online View Article Online Paper w Article Online Notes and references (10) Tm ¼ Tþ þ T ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Tþ þ T p 1 D. K. Pattadar and F. P. Zamborini, J. Am. Chem. Soc., 2018, 140, 14126–14133. 1 D. K. Pattadar and F. P. Zamborini, J. Am. Chem. Soc., 2018, 140, 14126–14133. Experimental data presented herein (Fig. 4) have shown that MD studies using the QSC force eld, while generally in agreement with the experimental data,3,18,19 consistently over- predict the melting points of all sizes of nickel nanoparticles studied – a predictable outcome as their system was shown to determine the melting point of bulk nickel to be 1487 C (32 C higher than the real bulk melting point). This QSC force eld has previously been used to accurately predict melting point depression in gold,20 copper,6 platinum21 and iron.22 It is of note that in each system the bulk melting point of each element was overpredicted. Finally we compared the normalised (DT ¼ T/T0) and undercooled (DT ¼ T0 T) QSC MD study and nd that, while closer to the experimental data they over- and under- predict the individual melting points respectively (ESI Fig. 5 and 6†). 2 W. Luo, W. Hu and S. Xiao, J. Phys. Chem. C, 2008, 112, 2359– 2369. 3 Y. Qi, T. Çain, W. L. Johnson and W. A. Goddard, J. Chem. Phys., 2001, 115, 385–394. 4 P. Buﬀat and J.-P. Borel, Phys. Rev. A, 1976, 13, 2287–2298. 5 K. Dick, T. Dhanasekaran, Z. Zhang and D. Meisel, J. Am. Chem. Soc., 2002, 124, 2312–2317. 6 H. H. Kart, H. Yildirim, S. Ozdemir Kart and T. Çaˇgin, Mater. Chem. Phys., 2014, 147, 204–212. y 7 J. Sun and S. L. Simon, Thermochim. Acta, 2007, 463, 32–40. 8 S. L. Lai, J. Y. Guo, V. Petrova, G. Ramanath and L. H. Allen, Phys. Rev. Lett., 1996, 77, 99–102. 9 H. J. Kim, I. K. Beak, K. H. Kim and S. P. Jang, Trans. Korean Soc. Mech. Eng. B, 2014, 38, 695–700. 10 P. Pawlow, Z. phys. Chem., 1909, 65, 545–548. 11 M. Takagi, J. Phys. Soc. Jpn., 1954, 9, 359–363. 2350 \| Nanoscale Adv., 2020, 2, 2347–2351 Conclusions 12 J. Zhang, Y. Zheng, D. Zhao, S. Yang, L. Yang, Z. Liu, R. Zhang, S. Wang, D. Zhang and L. Chen, J. Phys. Chem. C, 2016, 120, 10686–10690. In this study we have synthesized nickel nanoparticles of varying diameter via a reverse microemulsion synthesis and for the rst time we report experimental studies which complement and validate previous modelling of nickel nanoparticle melting point depression. We have used this method to report a detailed investigation of the melting point depression in nickel nano- particles. The method could be improved and expanded using alternative TEM grids, such as silicon nitride grids, as they are more stable than copper at elevated temperatures, allowing 13 M. Zhang, E. A. Olson, R. D. Twesten, J. G. Wen, L. H. Allen, I. M. Robertson and I. Petrov, J. Mater. Res., 2005, 20, 1802– 1807. 14 A. Kumar, A. Saxena, A. De, R. Shankar and S. Mozumdar, Adv. Nat. Sci.: Nanosci. Nanotechnol., 2013, 4, 25009. 15 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, 15 J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, g , , , , S. Saalfeld, B. Schmid, J. Y. Tinevez, D. J. White, This journal is © The Royal Society of Chemistry 2020 2350 \| Nanoscale Adv., 2020, 2, 2347–2351 Nanoscale Advances View Article Online Nanoscale Advances View Article Online Paper 20 Z. Qiao, H. Feng and J. Zhou, Phase Transitions, 2014, 87, 59– 70. V. Hartenstein, K. Eliceiri, P. Tomancak and A. Cardona, Nat. Methods, 2012, 9, 676–682. 16 C. T. Rueden, J. Schindelin, M. C. Hiner, B. E. DeZonia, 21 H. Akbarzadeh and G. A. Parsafar, Fluid Phase Equilib., 2009, 280, 16–21. A. E. Walter, E. T. Arena and K. W. Eliceiri, BMC Bioinf., 2017, 18, 1–26. 22 T. Shen, W. Meng, Y. Wu and X. Lu, Appl. Surf. Sci., 2013, 277, 7–14. 17 G. Guenther and O. Guillon, J. Mater. Sci., 2014, 49, 7915– 7932. 23 H. Chamati and K. Gaminchev, in J. Phys.: Conf. Ser., IOP Publishing, 2012, vol. 398, p. 012042. 18 T. D. Nguyen, C. C. Nguyen and V. H. Tran, RSC Adv., 2017, 7, 25406–25413. 24 K. K. Nanda, S. N. Sahu and S. N. Behera, Phys. Rev. A: At., Mol., Opt. Phys., 2002, 66, 132081–132088. 19 T. Çagin, Y. Qi, H. Li, Y. This journal is © The Royal Society of Chemistry 2020 Nanoscale Adv., 2020, 2, 2347–2351 \| 2351 Conclusions Kimura, H. Ikeda, W. L. Johnson and W. A. Goddard III, MRS Symposium Ser., 1999, vol. 554, p. 43. 25 K.-J. Hanszen, Z. Phys., 1960, 157, 523–553. Nanoscale Adv., 2020, 2, 2347–2351 \| 2351
https://openalex.org/W2589041862	https://e-journal.unair.ac.id/MKG/article/download/1359/2445	English	null	Antibacterial effects of 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on Aggregatibacter actinomycetemcomitans	Dental Journal	2,017	cc-by-sa	6,290	104 104 Dental Journal (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 Research Report Research Report abstract Background: Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is a Gram negative bacteria that form a subgingival plaque causing periodontitis. Nowadays, many natural resources can be used as a basic ingredient of drugs. One of the resources used as an antibacterial material is cacao bean. It contains of polyphenol flavonoids, such as catechin, epicatechin, anthocyanin, and proanthocyanidin. Chemical compounds contained in ethanol extract and water extract are different in quantity from those in cocoa beans. Purpose: This research aimed to find out difference in antibacterial activity between the 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on A. actinomycetemcomitans. Method: This research was an in vitro laboratory experiment. The serial dilutions was performed on the 70% ethanol and water extract of cacao beans a concentration of 100% to 3.125%. At each concentration, the 70% ethanol and water extract of cacao beans were added with grown bacterial suspension of A. actinomycetemcomitans. After they were incubated for 24 hours, the bacteria grown on Luria Berthani media were observed. Bacteria colonies then were measured in CFU/ml. Result: There were significant differences in bacterial colonies grown at the concentrations of 6.25% and 3.125% between the 70% ethanol extract of cacao beans and the water extract of cacao beans as p-value = 0.000 (p<0.05). Conclusion: 70% Ethanol beans and water extract of cacao beans have antibacterial activity against A. Actinomycetemcomitans. The concentrations of MIC and MBC extracts were 6.25% and 12.5% respectively. Keywords: Aggregatibacter actinomycetemcomitans; Cacao beans; antibacterial effect Correspondence: Hendrik Setia Budi, Department of Oral Biology, Faculty of Dental Medicine, Universitas Airlangga. Jln. Mayjend. Prof. Dr. Moestopo no. 47 Surabaya 60132, Indonesia. E-mail: hendriksetiabudi@gmail.com and Gram-negative bacteria. One of the Gram-negative anaerobic bacteria that plays a role in the formation of subgingival plaque causing periodontitis is Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans).1,4 Antibacterial effect of 70% ethanol and water extract of cacao beans (Theobroma cacao L.) on Aggregatibacter actinomycetemcomitans Ayu Rafania Atikah, Hendrik Setia Budi, and Tuti Kusumaningsih Departement of Oral Biology Faculty of Dental Medicine, Universitas Airlangga Surabaya - Indonesia Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 materials and method This study was an in vitro laboratory experimental research using randomized post test only control group design. This study was conducted at the Laboratory of Microbiology, Faculty of Dental Medicine, Universitas Airlangga from June to October 2015. Samples were A. actinomycetemcomitans bacteria taken from the microbiology laboratory, Faculty of Dental Medicine, Universitas Airlangga, and then cultured in Brain Heart Infusion Broth media (BHIB). Minimal sample size eligible to be analyzed is determined by Lemeshow formula, as many as five. Cocoa beans contain polyphenolic flavonoid of flavonol class, namely monomeric catechin and epicatechin flavonoids as well as proanthocyanidin polymerized flavonoids.10 Flavonols are classified into a flavonoid group composed of several molecules of phenols (polyphenols).11 Flavonols contained in cocoa stimulate peripheral blood mononuclear cells to secrete interleukin-5 (IL-5 ) and stimulate immunoglobulin A (IgA) production that protects the oral cavity from Streptococcus mutans.8 y Manufacture of 70% ethanol extract of cocoa beans and water extract of cocoa beans was conducted. The 70% ethanol extract of cocoa beans and the water extract of cocoa beans were obtained from Integrated Service Unit (UPT) Materia Medika Batu, East Java. Cocoa beans used were non-fermented cocoa beans and macerated using 70% ethanol and water solvent. Having obtained the 70% ethanol extract of cocoa beans and the water extract of cocoa beans, the preparation of A. actinomycetemcomitans bacteria then was performed by suspending the BHIB media until turbidity standard was equivalent to 0.5 Mc Farland (1.5 × 108 CFU/ ml). Manufacture of the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at various concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125% was performed using serial dilution method. Cocoa beans can also inhibit the growth of Streptococcus mutans at an effective concentration of 12.5%.12 The extract water of cocoa beans and the 70% ethanol extract of cocoa beans even have antibacterial activity against Escherichia coli and Bacillus subtilis. A research on cocoa beans extracted with the ethanol 70% shows a higher antimicrobial activity against Streptococcus mutans and C. albicans bacteria than with water.13 Cocoa, furthermore, will generate a different antibacterial activity when extracted with different polarities of its solvents.14 Extraction method varies depending on the samples to be tested. Several different solvents used include water, 70% ethanol, 70% methanol, acetonitrile, diethyl ether, and acetone. introduction Periodontal disease is an infectious disease of the oral cavity that can be suffered by teens and adults. Based on Health Research (RISKESDAS) in 2013, the prevalence of oral and dental problems, including periodontal disease reached 25.9%. The most common periodontal tissue diseases are gingivitis and periodontitis.1-3 A. actinomycetemcomitans are Gram-negative bacteria that have a small, non-motile, capnophilic, fermentative coccobacillus form. A. actinomycetemcomitans can be found in dental plaque, periodontal pockets, and buccal mucosa in 36% of patients in the normal population.1 A.actinomycetemcomitans release virulence factors, deliver adhesion on oral surfaces, inactivate the host immune response, as well as induce inflammation and tissue Periodontitis is an infectious disease resulting in inflammation of the tissues supporting teeth, gingival attachment loss progressively, and bone loss. The disease is caused by the induction of facultative anaerobic bacteria 105 105 Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 damage. A. actinomycetemcomitans are also frequently detected in periodontal tissues of healthy people and then can be grouped into a part of the normal flora in oral cavity.5 Polyphenol level, furthermore, will be higher when extracted with a lower solvent polarity. Solvent water will tend to dissolve inorganic compounds and salts of acids or bases.14 The use of water compared to the 70% ethanol as cosolvent can generate higher residual polyphenols, so the solvent water is considered to be better because it can maintain a high quantity of beneficial compounds.17 Currently, some herbal ingredients have been developed as an alternative to medicine ingredients, one of which is cocoa beans. Indonesia ranks on the third position in the world cocoa producers. Cocoa production in Indonesia has the potential to be improved, but it depends on political and local economic factors.6 The size of cocoa growing areas in Indonesia in 2002 has reached 776, 900 hectares scattered in all provinces, except in the capital city of Jakarta (DKI).7 This study aimed to determine whether there were differences in antibacterial activity between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria. Cocoa is composed of stems, fruits and seeds, flowers, roots, bark seeds, and leaves. introduction Cocoa (Theobroma cacao L.) has polyphenolic compounds, such as catechin, epicatechin, anthocyanin, proanthocyanidin, phenolic acid, condensed tannins, flavonoids, and other small compounds.8 Cocoa polyphenols are useful as an antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial.9 Generally, the majority of people consume cocoa only on the seeds alone. Several researches also have shown that cocoa bean skin and cocoa beans can be used as antibacterial ingredients.7 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 results 12,5% 12,5% dilution were planted in Luria Berthani agar media using streak technique as a cross-check to see the growth of bacterial colonies. It was necessary to do because of dark-colored extract material and turbidity occured at any concentration. This research used A. actinomycetemcomitan bacteria incubated for 1 x 24 hours at 37° C in the BHIB media, synchronized with the 0.5 McFarland standard. Materials used were non-fermented cocoa beans from Kota Batu, Malang. Next, cocoa extracts were made in UPT Materia Medika Batu in East Java by maceration method using 70% ethanol and water. Furthermore, based on the observation results on the number of bacterial colonies grown in the Luria Berthani media expressed in colony forming units (CFU), 70% ethanol extract of cocoa beans and water extract of cocoa beans could be able to inhibit and kill A. actinomycetemcomitan bacteria (Table 2). Based on the analysis results of chemical compounds contained in the 70% ethanol extract of cocoa beans as well as in the water extract of cocoa beans, there were different dosages of flavonoids, catechins, epicatechin, anthocyanin, and proanthocyanidin contained (Table 1). Catechin, epicatechin, and anthocyanin contained in the 70% ethanol extract of cocoa beans were higher than in the water extract of cocoa beans. Meanwhile, flavonoids and proanthocyanidin contained in the water extract of cocoa beans were higher than in the 70% ethanol extract of cocoa beans. Figure 2. a) Streak results of the 70% ethanol extract of cocoa b bacteria at any concentration as well as the controls grow results of the water extract of cocoa beans against A concentration as well as the controls grown in the Luria the 70% ethanol extract of cocoa beans against A. concentration as well as the controls grown in the Luria the water extract of cocoa beans against A. actinomycetem well as the controls grown in the Luria Berthani media. ure 2. a) Streak results of the 70% ethanol extract of cocoa bean bacteria at any concentration as well as the controls grown in results of the water extract of cocoa beans against A.act concentration as well as the controls grown in the Luria Ber the 70% ethanol extract of cocoa beans against A. acti concentration as well as the controls grown in the Luria Ber the water extract of cocoa beans against A. actinomycetemcom well as the controls grown in the Luria Berthani media. materials and method actinomycetemcomitan bacteria at a concentration o 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.125 o. Chemical compounds 70% ethanol extract of cocoa beans Water ex b Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% A B Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans Table 1 The analysis results of the chemical compounds contained ble 1 The analysis results of the chemical compounds contained in The reading of the results of antibacterial activity in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against the growth of A. actinomycetemcomitans bacteria was conducted by measuring the number of colonies grown on bacteria subculture as much as 0.1 ml of each tube as well as a positive control and a negative control on the Luria Berthani media. Planting on the Luria Berthani media was conducted using spreader, and incubated at 37 ° C for 24 hours. No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Proanthocyanidin 1.93% and the water extract of of cocoa beans o. Chemical compounds 70% ethanol extract of cocoa beans Water ex Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 The results showed 90% of A. actinomycetemcomitans bacterial growth was inhibited compared with the positive control used as MIC. The results also indicated 99.9% of A. materials and method 70% ethanol (ethyl alcohol) is a solvent which has a low boiling point and is widely used by industries. 70% ethanol has a boiling point of 70° C so that extraction temperature is used to attract all the components in the basic materials.15 70% ethanol is widely used as a solvent of various chemical or natural materials devoted to drugs. In in vitro research, 70% ethanol can disrupt transport ions, namely Na +, K +, and ATP. The use of 70% ethanol in antibacterial material has a risk to human health, especially in liver because of the residual concentration and ethanol exposure.16 The determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was started from the preparation of the test tube as much as 9 tube. Six test tubes were filled with bacterial suspensions of A. actinomycetemcomitans as much as 0.05 ml that had been standardized to 0.5 Mc Farland and mixed with BHIB media as well as the extracts at the various concentrations (100%, 50%, 25%, 12.5%, 6.25 %, and 3.125). The test tube K + (as positive control) containing 0.05 ml bacterial suspension of A. actinomycetemcomitans were planted in the BHIB media, while the test tube K- (as negative control) contained only media BHIB and control extracts. Control is useful to make sure that no bacterial contamination in the media. 106 Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% Table 1. The analysis results of the chemical compounds contained and the water extract of of cocoa beans Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentrati 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 materials and method actinomycetemcomitans bacterial death compared with the positive control was used as MBC. MIC and MBC were determined by measuring the number of colonies grown on the Luria Berthani media manually and expressed as CFU/ ml. The calculation was repeated five times by three different observers, and then the mean values were measured. B A A Processing data was conducted using a statistical analysis test for normality with Kolmogorov-Smirnov test to see whether the data obtained were normally distributed or not. Next, homogeneity test using Levene test was performed. A statistical test for antibacterial power difference in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration then was carried out using Independent t-test. Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentrati 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% ure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. actinomycetemcomitan bacteria at a concentration 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.12 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. materials and method Proanthocyanidin 1.93% 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% able 1. The analysis results of the chemical compounds contained in and the water extract of of cocoa beans gure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. actinomycetemcomitan bacteria at a concentration o 50% in tube 2; a concentration of 25% in tube 3; and a c concentration of 6.25% in tube 5; and a concentration of 3.125 o. Chemical compounds 70% ethanol extract of cocoa beans Water ex Flavonoids 1.32% 2 Catechins 2.01% 1 Epicatechin 1.36% 1 Anthocyanins 1.74% 1 Proanthocyanidin 1.93% 2 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% A B Figure 1. Serial dilution (A) the 70% ethanol extract of cocoa beans and (B) the water extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentration of 100% in tube 1; a concentration of 50% in tube 2; a concentration of 25% in tube 3; and a concentration of 12.5% in tube 4; a concentration of 6.25% in tube 5; and a concentration of 3.125% in tube 6. Table 1. The analysis results of the chemical compounds contained in 70% ethanol extract of cocoa beans and the water extract of of cocoa beans No. Chemical compounds 70% ethanol extract of cocoa beans Water extract of cocoa beans 1. Flavonoids 1.32% 2.18% 2. Catechins 2.01% 1.22% 3. Epicatechin 1.36% 1.08% 4. Anthocyanins 1.74% 1.67% 5. Proanthocyanidin 1.93% 2.51% Table 1. The analysis results of the chemical compounds contained and the water extract of of cocoa beans Figure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at a concentratio 50% in tube 2; a concentration of 25% in tube 3; and concentration of 6.25% in tube 5; and a concentration of 3 No. Chemical compounds 70% ethanol extract of cocoa beans Wate 1. Flavonoids 1.32% 2. Catechins 2.01% 3. Epicatechin 1.36% 4. Anthocyanins 1.74% 5. Proanthocyanidin 1.93% 3,125% 12,5% 25% 50% 3,125% 6,25% 25% 50% 100% 100% 6,25% able 1. The analysis results of the chemical compounds contained in and the water extract of of cocoa beans gure 1. Serial dilution (a) the 70% ethanol extract of cocoa beans and against A. results The comparative test on the MIC and MBC in the 70% ethanol extract cocoa beans and the water extract of cocoa beans was conducted using serial dilution method. The results showed that the extracts at the concentrations of 100%, 50%, 25%, and 12.5% contained no bacterial growth. Meanwhile, the mean bacterial growth in the 70% ethanol extract cocoa beans at the concentration of 6.25% was 11.4. On the other hand, the mean bacterial growth in the water extract of cocoa beans at the same concentration was 7.8 (Table 2). Serial dilution method is a method usually used in a comparative research of MIC and MBC in both the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A.actinomycetemcomitan bacteria (Figure 1). Before the antibacterial power difference analysis test on the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria was performed, the normality test using Kolmogorov- Smirnov test as well as the homogeneity test using Levene test were conducted in each group. The results of the During the observation of MIC and MBC in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at every concentration, the results of serial 107 107 ration of ube 4; a ration of ube 4; a Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 g y 50% in tube 2; a concentration of 25% in t concentration of 6 25% in tube 5; and a concen against A. actinomycetemcomitan bacteria at a 50% in tube 2; a concentration of 25% in t concentration of 6 25% in tube 5; and a concen Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results o the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration a 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% A Figure 2. results a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results o the water extract of cocoa beans against A actinomycetemcomitan bacteria at any concentration a 3,125% 12,5% 25% 50% 3,125% 6,25% 12,5% 25% 50% 100% 100% 6,25% B C D Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media. 1 1 B C D D Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomita bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) strea results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; d) replanting results o th t t t f b i t A ti t it b t i t t ti Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. results actinomycetemcomita bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) strea results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; c) replanting results o the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at an concentration as well as the controls grown in the Luria Berthani media; d) replanting results o Figure 2. a) Streak results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; b) streak results of the water extract of cocoa beans against A.actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; c) replanting results of the 70% ethanol extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media; d) replanting results of the water extract of cocoa beans against A. actinomycetemcomitan bacteria at any concentration as well as the controls grown in the Luria Berthani media. 11 well as the controls grown in the Luria Berthani media. 11 well as the controls grown in the Luria Berthani media. Table 2. The number of A. actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentrations of 70% ethanol extract of cocoa beans and water extract of cocoa beans Treatment Group N The means number of the bacterial colonies (x108 CFU/ml) 70% Ethanol extract Water extract Positive control Negative control At the concentration of 100% 5 0 0 109 0 At the concentration of 50% 5 0 0 At the concentration of 25% 5 0 0 At the concentration of 12.5% 5 0 0 At the concentration of 6.25% 5 11.4 7.8 At the concentration of 3.125% 5 27.2 18 Note: n: number of replication; 0: no growth on the negative control; 109: the number of bacterial colonies grown as the positive control 1 g 1 well as the controls grown in the Luria Berthani media. Table 2. The number of A. actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentrations of 70% ethanol extract of cocoa beans and water extract of cocoa beans g well as the controls grown in the Luria Berthani media. The number of A. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 discussion How to cultivate antibacterial materials, thus, is a thing that needs attention. Antibacterial material can be extracted by using various methods, one of which is by solvent extraction (maceration). Solvents widely used for polyphenol extraction method are water and 70% ethanol. Cocoa, on the other hand, will yield a different antibacterial activity when extracted with a different solvent polarity.14 This research was an in vitro research aimed to see the difference in antibacterial power beween the 70% ethanol extract of cocoa beans and the water extract of cocoa beans against A. actinomycetemcomitan bacteria. Antibacterial agents used in this research were non-fermented cocoa beans since non-fermented cocoa beans actually have higher antibacterial effects than fermented ones. Fermentation and drying processes, however, can reduce the active components of natural materials, such as polyphenol.18 This research also used a serial dilution method with the concentrations of 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%. A. actinomycetemcomitan bacteria grown on the BHIB media were considered as the positive control (+), whlie the bacteria grown on the BHIB media and the control extracts were considered as as the negative control (-). Serial dilution method was used because it can give the significant calculation results of microorganism colonies at small concentrations. Thus, if the serial dilution method is not conducted, there will be a number of colonies stacked so that the calculation of the colonies will be not accurate.19 Serial dilution method has widely been used for many years.20 Ethanol is a polar solvent that dissolves polar compounds, while water is an inorganic solvent that dissolves inorganic materials. In addition, cacao bean is an inorganic material because it is derived from plants. To dissolve the cocoa beans using ethanol, therefore, will take a long process because of evaporation process. The process of evaporation can affect the polyphenol content in the extract. Water, on the other hand, is a solvent that can maintain a high quantity of essential compounds contained in natural materials.17 Water solvent will dissolve inorganic compounds and salts of acids and bases.14 Cocoa beans diluted with water is more soluble and requires a shorter time than cocoa diluted with ethanol. The analysis results of chemical compounds in the 70% ethanol extract of cocoa beans contain flavonoids (1.32%), catechin (2.01%), epicatechin (1.36%), anthocyanin (1.74%), and proanthocyanidin (1.93%). The water extract of cocoa beans contain flavonoids (2.18%), catechin (1.22%), epicatechin (1.08%), anthocyanin (1.67%), and proanthocyanidin (2.51%). Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10.20473/j.djmkg.v49.i2.p104-109 results actinomycetemcomitan bacterial colonies grown in the Luria Berthani media at various concentration of 70% ethanol extract of cocoa beans and water extract of cocoa beans Table 3. The mean and standard deviation of the number of bacterial colonies of A. actinomycetemcomitans Treatment control Mean ± SD Significance 70% Ethanol extract Water extract At the concentration of 6.25% 11.4 ± 0.54772 a 7.8 ± 0.83666 b P = 0.000 At the concentration of 3.125% 27.2 ± 1.64317 a 18.0 ± 1.22474 b P = 0.000 Note: a b: significant difference Table 3. The mean and standard deviation of the number of bacterial colonies of A. actinomycetemcomitans normality and homogeneity tests showed that data obtained in the water extracts of cocoa beans at the concentrations of 6.25% and 3.125% were normally distributed and homogeneous (p>0.05). Whereas, data obtained in the 70% ethanol extract of cocoa beans at the concentration of 6.25% were not normally distributed (p <0.05), and at the concentration of 3.125% was not homogeneous (p<0.05). Next, Kruskal-Wallis test was carried out to test significant differences in antibacterial activities between the 70% ethanol extract of cocoa beans and the water extract of cocoa beans since there were groups which were not normally distributed and not homogeneous. The results showed value of p was less than 0.05 (p = 0.000), indicating that there was a difference in antibacterial activity between the Atikah, et al./Dent. J. (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 108 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentrations of 6.25% and 3.125% (Table 3). bacterial enzyme system. Proanthocyanidin, furthermore, is condensed tannins that can inhibit the formation of cell walls and lead to termination of the crosslinking peptide that combines the glycan chains of peptidoglycan in another chain, causing cell membrane damage.28 references 16. Darmono. Farmasi forensik dan toksikologi. Jakarta: UI Press; 2009. p. 23. 1. Carranza P, Klokkevold F, Newman M, Takei H. Carranza’s clinical periodontology. St. Louis, MO: Saunders Elsevier; 2009. p. 187- 192 17. Boakye S. Levels of selected pesticide residues in cocoa beans From Ashanti and Brong Ahafo Regions of Ghana. Dissertation. Ghana: Faculty of Physicial Science College of Science; 2012. 2. Riset Kesehatan Dasar (Riskesdas) Nasional. Jakarta: Badan Penelitan dan Pengembangan Kesehatan, Departemen Kesehatan Republik Indonesia; 2013. p. 113. 18. Smullen J, Koutsou GA, Foster HA, Zumbe A,Storey DM. The antibacterial activity of plant extract containing polyphenols against Streptococcus mutans. Caries Res 2007; 41: 342-9. 3. Chauhan VS, Chauhan RS, Devkar N, Vibhute A, More S. Gingival and periodontitis diseases in children and adolescents. J Dent & Allie Sci 2012 ; 1(1): 26. 19. Mejos Jay Al, CaraignJ.W, De Pano J, Labador AV, Macapagal EM. Bacterial colony isolation using serial dilution techniques. Quezon City: Institute of Biology, College of Science, University of the Phillipines; 2010. p. 6. 4. Newman M, Takei H, Klokkevold P, Carranza F. Carranza’s clinical periodontology. St. Louis, MO: Saunders Elsevier; 2012. p. 185- 93. 20. Seeley HW, VanDemark PJ. Selected exercises from microbes in action a laboratory manual of microbiology. 3 rd ed. Sanfrancisco: Freeman and Company; 1981. p. 37-41. 5. Umeda EJ, Priscila LL, Maria RL, Marcia PAM. Differential transcription of virulence genes in Aggregatibacter actinomycetemcomitans serotypes. Brazil: Departement of microbiology, Institute of Biomedical Sciences, University of Sao Paulo; 2013. p. 4-6. 21. V.S.T. Saito, T.F. dos Santos, C.G. Vinderola, C. Romano, J.R. Nicoli, L.S. Araújo, M.M. Costa, J.L. Andrioli, A.P.T. Uetanabaro. Viability and Resistance of Lactobacilli Isolated from Cocoa Fermentation to Simulated Gastrointestinal Digestive Steps in Soy Yogurt. Journal of food and science 2014; 79(2): doi. 10.1111/1750-3841.12326. 6. Gu F, Tan L , Wu H, Fang Y , Xu F , Chu Z, Wang Q. Comparison of cocoa beans from China, Indonesia and Papua New Guinea. Foods 2013; 2(2): 183-97. of food and science 2014; 79(2): doi. 10.1111/1750-3841.12326. 22. Kenneth T. Structure and function of bacterial cells. Online Textbook of Bacteriology2009; 5. 7. Misnawi. Pemanfaatan Biji Kakao sebagai Sumber Antioksidan Alami. Jember: Pusat Penelitian Kopi dan Kakao Indonesia; 2005. p. 6. 23. Zainal H, Made A, Kasno, Anggraini AD. Uji aktivitas antibakteri propolis lebah madu Trigona spp. Prosiding Seminar Nasional Indonesia, 2006; p. 204-14. 8. references Arlorio M, Coisson JD, Travaglia F, Varsaldi F, Miglio G, Lombardi G, Martelli A. Antioxidant and biological activity of phenolic pigments from Theobroma cacao hulls extracted with supercritical CO2. Food Research International 2005; 38(8-9): 1009-14. 24. Ariza BTS, Mufida D, Fatima N. In vitro antibacterial activity of cocoa ethanolic extract against Escherichia coli. International Food Research Journal 2014; 21(3): 935-40. CO2. Food Research International 2005; 38(8-9): 1009-14. 9. Hii CL, Law CL, Suzannah S, Misnawi, Cloke M. Polyphenols in cocoa (Theobroma cacao L.). As J Food Ag-Ind 2009; 2(04): 702- 22.f 25. Misnawi, Wulandari P, Suswati E, Rianul A. Antibacterial effect of ethanol extract cocoa beans (Theobroma cacao) on growth in vitro By Shigella dysentriae. Jurnal Medika Planta 2012; 1(5): 73. 10. Sulistyowati, Misnawi. Effects of alkali concentration and conching temperature on antioxidant activity and physical properties of chocolate. International Food Research Journal 2008; 15(3): 297- 304. 26. Sabir A. Aktivitas antibakteri flavonoid propolis Trigona sp terhadap bakteri Streptococcus mutans (in vitro). Majalah Kedokteran Gigi (Dental Journal) 2005; 38(2): 135-41. 26. Sabir A. Aktivitas antibakteri flavonoid propolis Trigona sp te 27. Chinami Hirao, Eisaku Nishimura, Masanori Kamei, Tomoko Ohshima, Nobuko Maeda. Antibacterial effects of cocoa on periodontal pathogenic bacteria. Journal of Oral Biosciences 2010; 52(3): 283-291. 11. Lee SY, Yoo SS, Lee MJ, Kwon IB, dan Pyun YR. Optimization of Nib Roasting in Cocoa bean processing with lotte-better taste and color process. Food Sci Biotechnol 2001; 10: 286-93. 12. Purnamasari DA, Munadziroh E, Yogiartono RM. Konsentrasi ekstrak biji kakao sebagai material alam dalam menghambat pertumbuhan Streptococcus mutans. Jurnal PDGI 2010; 59(1): 14-8. 28. Duskova M, Karpiskova R. Antimicrobial resistance of Lactobacilli Isolated from food. Czech J Food Sci 2013; 31(1): 27-32. 29. Surbhi Leekha, Christine L. Terrell, and Randall S. Edson. General principles of antimicrobial therapy. Mayo Clin Proc 2011;86(2): 156–167. 13. Gianmaria F. Ferrazzano, Ivana Amato, Aniello Ingenito, Antonino De Natale, Antonino Pollio. Anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia 2009; 80(5): 255–262 30. Akinyemi KO, Oluwa OK, Omomigbehin EO. Antimicrobial activity of crude extracts of three medicinal plants used in south-west Nigerian folk medicine on some food borne bacterial pathogens. African Journal of Traditional, Complementary and Alternative Medicines 2006; 3(4): 13-22. 14. John NA, Fang Z, Kebitsamang JM, Mohamed LB, Camel L. Quantification of Total Polyphenolic Content and Antimicrobial Activity of Cocoa (Theobroma cacao L.) Bean Shells. discussion (Majalah Kedokteran Gigi) 2016 June; 49(2): 104–109 109 109 15. Manuchair Ebadi. Pharmacodynamic basic of herbal medicine. 2nd ed. New york: Taylor & Francis; 2007.p. 647. discussion Proanthocyanidin contained in cocoa beans was 58%, catechin amounted to 37%, and anthocyanin amounted to 4%, thus indicating that the water extract of cocoa beans contain more useful chemical compounds than the ethanol extract of cocoa beans.10 In the antibacterial activity test, properties owned by the cell wall of bacteria may affect the ability of cocoa bean extracts to inhibit tested bacterial growth.21 A.actinomycetemcomitan bacteria are gram-negative bacteria that have a cell wall in the form of peptidoglycan surrounded by a membrane structure, called as outer membrane. Outer membrane of Gram-negative bacteria has lipopolysaccharide or endotoxin components that can prevent an antibacterial agent to penetrate into the cells.22,23 In the layers of lipopolysaccharide, Gram-negative bacteria have a system of selection against strange substances so that Gram negative bacteria are more resistant to strange substances than Gram positive bacteria.21 The presence of polyphenols, such as flavonoids in cocoa beans can penetrate the membrane of Gram-negative bacteria to mediate the cell response.24 In the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 6.25%, moreover, there was still bacterial colony growth on the Luria Berthani media after incubated for 1x24 hours. It means that not all bacteria die, or there are barriers to the growth of colonies by 90%.28 Nevertheless, in the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at the concentration of 12.5%, there was no colony growth. It indicates that the 70% ethanol extract of cocoa beans and the water extract of cocoa beans at this concentration could kill bacteria by 99.9% of the total average bacteria that managed to grow on the positive control and were considered as bactericide.29,30 Flavonoids have antibacterial activity through the barrier function of bacterial DNA gyrase so that replication capabilities and bacterial translation can be inhibited.25 Flavonoids may cause damage to the permeability of bacterial cell wall, microsomes, and lysosomes as a result of the interaction of flavonoids with bacterial DNA.26 Catechins, moreover, are natural polyphenolic compounds and secondary metabolites included in the constituent group of tannins.21 Catechins may damage cytoplasmic membrane causing the release of important metabolites that inactivate It can be concluded that water and 70% ethanol extract of cocoa beans have antibacterial activities against A. actinomycetemcomitans. Both the extracts also have the same MIC and MBC at concentrations of 6.25 and 12.5%. Atikah, et al./Dent. J. Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI 10 20473/j dj k 49 i2 104 109 references Pakistan Journal of Nutrition 2012; 11(7): 574-579 Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 56/DIKTI/Kep./2012. Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG DOI: 10 20473/j djmkg v49 i2 p104 109
https://openalex.org/W3005710798	https://academic.oup.com/nar/article-pdf/48/9/4658/33221338/gkaa229.pdf	English	null	Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of Erythropoietic Protoporphyria	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	12,354	ABSTRACT Erythropoietic protoporphyria (EPP; OMIM # 177000) is a rare autosomal recessive disorder (1), that is caused by deficiency in the heme biosynthesis enzyme ferrochelatase (FECH, EC 4.99.1.1) (Figure 1A) (2,3). During heme biosynthesis, FECH catalyses the incorporation of iron into protoporphyrin IX (PPIX) to form heme. With over 80% of heme produced in red blood cells progenitors, the bone marrow is the major site of biosynthesis. Kidneys and liver are also minor sites of heme biosynthesis (4). A lack of FECH means that PPIX continuously accumulates during erythroid maturation, resulting in PPIX circulating in red blood cells of the bloodstream. Upon short exposure to vis- ible light, PPIX produces reactive oxygen species leading to lipid peroxidation, cell membrane damage and inflamma- tion. This causes patients to suffer from burn-like injuries of the endothelial cells of the blood vessels and the surround- ing tissue, associated with acute severe pain; some will de- velop hepatic complications with deposits of PPIX crystals and are at risk of liver failure (5,6). Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute photo- toxic reactions after sunlight exposure. It is caused by a deﬁciency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss- of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthe- sis, whereby an increase of a few percent would pro- vide therapeutic beneﬁt. However, successful appli- cation of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl- chemistry increase FECH levels in cells. We conju- gated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution proﬁles, with in- creased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of cor- rectly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splic- ing dysregulation in the bone marrow. ( ) EPP in almost all patients is associated with an alteration in both FECH alleles. 4658–4671 Nucleic Acids Research, 2020, Vol. 48, No. 9 doi: 10.1093/nar/gkaa229 4658–4671 Nucleic Acids Research, 2020, Vol. 48, No. 9 doi: 10.1093/nar/gkaa229 Published online 20 April 2020 C⃝The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria Franc¸ois Halloy1, Pavithra S. Iyer1, Paulina ´Cwiek1, Alice Ghidini1, Jasmin Barman-Aks¨ozen2, Nicole Wildner-Verhey van Wijk3, Alexandre P.A. Theocharides3, Elisabeth I. Minder4, Xiaoye Schneider-Yin2, Daniel Sch¨umperli 1,* and Jonathan Hall 1,* 1Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland, 2Institute of Laboratory Medicine, Triemli Hospital, Zurich, Switzerland, 3Department of Medical Oncology and Hematology, University Hospital and University of Zurich, Zurich, Switzerland and 4Department of Internal Medicine, Triemli Hospital, Zurich, Switzerland eceived February 14, 2020; Revised March 25, 2020; Editorial Decision March 26, 2020; Accepted April 09, 2020 To whom correspondence should be addressed. Tel: +41 44 633 74 35; Fax: +41 44 633 13 69; Email: jonathan.hall@pharma.ethz.ch Correspondence may also be addressed to Daniel Sch¨umperli. Email: daniel.schuemperli@pharma.ethz.ch ABSTRACT This aberrant splicing is increased by the c.315-48C variant which is present in trans to a hypomorphic FECH allele in 95% of EPP patients and in a few percent of the general population. (C) The transgenic murine Emi model shows the aberrant splicing of human patients, but additionally skips exon 3 in most of the FECH transcripts. is a viable means to restore sufficient FECH expression for haplo-sufficiency in patients. SSOs composed of LNA (locked nucleic acids) and morpholino chemistries are re- ported to correct the aberrant splicing of the FECH mRNA ex vivo in patients’ blood cells (8,9), but this approach has not yet been described in vivo. and in the Emi mouse. Using different linker strategies, we conjugated the SSO to three targeting moieties: thio- cholesterol (21,22), stearic acid (23) and a bone marrow- homing peptide (24). We first assessed their metabolic sta- bility in vitro, and then their distribution, metabolism and splice-switching activity in Emi/wt heterozygous mice us- ing mass spectrometry and chemical ligation PCR methods. Whereas the oligonucleotide moiety was metabolically sta- ble, the conjugated groups underwent degradation in vitro and in vivo, albeit through different pathways. The conju- gates also altered in vivo-distribution and splice-switching activity of the SSO. The parent SSO was detected in all tis- sues tested, and showed splice-switching of FECH RNA in liver, kidney and lung. The cholesterol group increased liver transport by seven-fold, but this SSO was not more potent than the parent SSO. The stearic-acid conjugate increased liver delivery by a lesser degree, but raised FECH transcript levels there by 60%. Notably, the homing-peptide increased bone marrow accumulation by seven-fold, but it hardly af- fected FECH splicing, whereas the cholesterol group, which did not improve transport to bone marrow, increased levels of the correct FECH transcript by 80%. y SSOs are oligonucleotides of about 20 nucleotides (nt) in length, that bind to a pre-mRNA and modu- late its splicing (10,11). Several are in development to treat rare diseases caused by aberrant RNA maturation, reading-frame shifts, or exon skipping. The most promi- nent example is nusinersen, an approved treatment of spinal muscular atrophy (SMA) that mediates exon in- clusion in the SMN2 pre-mRNA (12,13). Nusinersen is composed of phosphorothioated (PS)-2′-O-methoxyethyl (MOE)-ribonucleotides (14), which endow the drug with high metabolic stability and extended distribution in vivo. ABSTRACT On one, FECH expression is atten- uated by a deleterious mutation (7); on the other, a single nucleotide polymorphism (SNP) in intron 3 (c.315-48T>C) bolsters the use of a cryptic 3′ splice site, that produces an aberrant mRNA incorporating additional sequence preced- ing exon 4 (Figure 1B). Stop codons in this sequence re- sult in degradation of the transcript by nonsense-mediated mRNA decay (NMD) (3). The combined effects of these al- leles are reduced levels (>65%) of FECH activity, which ren- ders patients symptomatic. As the c.315-48C allele turns a carrier of a loss-of-function (LoF) allele from asymptomatic to symptomatic, a splice-switching oligonucleotide (SSO) Nucleic Acids Research, 2020, Vol. 48, No. 9 4659 Figure 1. A single-nucleotide polymorphism causes aberrant splicing of FECH mRNA. (A) Biosynthesis of heme from glycine and succinyl CoA comprises eight sequential steps occurring in the cytosol and in the mitochondria. FECH is the last enzyme of the pathway and catalyzes the incorporation of iron into protoporphyrin IX. EPP patients present a dramatic decrease of FECH, resulting in reduced heme production and accumulation of protoporphyrin IX in cells. (B) FECH pre-mRNA contains a weak cryptic splice site in intron 3 (c.315-63.) close to a single nucleotide polymorphism (c.315-48T in healthy individuals). Utilization of this splice site yields a transcript which is a substrate for NMD. This aberrant splicing is increased by the c.315-48C variant which is present in trans to a hypomorphic FECH allele in 95% of EPP patients and in a few percent of the general population. (C) The transgenic murine Emi model shows the aberrant splicing of human patients, but additionally skips exon 3 in most of the FECH transcripts. Figure 1. A single-nucleotide polymorphism causes aberrant splicing of FECH mRNA. (A) Biosynthesis of heme from glycine and succinyl CoA comprises eight sequential steps occurring in the cytosol and in the mitochondria. FECH is the last enzyme of the pathway and catalyzes the incorporation of iron into protoporphyrin IX. EPP patients present a dramatic decrease of FECH, resulting in reduced heme production and accumulation of protoporphyrin IX in cells. (B) FECH pre-mRNA contains a weak cryptic splice site in intron 3 (c.315-63.) close to a single nucleotide polymorphism (c.315-48T in healthy individuals). Utilization of this splice site yields a transcript which is a substrate for NMD. ABSTRACT PS-MOE oligonucleotides traffic to the liver, kidneys and spleen at therapeutically-useful concentrations after sys- temic administration (15,16). They have also been de- tected in bone marrow in rodents, though SSOs of vari- ous chemistries were inactive in the bone marrow of a re- porter mouse model (17), possibly due to poor cellular- uptake and/or to the high turnover of red blood cell progen- itors (18). This obstacle impacts an SSO strategy for EPP, but may be addressable thanks to advances in the use of conjugated groups for organ-targeted delivery (e.g. GalNAc sugars (19)). This study of splice-switching oligonucleotide conjugates complements recent investigations of the pharmacokinetic and pharmacodynamic properties of antisense and siRNA conjugates (25,26). The results reinforce the notion that increased delivery does not necessarily translate to im- proved potency. They also advance the prospect of an oligonucleotide-based therapy for correction of the FECH splicing defect at the origin of EPP. We recently developed a transgenic mouse model for human EPP (Emi mouse) (20), incorporating the c.315- 63 cryptic splice site of FECH from human intron 3. The mouse displays the aberrant splicing of exon 4; however, for reasons unknown, exon 3 is deleted from most transcripts (Figure 1C). Hence, SSOs that correct the aberrant splic- ing of the transgene will not increase levels of FECH ac- tivity, nor attenuate levels of circulationg PPIX. Here, we describe a 22-nt PS-MOE SSO, which binds to the c.315- 48 SNP and corrects splicing of FECH transcripts in cells Small scale synthesis of MOE-PS splice-switching oligonu- cleotides Small scale synthesis of MOE-PS splice-switching oligonu- cleotides Small scale oligonucleotide synthesis was conducted on a MerMade 12 synthesizer (BioAutomation Inc) on a 50 nmol scale on 500 ˚A UnyLinker CPG (ChemGenes) 4660 Nucleic Acids Research, 2020, Vol. 48, No. 9 with standard synthesis conditions. DMT cleavage was accomplished with a solution of 3% dichloroacetic acid in dichloromethane (V/V). 2′-O-MOE phosphoramidites (ThermoFisher Scientific) were prepared at 0.08 M con- centration in dry acetonitrile. 5-(Benzylthio)-1H-tetrazole (Carbosynth) was used at 0.24 M concentration in dry acetonitrile to activate the phosphoramidites for cou- pling. Sulfurization was carried out with a 0.05 M so- lution of 3-((N,N-dimethylaminomethylidene)amino)-3H- 1,2,4-dithiazole-5-thione (Glen Resarch #40-4037) in a 1:1 mixture of pyridine/acetonitrile. Capping of failed se- quences was achieved with acetic anhydride in tetrahydrofu- ran (THF) and 16% N-methylimidazole in THF. Following cycle parameters were used: Deblock 2 × 60 s; Coupling 2 × 180 s; Sulfurization 1 × 150 s; Capping 2 × 50 s. Analytical data are provided in Supplementary Figure S1. TCT CTA CCT GGT GTG TGG-3′, Reverse 5′-GAC AAT TCA TCC AGC AGC TTC-3′. A PCR mix containing 1 l FW primer (10 M stock), 1 l RV primer (10 M stock), 1 l dNTPs mix (Promega #U1515), 5 l 10× DreamTaq Buffer (ThermoFisher #EP0703), 0.25 l DreamTaq DNA Polymerase (ThermoFisher #EP0703) and 40.75 l ultra- pure water was prepared and mixed with 1 l of cDNA solu- tion diluted 1/3 in water. PCR program: 95◦C, 1 min; 30 cy- cles (95◦C, 30 s – 60◦C, 30 s – 68◦C, 30 s); 68◦C, 5 min. PCR products were loaded on a 2% agarose gel and bands quanti- fied using ImageJ software. Ratios were expressed as: inten- sity of the aberrant amplicon band / (intensities of aberrant + correct bands). Large scale synthesis of ORN1 and of conjugates ORNst, ORN84 and ORNch Cells were washed with DPBS and RNA extraction was carried out using Qiagen RNEasy kit according to manufacturer’s instructions (Qiagen #74106, #79254). cDNA was generated using PrimeScript RT kit (Takara #RR037A). For semi-quantitative RT-PCR; PCR primers specific for human FECH exon 2–exon 4 transcripts were used: Forward 5′-CAC AGA AAC AGC CCA GCA TG- 3′, Reverse 5′-GAC AAT TCA TCC AGC AGC TTC- 3′. A PCR mix containing 1.25 l FW primer (10 M stock), 1.25 l RV primer (10 M stock), 0.125 l dNTPs mix (Promega #U1515), 0.25 l 5× Q5 Polymerase Buffer (NEB #M0491), 0.25 l Q5 DNA Polymerase (NEB #M0491) and 15.75 l ultrapure water was prepared and mixed with 1 l of cDNA solution. PCR program: 98◦C, 30 s; 33 cycles (98◦C, 10 s – 58◦C, 20ms – 72◦C, 2 s); 72◦C, 2 min. PCR products were loaded on a 2% agarose gel and bands quantified using ImageJ software. Ratios were expressed as: intensity of the aberrant amplicon band/(intensities of aberrant + correct bands). Biological replicates are presented in Supplementary Figure S4. For RT-qPCR, a primer pair for correctly spliced FECH tran- script was used. A mix containing 4 l SYBR Mix 2×, 0.33 l primer pair (stock of 10 M each in water) and 3.17 l ultrapure water was prepared and mixed with 0.5 l cDNA solution. Primers were purchased from Microsynth (Balgach, Switzerland) and stored as pairs at 10 M each. Samples were run in technical triplicates and GAPDH was used as housekeeping control. Fold change calculations for the correctly spliced FECH transcript were made by com- paring each treatment group with the mock group. FECH FW primer: 5′-CCT ATT CAG AAT AAG CTG GCA CC-3′; FECH RV primer: 5′-CCT GCT TGG AAG TCC ATA TCT TG-3′. GAPDH FW primer: 5′-AGG TCG GTG TGA ACG GAT TTG-3′; GAPDH RV primer: 5′-TGT Large scale synthesis was conducted on a MerMade 12 syn- thesizer with minor changes compared to the small-scale procedure: sulfurization reagent was prepared at 0.1 M in- stead of 0.05 M. Modified cycle parameters were used: De- block 2 × 60 s; Coupling 1 × 600 s; Sulfurization 1 × 600 s; Capping 2 × 100 s (see also Supplementary Methods). An- alytical data of batches used for in vivo work are provided in Supplementary Figure S3. Synthesis of chemical ligation-qPCR primers Primers were synthesized on a MerMade 12 synthesizer fol- lowing a similar procedure with minor modifications to that described in the initial publication (27). Analytical data are provided in Supplementary Figure S2. Endogenous splicing correction and correct FECH transcript evaluation in K562 cells K562 cells were kept in culture at 37◦C, 5% CO2. Cells were plated at 200 000 cells/well in a 24-well plate in 0.5 ml IMDE (Sigma #I3390) containing 10% fetal bovine serum and 4 mM L-glutamine (Sigma #G7513). Oligonucleotides were transfected 1.5 h after plating using Lipofectamine™ 2000 (ThermoFisher, #11668027) following manufacturer’s instructions. Emetine (Sigma-Aldrich #E2375) was added in each desired well at a final concentration of 3 M 2.5 h hours after oligonucleotide treatment and 22 h before lysis and RNA extraction. Stability assay in mouse plasma 5 nmol of in-vivo ready oligonucleotides (UV purity > 99%, sodium salt) were spiked in 200 l of mouse K2-EDTA plasma (BioIVT #MSE02PLK2Y2N) and 20 l aliquots were taken immediately after mixing, and after 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6.5 h, 22 h and 26 h incuba- tion and gentle shaking at 37◦C. Aliquots were immedi- ately quenched with 350 l OTX Clarity Lysis buffer (Phe- nomenex #AL0-8579) and frozen at –20◦C. Clean-up was conducted all at once using the Clarity® OTX Extraction kit (Phenomenex #KS0-9253) according to manufacturer’s procedure for biological fluids with the following adjust- ments: formulation of all buffers with ammonium acetate in substitution of sodium phosphate; dissolution of the final eluate after evaporation to dryness in 100 l of TE buffer pH 8.0. Reconstituted samples were analysed qualitatively by LC–MS (Agilent 1200/6130 system) equipped with a Waters Acquity OST C-18 column. Oligonucleotide screening in the COS-7 FECH-C minigene system COS-7 cells were kept in culture at 37◦C, 5% CO2. Cells were plated at 140 000 cells/well in a 24-well plate in 1 ml DMEM-GlutaMax (ThermoFisher #10566016) containing 10% fetal bovine serum for 18 h prior to plasmid trans- fection. 700 ng of the FECH-C minigene (28) was trans- fected using XtremeGENE HP DNA transfection reagent (Sigma-Aldrich #XTGHP-RO) as per manufacturer’s in- structions with a ratio of 2.5 l transfection reagent/g plasmid DNA. Oligonucleotides were transfected 4 h af- ter the plasmid using Lipofectamine™2000 (ThermoFisher, #11668027) following manufacturer’s instructions. 24 h prior to lysis and RNA extraction, emetine (Sigma-Aldrich #E2375) was added in each desired well at a final concen- tration of 3 M. Cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and RNA extraction was carried out using Qiagen RNEasy kit according to manufacturer’s instruc- tions with the inclusion of two DNAse-treatment steps (Qi- agen #74106, #79254). cDNA was prepared using the High Capacity cDNA RT kit (Applied Biosystems #4374966) in accordance with the manufacturer’s instructions. PCR primers specific for the minigene were used: Forward 5′-GC Nucleic Acids Research, 2020, Vol. 48, No. 9 4661 AGA CCA TGT AGT TGA GGT CA-3′. Statistical anal- ysis (multiple t-tests) was conducted with GraphPad Prism v7. software. AGA CCA TGT AGT TGA GGT CA-3′. Statistical anal- ysis (multiple t-tests) was conducted with GraphPad Prism v7. software. Animal injections The oligonucleotides and oligonucleotide conjugates were injected subcutaneously into mice of the B6.Cg- Fechtm1.1(FECH)Emi line (Emi for short) of Emi/wt genotype at a dose of 50 mg/kg. Four doses were administered over a period of two weeks. Weight of the mice was de- termined prior to each injection to monitor any adverse weight loss due to oligonucleotide injection. 24 h after the last injection, the mice were weighed and euthanized by carbon dioxide inhalation. Blood was withdrawn from the posterior vena cava of each mouse and used to prepare heparinized plasma. Liver, spleen, kidneys, lungs, heart, brain, quadriceps muscle were collected and snap frozen in liquid nitrogen. Bone marrow was isolated from femurs and tibiae by flushing the bones with cell culture medium (RPMI +10% FBS). Bone marrow cell pellets were frozen at –80◦C till used for uptake studies or RNA isolation. Tissue samples were ground into a fine powder in liquid nitrogen and separate samples were immediately aliquoted for dissolution in OTX Lysis buffer (Phenomenex #AL0-8579) for uptake and metabolite study, or in Trizol (ThermoFisher #1559606) for RNA extraction. All animal experiments were conducted in accordance with local laws under the license number ZH115/16. Western blotting experiments in K562 cells K562 cells were grown as above. Cells were plated at 100 000 cells/well in a 24-well plate in 1 ml medium and oligonucleotides transfected 1.5 h after plating using Lipo- fectamine™2000. K562 cells were lysed 24 or 48 h af- ter treatment in 100 l RIPA buffer (ThermoFisher Sci- entific) supplemented with protease inhibitor (cOmplete, Roche). Lysates were incubated on ice for 30 min prior to brief needle sonication (25% amplitude, 6 s pulse), fol- lowed by centrifugation at 12 000 g for 15 min. Super- natants were collected and protein concentration was as- sessed with the Pierce BCA protein assay kit (ThermoFisher Scientific). 10 g of protein from each sample was de- naturated in Laemmli buffer (BioRad) containing 7% - mercaptoethanol, and loaded onto 4–20% pre-cast TGX gels (BioRad) for electrophoresis. Protein transfer onto ni- trocellulose membranes (GE Life Sciences) was carried out for 90 min at 25 W in a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad). Membranes were blocked for 3 h with 5% (w/v) nonfat dry milk in 0.05% Tween 20 in PBS (PBS-T) and were incubated overnight at 4◦C with the pri- mary antibody. Membranes were washed three times with 0.05% PBS-T, followed by incubation with the secondary antibody for 1 h in 5% milk in 0.05% PBS-T. After washing with 0.05% PBS-T, signal was detected with the ECL Prime reagent (GE Life Sciences) in a ChemiDoc XRS+ station (BioRad).i CL-qPCR in tissues The ground tissue powder was suspended in 10 volumes/weight OTX Lysis Buffer, briefly needle-sonicated and centrifuged for 30 s at 14 000 rpm. Supernatants were collected, diluted at 1/750 in ultrapure water, and used for analysis. CL-qPCR was carried out following the protocol from Boos et al. (27), with minor modifications. For each tissue and each compound, a calibration curve was gener- ated and run along with the samples. Mix composition for Chemical Ligation: 0.1 l PS primer (10 M stock), 0.1 l BPS primer (10 M primer), 1 l Poly A (GE Healthcare #27411001, 1 mg/ml), 1 l 10× Buffer with MgCl2 (Roche #12032902001) and 5.8 l ultrapure water, mixed with 2 l diluted lysate. Chemical ligation was run for 1 h at 33◦C. qPCR was run on a LC480 device (Roche). A qPCR mix containing 0.15 l FW primer (10 M stock), 0.15 l RV primer (10 M stock), 0.13 l BHQ primer (28.28 M stock), 0.15 l dNTPs mix (ThermoFisher #10297018), 1 l 10× Buffer with MgCl2 (Roche #12032902001), 0.1 l 10× FastTaq Polymerase (Roche #12032902001) and 6.32 l ultrapure water was prepared and mixed with 2 l of chemical ligation reaction mixture. qPCR program was as follows: 10 min, 95◦C, 50 cycles (3 s/95◦C – 30 s/55◦C – 10 s/72◦C). Concentrations in tissues were obtained through interpolation from the calibration curves and expressed in ng oligonucleotide/mg tissue for each mouse. Chemiluminescence was quantified using the ImageJ software. Ratios were calculated and graphical output and statistical analysis (one-way ANOVA) was conducted with GraphPad Prism v7. software. Biological replicates are pre- sented in Supplementary Figure S5. List of primary anti- bodies used: FECH (mouse, SantaCruz, sc-377377, 1/500 dilution), GADPH (mouse, Proteintech, 60004-1-Ig, 1/10 000 dilution) and the corresponding secondary antibody: anti-mouse (goat, Seracare, 5220-0341, 1/10 000 dilution). Oligonucleotide extraction from tissue lysates for LC–MS analysis Ground tissues were suspended in 10 volumes/weight OTX Lysis Buffer, briefly needle-sonicated and centrifuged for 30 s at 14 000 rpm. Supernatants were collected for oligonu- cleotide clean-up and directly processed with the Clarity® OTX Extraction kit (Phenomenex #KS0-9253) according 4662 Nucleic Acids Research, 2020, Vol. 48, No. 9 FECH-C variant in COS-7 cells, which we have described previously (20). For each SSO, we calculated the fraction of ‘correct’ (desired) transcripts on agarose gels after work- up using RT-PCR. Since the aberrant transcript, at least for the complete FECH gene, is degraded through non- sense mediated mRNA decay (NMD) (3), we pre-treated cells with emetine, a global inhibitor of translation that pre- vents NMD (34) to ensure reliable measurements of aber- rant transcript levels. Most SSOs showed some effect on tar- get splicing, changing the fraction of correct transcripts by a few percent in the desired direction (Figure 2B). Several SSOs binding between residues 40–66 produced almost full splice correction, and activity dropped away with sequences binding upstream of position –50. In a round of optimiza- tion for increased potency, we identified two 22-nt- and one 25-nt SSOs displaying equal or better activity than the afor- mentioned LNA SSO (8), which we used as a control (Fig- ure 2C, D). From these, ORN1 was selected as the lead SSO. to manufacturer’s procedure for tissues with the follow- ing adjustments: formulation of all buffers with ammo- nium acetate in substitution of sodium phosphate; dis- solution of the final eluate after evaporation to dryness in 100 l of TE buffer pH 8.0 instead of water. Pro- teinase K pre-treatment was necessary for successful recov- ery of the oligonucleotide with liver samples coming from mice treated with cholesteryl conjugate ORNch; this pre- treatment was used for all subsequent ORNch tissues. RNA extraction from mouse tissues and RT-qPCR for correct FECH transcript RNA extraction from mouse tissues and RT-qPCR for correct FECH transcript The ground tissue powder for each sample was dissolved in Trizol reagent (10–50 mg tissue/ml Trizol) and RNA was extracted following manufacturer’s instructions. RNA sam- ples were subjected to an extra clean-up step and DNase treatment using Qiagen RNeasy kit as per manufacturer’s instructions (Qiagen #74106, #79254). cDNA was gener- ated using PrimeScript RT kit (Takara #RR037A). qPCR was run on a LC480 device (Roche) with the KAPA SYBR Fast qPCR kit (#KK4618). A qPCR mix containing 4 l SYBR Mix 2×, 0.33 l primer pair (stock of 10 M each in water) and 3.17 l ultrapure water was prepared and mixed with 0.5 l cDNA solution. Primers were purchased from Microsynth (Balgach, Switzerland) and stored as pairs at 10 M each. Samples were run in technical triplicates and GAPDH was used as housekeeping control. Fold change calculations for the FECH correctly spliced transcript were made by comparing each treatment group with the saline group. FECH FW primer: 5′-CCT ATT CAG AAT AAG CTG GCA CC-3′; FECH RV primer: 5′-GGG GAT CCG CCT CCA ATC-3′. GAPDH FW primer: 5′-AGG TCG GTG TGA ACG GAT TTG-3′; GAPDH RV primer: 5′- TGT AGA CCA TGT AGT TGA GGT CA-3′. Statisti- cal analysis (multiple t-tests) and outlier removal (ROUT method) were conducted with GraphPad Prism v7. soft- ware. Binding the c.315-48C SNP but not the aberrant splice site is sufficient for splicing correction We noted from the initial screen that the most active SSOs covered the –48 SNP, but not necessarily the –63 aberrant splice site. We tested a library of shorter (10-nt) MOE- PS oligonucleotides (Figure 2E; Supplementary Table S1) binding to the –63 and the –48 sites of FECH separately, in order to pinpoint the nucleotides important for the activity of the SSOs in the region of the SNP. One sequence bind- ing at positions –45–54 altered the course of splicing signifi- cantly, and three-nucleotide shifts upstream or downstream resulted in loss of activity (Figure 2E). Co-treatments of cells with SSOs blocking simultaneously the splice site (– 57–66) and the SNP (–45–54) did not produce synergistic effects. We and others (3) have observed that ratios of aber- rant:correct FECH transcripts vary according to the geno- type, the cell type and iron availability (35). With emetine treatment to inhibit NMD of mRNA in lymphoblasts im- mortalized from donors, we found approximately 30% and 60% of the FECH transcripts were aberrantly spliced for the T/T (c.315-48T)- and C/C (c.315-48C)-homozygotes, respectively (not shown). We observed the same ratios in COS-7 cells transfected with the minigenes (Supplemen- tary Figure S6) and approximately 15% aberrant splicing in the K562 cell line, which is bone-marrow derived, and has erythroid features (36); we confirmed the T/T genotype of these cells by Sanger sequencing (Supplementary Figure S7). Taken together, this implies that the outcome of FECH splicing may depend upon a finely-tuned balance of pos- itive and negative splicing regulatory elements. According to Human Splicing Finder (37), the -48 SNP is embedded in predicted binding sites for hnRNPA1, SRSF1 and SRSF2 alternative splice factors, and the T>C polymorphism may create an Exon Inclusion Element (EIE) (38). An oligonu- cleotide binding to the –45–54 locus might lessen this activa- tion, in a similar fashion to that described for the cblE type of homocystinuria, where the c.309+469T>C mutation cre- ates an EIE and promotes the inclusion of a pseudo-exon (39). Identification of a lead SSO for FECH RNA splicing correc- tion In panels (A), (C) and (E), the ag labeled in red indicates the cryptic 3′ acceptor site, and the C labeled in red 315 48 i t Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 wnloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 ticle/48/9/4658/5822958 by guest on 24 October 2024 Figure 2. Identification of splice-switching oligonucleotides (SSOs) targeting intron 3 of FECH pre-mRNA. (A) Binding sites for twenty 19-nt MOE-PS SSOs in the region of the FECH c.315-48 polymorphism and the c.315-63 aberrant splice site. (B) Agarose gel showing relative amounts of aberrant and correct transcripts after treatment with 50 nM concentrations of SSOs in COS-7 cells expressing a FECH-C minigene; LNA45-63 (9) is a positive control. (C) Lead optimization: binding sites for 22 and 25-nt SSO analogs of selected SSOs from b). (D) Activity of ORNs 1–3 showing complete splice correction at 10 nM concentration in the same assay. (E) Binding sites for ten 10-nt SSOs in the regions of the –63-splice site and the –48 polymorphism, and agarose gels showing relative amounts of aberrant and correct transcripts from treatment at 50 nM concentration of COS-7 cells expressing a FECH-C minigene with short SSOs; positive control is -45-63 LNA-SSO. Splicing correction obtained in the minigene experiment with single oligonucleotide treatment (left- hand side) or cotreatments (right-hand side). In panels (A), (C) and (E), the ag labeled in red indicates the cryptic 3′ acceptor site, and the C labeled in red the c.315-48 variant. Figure 2. Identification of splice-switching oligonucleotides (SSOs) targeting intron 3 of FECH pre-mRNA. (A) Binding sites for twenty 19-nt MOE-PS SSOs in the region of the FECH c.315-48 polymorphism and the c.315-63 aberrant splice site. (B) Agarose gel showing relative amounts of aberrant and correct transcripts after treatment with 50 nM concentrations of SSOs in COS-7 cells expressing a FECH-C minigene; LNA45-63 (9) is a positive control. (C) Lead optimization: binding sites for 22 and 25-nt SSO analogs of selected SSOs from b). (D) Activity of ORNs 1–3 showing complete splice correction at 10 nM concentration in the same assay. Identification of a lead SSO for FECH RNA splicing correc- tion Identification of a lead SSO for FECH RNA splicing correc- tion As of yet, it is difficult to predict potent SSO sequences us- ing computational methods (29,30). Therefore, the search for an effective SSO typically begins by ‘walking’ empiri- cally a set of overlapping sequences across exonic and in- tronic regions in the region of the pathogenic mutation. This approach identified nusinersen, which binds the ISS-N1 site on SMN2 (13), and more recently, an effective SSO as a po- tential treatment for familial dysautonomia (31). y ( ) We scanned regions on the FECH pre-mRNA close to the c.315-48 polymorphism (Figure 2A; sequences in Sup- plementary Information) for splice-switching activity by us- ing a set of sequence-overlapping 19-nt MOE-PS oligonu- cleotides. We elected to use MOE-PS SSOs because of their successful use in clinical programs (www.ionispharma.com) (32), their excellent safety record (32,33), their efficient dis- tribution in vivo (15), the ease with which they can be elab- orated with targeting groups or stereopure linkages (28). We employed a mini-gene assay transiently expressing the Nucleic Acids Research, 2020, Vol. 48, No. 9 4663 ure 2. Identification of splice-switching oligonucleotides (SSOs) targeting intron 3 of FECH pre-mRNA. (A) Binding sites for twenty 19-nt MOE-PS Os in the region of the FECH c.315-48 polymorphism and the c.315-63 aberrant splice site. (B) Agarose gel showing relative amounts of aberrant and rect transcripts after treatment with 50 nM concentrations of SSOs in COS-7 cells expressing a FECH-C minigene; LNA45-63 (9) is a positive control. Lead optimization: binding sites for 22 and 25-nt SSO analogs of selected SSOs from b). (D) Activity of ORNs 1–3 showing complete splice correction 0 nM concentration in the same assay. (E) Binding sites for ten 10-nt SSOs in the regions of the –63-splice site and the –48 polymorphism, and agarose showing relative amounts of aberrant and correct transcripts from treatment at 50 nM concentration of COS-7 cells expressing a FECH-C minigene h short SSOs; positive control is -45-63 LNA-SSO. Splicing correction obtained in the minigene experiment with single oligonucleotide treatment (left- d side) or cotreatments (right-hand side). Distribution and metabolism of FECH SSOs in mouse tissues and organs Recently, we engineered a murine model of EPP (Emi), in which intron 3 of the mouse gene was replaced by the corre- sponding human sequence bearing the c.315-48C polymor- phism (20) (Figure 1C). Homozygous Emi/Emi mice were not viable. However, viable Emi/wt mice contain one wild- type allele and one allele of the EPP genotype which pro- duces the same aberrant/correct splicing products as the human c.315-48C allele. Unfortunately, this splicing pheno- type was accompanied by an additional unanticipated splic- ing defect - an almost complete skipping of exon 3, and thus loss of FECH protein. Hence, the Emi model is unsuitable for studying effects of SSOs on the disease phenotype, but is amenable to investigations of the pharmacokinetics proper- ties of SSOs and their effects on the amount of FECH tran- scripts containing the extra 63 nucleotide sequence (Figure 1C). Synthesis of MOE-PS SSOs conjugated with groups for tar- geted delivery MOE-PS oligonucleotides are delivered to bone marrow after systemic administration in rodents (15,17) and in man (see refs in (16)), however, MOE-PS SSOs were in- active when tested in a mouse model for splicing of a re- porter gene (17). Therefore, we conjugated ORN1 with three prototypical functionalities that are reported to en- hance delivery to selected tissues in vivo (Figure 4A): stearic acid which increases circulation times due to al- bumin binding (23) (ORNst); a heptapeptide residue that homes to bone marrow and binds primitive stem cells (24) (ORN84); and cholesterol which delivers single-stranded and double-stranded oligonucleotides to bone marrow (21,40,41) (ORNch). Stearic acid was conjugated as an amide whereas the peptide and the cholesterol groups were conjugated via thiol-maleimide chemistry (see Supplemen- tary Methods) (42,43). from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 SSOs were administered to Emi/wt mice twice a week over two weeks similar to other published protocols us- ing MOE-PS oligonucleotides (15,50–52). Mice were sac- rificed 24 h after the last injection. No signs of toxicity were apparent from measurements of total body weight, liver/body weight and spleen/body weight ratios as well as assessment of the general condition (not shown). Ac- cumulation of SSOs in selected tissues was quantified us- ing chemical ligation––qPCR (CL-qPCR) (27,53,54) (Fig- ure 5A). This technique is particularly useful for quantifi- cation of SSOs that are heavily modified and are not ampli- fiable by polymerases. Standard calibration curves for the four SSOs were generated in diluted lysate (Supplementary Figure S8) and used to determine SSO concentrations in the tissues of treated mice. 4664 Nucleic Acids Research, 2020, Vol. 48, No. 9 4664 Nucleic Acids Research, 2020, Vol. 48, No. 9 products with the same mass (9411: ORNch+18), which we assigned as ring-opened regioisomers (Y1 and Y2) (Figure 4C). The stearic acid conjugate ORNst showed only trace amounts of degradation after 26 h (Figure 4D). It has not yet been possible to test ORN1 for its effects on native FECH mRNA and protein, since transfectable patient-derived (C/C)-cells expressing low levels of FECH protein are unavailable, and we have so far been unable to generate a model cell line using CRISPR-Cas based meth- ods. Therefore, we assayed ORN1 in K562 erythroleukemia cells, expressing the T-variant of FECH, in which ORN1 binds its target with a G–U wobble at the site of the poly- morphism. We included a fully complementary version of the SSO (ORN1T) in the assay. Both SSOs showed splice- switching activity (Figure 3A) and increased the levels of the correctly spliced transcript by 25–30% (Figure 3B) and FECH protein by ∼50% (Figure 3C). In vitro characterization of conjugate stability in plasma and on albumin binding MOE-PS oligonucleotides are highly resistant to nuclease- mediated degradation in vivo (44,45), but less is known about the metabolic stability of the conjugates and their linkers. The thiosuccinimide linker of ORN84 and ORNch has been widely used in the field of antibody drug conju- gates but may undergo thiol-exchange reactions with cys- teines of serum albumin (46) or glutathione (47). The pep- tide of ORN84 is not modified to convey stability against hy- drolases. Therefore, we examined the stability of the SSOs in a plasma stability assay that mimics conditions in the blood- stream (48,49). We incubated the SSOs in mouse plasma and removed aliquots periodically for analysis by liquid chromatography–mass spectrometry (LC–MS). ORN1 was detected in all tissues except for brain and muscle (Figure 5B). Consistent with previous studies (55), the highest levels were found in kidney (178 ± 14 ng/mg tis- sue) and liver (46±8 ng/mg tissue), followed by bone mar- row (35 ± 12 ng/mg tissue), spleen (20 ± 3 ng/mg tissue) and lung (10 ± 3 ng/mg tissue). Furthermore, the abso- lute concentrations of ORN1 measured using CL-qPCR are in accordance with those determined using radio-labeled oligonucleotides (15). ORNst showed a 2-fold higher uptake in liver (84 ± 16 ng/mg tissue), in spleen (40 ± 12 ng/mg tissue) and lung (17 ± 4 ng/mg tissue), and a slightly re- duced accumulation in kidney. These findings are again con- sistent with reports that siRNAs conjugated to hydropho- bic fatty acids are transported to the liver at the expense of kidney (25). ORNch showed 7-fold higher levels in liver (341 ± 50 ng/mg tissue), a 2-fold increase in spleen (54 ± 12 ng/mg tissue) and a 60% decrease in kidney compared to ORN1; the presence of ORNch in lung tissue was be- low accurate detection. Again, this is consistent with the findings of other groups using cholesteryl-siRNAs (23,56) and single-stranded antisense–cholesteryl conjugates (57). ORN84 showed an approximate 2-fold increase in liver (78 g p y p y ( ) ORN1 was highly high stable in plasma, though trace amounts of a 3′ (n – 1) metabolite was detected after 26 h (not shown). In contrast, all of the conjugates suffered some degradation. Identification of a lead SSO for FECH RNA splicing correc- tion (E) Binding sites for ten 10-nt SSOs in the regions of the –63-splice site and the –48 polymorphism, and agarose gels showing relative amounts of aberrant and correct transcripts from treatment at 50 nM concentration of COS-7 cells expressing a FECH-C minigene with short SSOs; positive control is -45-63 LNA-SSO. Splicing correction obtained in the minigene experiment with single oligonucleotide treatment (left- hand side) or cotreatments (right-hand side). In panels (A), (C) and (E), the ag labeled in red indicates the cryptic 3′ acceptor site, and the C labeled in red the c.315-48 variant. In vitro characterization of conjugate stability in plasma and on albumin binding (A) Activity of ORN1, ORN1T and two randomized versions of ORN1 in K562 cells 24 h after transfection (ORN1 and ORN1T bind FECH mRNA in K562 cells with a G-U wobble and with perfect complementarity, respectively). Aberrant transcripts were stabilized with 3 M emetine treatment. (B) ORN1 and ORN1T selectively increase levels of endogenous correctly spliced FECH transcripts in K562 cells as assayed by SYBR Green qPCR. n = 3 biological replicates; statistical analysis (t tests) was conducted with GraphPad Prism software: P < 0.05; P < 0.01. Data expressed as mean value ± s.e.m. (C) Representative Western Blot of FECH and GAPDH after a single transfection at 500 nM in K562 cells after 24 or 48 h treatments; corresponding graph plot from n = 3 biological replicates; statistical analysis (one-way Anova) was conducted with GraphPad Prism software: P < 0.05 compared to mock treatments. Data expressed as mean value ± s.e.m. p p Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by gues Figure 3. Endogenous activity of the lead SSO ORN1 at RNA and protein levels in erythroleukemic K562 cells. (A) Activity of ORN1, ORN1T and two randomized versions of ORN1 in K562 cells 24 h after transfection (ORN1 and ORN1T bind FECH mRNA in K562 cells with a G-U wobble and with perfect complementarity, respectively). Aberrant transcripts were stabilized with 3 M emetine treatment. (B) ORN1 and ORN1T selectively increase levels of endogenous correctly spliced FECH transcripts in K562 cells as assayed by SYBR Green qPCR. n = 3 biological replicates; statistical analysis (t tests) was conducted with GraphPad Prism software: P < 0.05; P < 0.01. Data expressed as mean value ± s.e.m. (C) Representative Western Blot of FECH and GAPDH after a single transfection at 500 nM in K562 cells after 24 or 48 h treatments; corresponding graph plot from n = 3 biological replicates; statistical analysis (one-way Anova) was conducted with GraphPad Prism software: P < 0.05 compared to mock treatments. Data expressed as mean value ± s.e.m. ± 22 ng/mg tissue) and lung (27 ± 8 versus 10 ± 3 ng/mg tissue), and markedly improved delivery to bone marrow (224 ± 87 versus 35 ± 12 ng/mg tissue) in line with its pre- viously described properties (24). sues (metabolite B), together with minor amounts of the 3′(n – 1)-metabolite (C). In vitro characterization of conjugate stability in plasma and on albumin binding Since the stearoyl group of this SSO was stable in plasma (Figure 4D), it seems possible that its metabolism occurred at the site of accumulation, or intra- cellularly. Similar to ORNst, ORN84 was not found intact in any tissues and the majority of its metabolites derived from progressive cleavage of the peptide. The major metabolite in liver, kidney, lung and bone marrow was the SSO attached to the terminal cysteine residue (H and I), whereas partial cleavage of the peptide occurred in spleen (metabolite G, possibly with E and F). ORNch was the most resistant of the series, and delivered a strong signal for intact ORNch from kidney, liver and spleen samples; the SSO was also de- tectable in lung and in bone marrow in lower amounts. In- terestingly, cleavage of the PS-linkage to yield ORN1 and its 3′-truncated fragment (A) was observed in samples from kidney, liver and spleen as well as bone marrow. We investigated the metabolic fate of the four SSOs after repeated administration in vivo. Tissue samples were pro- cessed using an OTX Clarity Kit and eluates were collected from kidney, liver, spleen, lung, brain and bone marrow. The extraction protocol worked poorly for samples con- taining ORNch, which was remedied by pre-treatment with proteinase K. Reference LC–MS spectra were generated by spiking SSOs into eluates collected from tissues from saline- treated mice. This permitted unambiguous identification and qualitative analysis of oligonucleotide-derived peaks in the chromatograms from samples from treated animals (Figure 6A). Deconvolution of the mass spectra returned molecular weights from which the structures of metabolites were inferred (Figure 6B; Supplementary Figures S9–S12). Intact ORN1 was detected in all the tissues except the brain, accompanied by 5–10% of the (n – 1)-metabolite cor- responding to loss of the 3′-terminal nucleotide (metabo- lite A). In contrast, all of the SSO conjugates showed frag- mentation. Despite its high stability in mouse plasma, fully intact ORNst was only detected in trace amounts in the liver sample: hydrolysis of the stearoyl group was seen in all tis- In vitro characterization of conjugate stability in plasma and on albumin binding The peptide moiety of ORN184 underwent se- quential proteolysis from the C-terminus: metabolites miss- ing one (B) and two amino-acids (C) were seen after 6.5 h, and (D) and (E) were identified after 26 h (Figure 4B). The linker also underwent ring opening of the succinimide (metabolite Y) after 1 h, and cleavage of the amide was apparent after 6.5 h (metabolite Z). ORNch underwent a similar albeit slower hydrolysis of the linker, producing two g p y p y ( ) ORN1 was highly high stable in plasma, though trace amounts of a 3′ (n – 1) metabolite was detected after 26 h (not shown). In contrast, all of the conjugates suffered some degradation. The peptide moiety of ORN184 underwent se- quential proteolysis from the C-terminus: metabolites miss- ing one (B) and two amino-acids (C) were seen after 6.5 h, and (D) and (E) were identified after 26 h (Figure 4B). The linker also underwent ring opening of the succinimide (metabolite Y) after 1 h, and cleavage of the amide was apparent after 6.5 h (metabolite Z). ORNch underwent a similar albeit slower hydrolysis of the linker, producing two Nucleic Acids Research, 2020, Vol. 48, No. 9 4665 re 3. Endogenous activity of the lead SSO ORN1 at RNA and protein levels in erythroleukemic K562 cells. (A) Activity of ORN1, ORN1T and two omized versions of ORN1 in K562 cells 24 h after transfection (ORN1 and ORN1T bind FECH mRNA in K562 cells with a G-U wobble and with ect complementarity, respectively). Aberrant transcripts were stabilized with 3 M emetine treatment. (B) ORN1 and ORN1T selectively increase levels ndogenous correctly spliced FECH transcripts in K562 cells as assayed by SYBR Green qPCR. n = 3 biological replicates; statistical analysis (t tests) conducted with GraphPad Prism software: P < 0.05; P < 0.01. Data expressed as mean value ± s.e.m. (C) Representative Western Blot of FECH GAPDH after a single transfection at 500 nM in K562 cells after 24 or 48 h treatments; corresponding graph plot from n = 3 biological replicates; stical analysis (one-way Anova) was conducted with GraphPad Prism software: P < 0.05 compared to mock treatments. Data expressed as mean e ± s.e.m. Downloaded from https://academic.oup.com/nar/article/48/9/4658/58 Figure 3. Endogenous activity of the lead SSO ORN1 at RNA and protein levels in erythroleukemic K562 cells. Effects of SSOs on FECH RNA splicing in mouse tissues and organs As discussed above, splice-switching activity of the SSOs in treated Emi/wt mice could not be assayed at the protein level, as the predominant transcript III (Figure 1c) from the humanized allele lacks exon 3. Therefore, we assayed 4666 Nucleic Acids Research, 2020, Vol. 48, No. 9 SO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N ate ORN84 and the cholesterol conjugate (ORN h) were generated in two steps using a 5′ caged maleimide Figure 4. Synthesis of SSO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. The ORNst was obtained in two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N-hydr (A); the peptide conjugate ORN84 and the cholesterol conjugate (ORNch) were generated in two steps using a 5′-caged maleimide modi and reaction with the cysteine-modified CD84-peptide (B) or thiocholesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: m (B–D) ORN84, ORNch and ORNst were incubated in mouse plasma for 26 h at 37◦C. Aliquots of plasma were removed periodica ysed by LC–MS. ORN184 underwent hydrolysis at the peptide and the succinimide linker (b). ORN1ch underwent hydrolysis-media ORN1st was barely affected under the conditions (D). Red arrows indicate inferred sites of cleavage, based upon the masses of frag chromatagrams. s of SSO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. The stearic acid conjugate ed in two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N-hydroxy succinimide ester njugate ORN84 and the cholesterol conjugate (ORNch) were generated in two steps using a 5′-caged maleimide modifier phosphoramidite the cysteine-modified CD84-peptide (B) or thiocholesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: monomethoxy-trityl). RNch and ORNst were incubated in mouse plasma for 26 h at 37◦C. Aliquots of plasma were removed periodically, cleaned and anal- ORN184 underwent hydrolysis at the peptide and the succinimide linker (b). ORN1ch underwent hydrolysis-mediated ring opening (C). y affected under the conditions (D). Red arrows indicate inferred sites of cleavage, based upon the masses of fragments in the LC–MS Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 plasma. (A) Synthesis routes of MOE-PS SSO conjugates. Effects of SSOs on FECH RNA splicing in mouse tissues and organs The stearic acid conjugate hosphoramidite linker, followed by reaction with stearic N-hydroxy succinimide ester ch) were generated in two steps using a 5′-caged maleimide modifier phosphoramidite olesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: monomethoxy-trityl). for 26 h at 37◦C. Aliquots of plasma were removed periodically, cleaned and anal- e succinimide linker (b). ORN1ch underwent hydrolysis-mediated ring opening (C). dicate inferred sites of cleavage, based upon the masses of fragments in the LC–MS Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on 24 October 2024 Figure 4. Synthesis of SSO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. The stearic acid conjugate ORNst was obtained in two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N-hydroxy succinimide ester (A); the peptide conjugate ORN84 and the cholesterol conjugate (ORNch) were generated in two steps using a 5′-caged maleimide modifier phosphoramidite and reaction with the cysteine-modified CD84-peptide (B) or thiocholesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: monomethoxy-trityl). (B–D) ORN84, ORNch and ORNst were incubated in mouse plasma for 26 h at 37◦C. Aliquots of plasma were removed periodically, cleaned and anal- ysed by LC–MS. ORN184 underwent hydrolysis at the peptide and the succinimide linker (b). ORN1ch underwent hydrolysis-mediated ring opening (C). ORN1st was barely affected under the conditions (D). Red arrows indicate inferred sites of cleavage, based upon the masses of fragments in the LC–MS chromatagrams. Figure 4. Synthesis of SSO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. The stearic acid conjugate ORNst was obtained in two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N-hydroxy succinimide ester (A); the peptide conjugate ORN84 and the cholesterol conjugate (ORNch) were generated in two steps using a 5′-caged maleimide modifier phosphoramidite and reaction with the cysteine-modified CD84-peptide (B) or thiocholesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: monomethoxy-trityl). (B–D) ORN84, ORNch and ORNst were incubated in mouse plasma for 26 h at 37◦C. Aliquots of plasma were removed periodically, cleaned and anal- ysed by LC–MS. ORN184 underwent hydrolysis at the peptide and the succinimide linker (b). ORN1ch underwent hydrolysis-mediated ring opening (C). ORN1st was barely affected under the conditions (D). Red arrows indicate inferred sites of cleavage, based upon the masses of fragments in the LC–MS chromatagrams Nucleic Acids Research, 2020, Vol. 48, No. 9 4667 Figure 5. Effects of SSOs on FECH RNA splicing in mouse tissues and organs The hu- man aberrant FECH transcript is a substrate for NMD and therefore is unstable in vivo (3). Nevertheless, we assayed the aberrant/correct splice ratios in the liver sample using RT-PCR. The data correlated with that from the RT-qPCR measurements for three out of four SSOs (ORN1, ORNst, ORNch) (Supplementary Figure S13). SSO activity by the increase in the correctly-spliced tran- script I by using SBYR Green RT-qPCR. Splice correc- tion of the FECH transgene by SSOs was observed in kid- ney, liver, bone marrow, spleen and lung, with varying effi- ciency; we detected no increases in the correct transcript in spleen, heart and gastrocnemius muscle (Figure 7). The hu- man aberrant FECH transcript is a substrate for NMD and therefore is unstable in vivo (3). Nevertheless, we assayed the aberrant/correct splice ratios in the liver sample using RT-PCR. The data correlated with that from the RT-qPCR measurements for three out of four SSOs (ORN1, ORNst, ORNch) (Supplementary Figure S13). Effects of SSOs on FECH RNA splicing in mouse tissues and organs Characterization of SSOs and their accumulation in selected tissues. (A) Principle of CL-qPCR (53). Two chemically modified PCR primers ‘PS’ and ‘PBS’ are added to a sample containing the chemically modified SSO and incubated for 1 h at 30◦C. Hybridization of the primers to the SSO lead to formation of a DNA strand, which serves as template for quantitative PCR. (B) Quantification using CL-qPCR of target oligonucleotides ORN1, ORNch, ORNst and ORN84 in kidney, liver, bone marrow, spleen and lung. Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. n = 4; data expressed as mean value ± s.e.m.; p = present (detectably higher than NTC, but not quantifiable). Downloaded from https://academic.oup.com/nar/a Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on Figure 5. Characterization of SSOs and their accumulation in selected tissues. (A) Principle of CL-qPCR (53). Two chemically modified PCR primers ‘PS’ and ‘PBS’ are added to a sample containing the chemically modified SSO and incubated for 1 h at 30◦C. Hybridization of the primers to the SSO lead to formation of a DNA strand which serves as template for quantitative PCR (B) Quantification using CL qPCR of target oligonucleotides ORN1 ORN h Figure 5. Characterization of SSOs and their accumulation in selected tissues. (A) Principle of CL-qPCR (53). Two chemically modified PCR primers ‘PS’ and ‘PBS’ are added to a sample containing the chemically modified SSO and incubated for 1 h at 30◦C. Hybridization of the primers to the SSO lead to formation of a DNA strand, which serves as template for quantitative PCR. (B) Quantification using CL-qPCR of target oligonucleotides ORN1, ORNch, ORNst and ORN84 in kidney, liver, bone marrow, spleen and lung. Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. n = 4; data expressed as mean value ± s.e.m.; p = present (detectably higher than NTC, but not quantifiable). mRNA strongly suggests a successful uptake of the SSO by erythroblasts. SSO activity by the increase in the correctly-spliced tran- script I by using SBYR Green RT-qPCR. Splice correc- tion of the FECH transgene by SSOs was observed in kid- ney, liver, bone marrow, spleen and lung, with varying effi- ciency; we detected no increases in the correct transcript in spleen, heart and gastrocnemius muscle (Figure 7). DISCUSSION Loss-of-function (LoF) mutations that result in reduced or abolished protein function represent a common cause of ge- netic disorders. Many of these result from defective splic- ing events (59). It is difficult to discover or design conven- tional small-molecule drugs to correct defective splicing in a sequence-selective manner. However, oligonucleotides are well suited to modulate mRNA splicing since they bind with high selectivity directly to the disease-causing element. Con- sequently, there is considerable excitement for a wave of SSOs that are currently in pre-clinical and clinical develop- ment (31,60–62). For single-stranded PS oligonucleotides, kidney is the main site of accumulation (Figure 5B) (15,58). The stearoyl conjugate ORNst was most effective in liver (1.6-fold) and in lung, two tissues in which the conjugated group increased accumulation (Figure 5B). ORN84, which was found at 6.5- fold higher levels in bone marrow (Figure 5B), did not show significantly better splice-modulating activity than ORN1 (Figure 7). Splice-switching mechanisms differ fundamentally from terminating mechanisms for oligonucleotide therapies. For the latter, the drug is typically optimized to provide a max- imum inhibition of the target; for the former, the objective is to increase expression of the target to counter a loss of function. For many LoF diseases, even a small increase in levels of the target protein can be therapeutically beneficial (63,64). This applies to EPP where it is thought that rais- ing FECH protein/enzyme activity by 10–20% may be suf- ficient to provide therapeutic benefit to patients (9). The de- velopment of an oligonucleotide therapy for EPP is espe- cially desirable since nearly all patients exhibit the same in- tronic SNP variant. On the other hand, an oligonucleotide therapy for EPP requires the delivery of the SSO into ery- ( g ) From the three SSO conjugates, ORNch provided the most promising results. Surprisingly, it did not exhibit supe- rior activity to that of ORN1 in liver nor in kidney. This was unexpected since cholesterol conjugates are a well-known means to boost the activities of antimiR oligonucleotides in liver (21) (ORNch exhibited a 7-fold increase in liver; Figure 5B). Most importantly, however, ORNch showed a 1.8-fold increase in the FECH transcript in bone marrow compared to ORN1, with a similar level of accumulation. Since most of the FECH transcripts in the bone marrow likely originate from erythroblasts, the increase in correctly spliced FECH 4668 Nucleic Acids Research, 2020, Vol. 48, No. DISCUSSION 9 Metabolites of SSOs ORN1, ORNst, ORN84 and ORNch from tissues after administration to Emi/wt mice. (A) Animals received four injections kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. Oligonucleotides were extracted from the tissue matrix using the Phe- Clarity OTX kit. Tissues from mice treated with ORN1ch were subjected to a proteinase K digestion before processing. Detected oligonucleotide e marked in red, next to their associated masses determined by LC–MS (see also Supplementary Figures S9–S12). (B) Calculated molecular parent SSOs and their inferred metabolites from LC–MS data shown in a) (C17 is the stearoyl chain; chol is the thiocholesteryl group). Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on Figure 6. Metabolites of SSOs ORN1, ORNst, ORN84 and ORNch from tissues after administration to Emi/wt mice. (A) Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. Oligonucleotides were extracted from the tissue matrix using the Phe- nomenex Clarity OTX kit. Tissues from mice treated with ORN1ch were subjected to a proteinase K digestion before processing. Detected oligonucleotide species are marked in red, next to their associated masses determined by LC–MS (see also Supplementary Figures S9–S12). (B) Calculated molecular weights of parent SSOs and their inferred metabolites from LC–MS data shown in a) (C17 is the stearoyl chain; chol is the thiocholesteryl group). Figure 6. Metabolites of SSOs ORN1, ORNst, ORN84 and ORNch from tissues after administration to Emi/wt mice. (A) Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. Oligonucleotides were extracted from the tissue matrix using the Phe- nomenex Clarity OTX kit. Tissues from mice treated with ORN1ch were subjected to a proteinase K digestion before processing. Detected oligonucleotide species are marked in red, next to their associated masses determined by LC–MS (see also Supplementary Figures S9–S12). (B) Calculated molecular weights of parent SSOs and their inferred metabolites from LC–MS data shown in a) (C17 is the stearoyl chain; chol is the thiocholesteryl group). Figure 6. Metabolites of SSOs ORN1, ORNst, ORN84 and ORNch from tissues after administration to Emi/wt mice. (A) Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last dose. DISCUSSION We conjugated the SSO to three classes of targeting groups (cholesterol, fatty acids, peptides) and administered them to a mouse model of EPP in order to study their distribution, their metabolic stability and their FECH splice-switching ability. The conjugated groups exhibited distinct distribu- tion profiles, notably increasing SSO delivery to liver, kid- ney, bone marrow and lung. A variety of sensitive tech- niques have emerged recently for the detection and quantifi- cation of oligonucleotides in biological samples (27,65,66). We employed CL-qPCR to quantify the SSOs in tissues and recorded similar values to those reported using other methods (10’s-100’s ng/mg tissue). Whereas the PS-MOE oligonucleotide was highly stable, the conjugates were heav- ily metabolized in vivo, particularly the amide linkage of ORNst, which was unexpected given its common use in drug conjugates (67–69). This metabolism likely occurred after clearance from circulation, since amide cleavage was not observed in the mouse plasma assay. The most robust SSO in vivo was ORNch, which did not boost the accumulation in bone marrow but nevertheless increased levels of cor- rectly spliced FECH transcript by 80%. It is presently un- known whether a 1.8-fold increase in FECH transcript lev- els would produce a therapeutically-meaningful outcome, i.e. would lower circulating PPIX. However, the experi- ments performed in the K562 cells are encouraging, where a 30% increase of correctly spliced RNA raised levels of FECH protein by 50% (Figure 3B, C). Clarity on this ques- In this study, we quantified SSO accumulation and splice switching data in six tissues of the mouse by four SSOs. In order to affect splice switching, an SSO must reach the target tissue, be taken up by the appropriate cells, escape from intracellular compartments and traffic to the nucleus so as to bind the target pre-mRNA. One expects that the conjugate groups––and their metabolism––will affect these individual steps in various ways. Therefore, it was not un- expected that for these four structurally-distinct SSOs tis- sue accumulation did not correlate collectively with splice- switching activity. The recent approval of several breakthrough oligonu- cleotide therapeutics signifies that RNA can today be con- sidered as a mainstream class of drugs and drug targets. The oligonucleotides are single- or double-stranded, of various chemistries and they elicit their action upon well-validated mRNA targets expressed in the liver and the central ner- vous system. DISCUSSION Oligonucleotides were extracted from the tissue matrix using the Phe- nomenex Clarity OTX kit. Tissues from mice treated with ORN1ch were subjected to a proteinase K digestion before processing. Detected oligonucleotide species are marked in red, next to their associated masses determined by LC–MS (see also Supplementary Figures S9–S12). (B) Calculated molecular weights of parent SSOs and their inferred metabolites from LC–MS data shown in a) (C17 is the stearoyl chain; chol is the thiocholesteryl group). Nucleic Acids Research, 2020, Vol. 48, No. 9 4669 Figure 7. Treatment of Emi/wt mice with SSOs modulates splicing in selected tissues. Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last injection. Levels of correct FECH transcript were assessed by SYBR green RT-qPCR and normalized to GAPDH and to the saline group. n = 3 animals for the saline group; n = 4 animals for oligonucleotide treated groups; statistical analysis (t tests) was conducted with GraphPad Prism software: P < 0.05; P < 0.01 (whiskers indicate lowest and highest values; middle line represents the median). Downloaded from https://academic.oup.com/nar/article/48/9/4658/5822958 by guest on Figure 7. Treatment of Emi/wt mice with SSOs modulates splicing in selected tissues. Animals received four injections at 50 mg/kg in 0.9% saline over 2 weeks and were sacrificed 24 h after the last injection. Levels of correct FECH transcript were assessed by SYBR green RT-qPCR and normalized to GAPDH and to the saline group. n = 3 animals for the saline group; n = 4 animals for oligonucleotide treated groups; statistical analysis (t tests) was conducted with GraphPad Prism software: P < 0.05; **P < 0.01 (whiskers indicate lowest and highest values; middle line represents the median). throcyte precursors in the bone marrow and with an effi- ciency to counter the rapid pace of erythrocyte biogenesis.i tion will be available from an improved mouse model of EPP (ongoing), i.e. expressing the same FECH transcripts (I and II in Figure 1C) present in EPP patients.i In this study, we identified PS-MOE SSOs that bind to the c.315-48 SNP and redirect the splicing of FECH, al- most completely to the correct form in cell models. It is likely that the SSO blocks an EIE at the site of the SNP. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. DISCUSSION Extending the use of SSO therapies to the bone marrow, for example for EPP or other disorders, would sig- nificantly advance the oligonucleotide field. REFERENCES 1. Lecha,M., Puy,H. and Deybach,J.-C. (2009) Erythropoietic protoporphyria. Orphanet. J. Rare. Dis., 4, 1–10. 23. Wolfrum,C., Shi,S., Jayaprakash,K.N., Jayaraman,M., Wang,G., Pandey,R.K., Rajeev,K.G., Nakayama,T., Charrise,K., Ndungo,E.M. et al. (2007) Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotech., 25, 1149–1157. 2. Gouya,L., Puy,H., Lamoril,J., Da Silva,V., Grandchamp,B., Nordmann,Y. and Deybach,J.-C. (1999) Inheritance in erythropoietic Protoporphyria: A common Wild-Type ferrochelatase allelic variant with low expression accounts for clinical manifestation. 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https://openalex.org/W2807295999	https://hal.science/hal-01910318/file/Togo2018.pdf	English	null	Draft Genome and Description of Eisenbergiella massiliensis Strain AT11T: A New Species Isolated from Human Feces After Bariatric Surgery	Current microbiology	2,018	cc-by	4,875	Draft Genome and Description of Eisenbergiella massiliensis Strain AT11(T): A New Species Isolated from Human Feces After Bariatric Surgery Amadou H. Togo, Awa Diop, Matthieu Million, Marie M. Maraninchi, Jean-Christophe J.-C. Lagier, Catherine Robert, Fabrizio Di Pinto, Didier Raoult, Pierre-Edouard Fournier, Fadi Bittar To cite this version: Amadou H. Togo, Awa Diop, Matthieu Million, Marie M. Maraninchi, Jean-Christophe J.-C. Lagier, et al.. Draft Genome and Description of Eisenbergiella massiliensis Strain AT11(T): A New Species Isolated from Human Feces After Bariatric Surgery. Current Microbiology, 2018, 75 (10), pp.1274- 1281. 10.1007/s00284-018-1520-2. hal-01910318 Distributed under a Creative Commons Attribution 4.0 International License Abstract A novel strain of a Gram-stain negative, non-motile, non-spore forming rod-shaped, obligate anaerobic bacterium, des- ignated AT11T, was isolated from a stool sample of a morbidly obese woman living in Marseille, France. This bacterium was characterized using biochemical, chemotaxonomic, and phylogenetic methods. The 16S rRNA gene sequence analysis showed that strain AT11T had a 97.8% nucleotide sequence similarity with Eisenbergiella tayi strain B086562T, the closest species with standing in nomenclature. The major cellular fatty acids of the novel isolate were C16:0 followed by saturated or unsaturated C18 fatty acids (C18:1n9, C18:1n5 and C18:0). The draft genome of strain AT11T is 7,114,554 bp long with 48% G+C content. 6176 genes were predicted, including 6114 protein-coding genes and 62 were RNAs (with 2 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes, and 56 tRNA genes). The digital DNA–DNA hybridization (dDDH) related- ness between the new isolate and E. tayi strain B086562T was 23.1% ± 2.2. Based on the phenotypic, chemotaxonomic, genomic, and phylogenetic characteristics, Eisenbergiella massiliensis sp. nov., is proposed. The type strain is AT11T (= DSM 100838T = CSUR P2478T). Draft Genome and Description of Eisenbergiella massiliensis Strain AT11T: A New Species Isolated from Human Feces After Bariatric Surgery Amadou H. Togo1 · Awa Diop2 · Matthieu Million1 · Marie Maraninchi3 · Jean‑Christophe Lagier1 · Catherine Robert2 · Fabrizio Di Pinto1 · Didier Raoult1 · Pierre‑Edouard Fournier2 · Fadi Bittar1,4 HAL Id: hal-01910318 https://hal.science/hal-01910318v1 Submitted on 26 Jul 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License * Fadi Bittar fadi.bittar@univ-amu.fr * Fadi Bittar fadi.bittar@univ-amu.fr 1 Aix Marseille Univ, IRD, APHM, MEPHI, IHU- Méditerranée Infection, Marseille, France 2 Aix Marseille Univ, IRD, APHM, SSA, VITROME, IHU-Méditerranée Infection, Marseille, France 3 Aix Marseille Univ, NORT “Nutrition, Obesity and Risk of Thrombosis”, INSERM1062, INRA1260, 13385 Marseille, France 4 IHU-Méditerranée Infection, 19-21 Bd Jean Moulin, 13005 Marseille, France 4 IHU-Méditerranée Infection, 19-21 Bd Jean Moulin, 13005 Marseille, France Introduction induces a sustainable weight loss, improves complications related to obesity, and increases the diversity of the gut flora [14, 34]. The number of people suffering from obesity has increased in recent decades [25]. It has been well established that the gut microbiota contributes to the development of human metabolic disorders such as obesity [18, 24]. Bariatric sur- gery is the most effective treatment for morbid obesity. It We conducted a study comparing the gut microbiota from obese patients before and after bariatric surgery using a new microbial high-throughput culture approach known as culturomics [16]. This new approach makes it possible to isolate and describe the living microbial diversity of any environmental and clinical sample. Using culturomics, we isolated a new anaerobic bacterium, strain AT11T, from a stool sample harvested following bariatric surgery. The dis- covery of this bacterium has been previously reported as a new species announcement without a thorough description [31]. Herein, strain AT11T was analyzed by a polyphasic approach in order to describe it as a new bacterial taxon. This combines phenotypic characteristics, the matrix laser desorption ionization-time of flight mass spectrometry (MALDI-TO MS) spectrum, and genomic properties known as taxono-genomics [27]. Here, we propose a classification and a set of pheno- typic, chemical, and chemotaxonomic characteristics of a new bacterial species: strain AT11T, which belongs to 4 IHU-Méditerranée Infection, 19-21 Bd Jean Moulin, 13005 Marseille, France 1 the genus Eisenbergiella [1], together with the descrip- tion of the complete genome sequencing, annotation, and genomic comparison. To date, this genus includes only one species Eisenbergiella tayi, the type strain B086562T (= LMG 27400T = DSM 26961T = ATCC BAA-2558T) as reported in List of Prokaryotic Names with Standing in Nomenclature (http://www.bacte rio.net/rumin ococc us.html). .cgi for the taxonomic assignation. Pairwise sequence simi- larities were calculated using the method recommended by Meier-Kolthoff et al. [23] and as described previously [33]. Sequences were aligned using ClustalW with default param- eters and phylogenies were inferred using the GGDC web server available at http://ggdc.dsmz.de/ using the DSMZ phylogenomics pipeline. Materials and Methods Different growth temperatures (room temperature, 28, 37, 45, and 55 °C) were tested on sheep blood-enriched Colum- bia agar (bioMérieux) under anaerobic conditions using GENbag anaer system (bioMérieux), microaerophilic con- ditions using GENbag microaer system (bioMérieux), and aerobic conditions, with or without 5% CO2. Ethics and Sample Collection Once informed consent had been obtained, stool samples were collected before and after surgery. These samples were obtained from a 56-year-old obese French woman follow- ing bariatric surgery on April 27, 2011. All samples were stored at − 80 °C before culturing. The study and the assent procedure were approved by the local ethics committee of IFR 48, under ascent number 09-022, 2010. Phenotypic and biochemical characteristics were per- formed as described elsewhere [32]. In addition to the three API gallery systems (API® ZYM, API® 20A, and API® 50 CH) usually used in our laboratory, API® Rapid ID 32A gallery system was added and the tests were done according to the manufacturer’s instructions (bioMérieux). Isolation and Identification of the Strain E test strips for Amikacin 0.016–256 µg/ml, Vancomy- cin 0.016–256 µg/ml, Imipenem 0.002–32 µg/ml, Ceftriax- one 0.016–256 µg/ml, Rifampicin 0.002–32 µg/ml, Benzyl penicillin 0.002–32 µg/ml, Amoxicillin 0.016–256 µg/ml, Minocycline 0.016–256 µg/ml, Teicoplanin 0.016–256 µg/ ml, Erythromycin 0.016–256 µg/ml, and Daptomycin 0.016–256 µg/ml (bioMérieux) were used for the antimicro- bial agent susceptibility of strain AT11T as recommended by EUCAST [4, 22]. Breakpoint tables for the interpretation of MICs and inhibition zone diameters, version 7.1, 2017, were used to interpret the results: these are available at http:// www.eucas t.org. Strain AT11T was first grown on July 22, 2015. One gram of stool was pre-incubated in BD BACTEC™ Lytic/10 Anaerobic/F Culture Vials media culture bottles (Bec- ton, Dickenson and Company, Le Pont de Claix, France) enriched with 4 ml of filtered rumen juice and 4 ml of sheep blood. The pre-incubated product was cultured on 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l’Etoile, France) as described elsewhere [32]. This strain was isolated 21 days after pre-incubation. The resulting colonies were then identified using MALDI- TOF mass spectrometry (Bruker Daltonics, Leipzig, Ger- many) as previously described [29]. When the spectra of a bacterium are not identified by MALDI-TOF MS screening, 16S rRNA gene amplification and sequencing is performed. Cellular fatty acid methyl ester (FAME) analysis of this was then performed using gas chromatography/mass spec- trometry (GC/MS) as described by Dione et al. [5]. Phylogenetic Analysis The genomic DNA of strain AT11T was sequenced and assembled as described in previous studies [33]. It was quantified by a Qubit assay using the high sensitivity kit (Life Technologies, Carlsbad, CA, USA) to 107.7 ng/µl and mechanically sheared with a circular shear to small frag- ments with an optimal length of 1401 bp using the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). A High Sensitivity Bioanalyzer LabChip (Agilent Technolo- gies Inc, Santa Clara, CA, USA) was used to visualize the library profile and the final concentration library was meas- ured at 34.4 nmol/l. The libraries were then normalized The 16S rRNA gene amplification PCR and sequencing were performed using GeneAmp PCR System 2720 ther- mal cyclers (Applied Bio systems, Bedford, MA, USA) and ABI Prism 3130xl Genetic Analyzer capillary sequencer (Applied Bio systems), respectively, as described by Dran- court et al. [6]. The CodonCode Aligner was used to correct sequences and BLASTn searches were performed on the NCBI (National Centre for Biotechnology Information) web server at http://blast .ncbi.nlm.nih.gov.gate1 .inist .fr/Blast 2 and pooled at 2 nM. After a denaturation step and dilu- tion at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and a sequenc- ing run were performed in a single 2 × 251-bp run. A total of 5.6 Gb of information was obtained from the 589 K/ mm2 cluster density with a cluster passing quality control filters of 96.1% (11,444,000 passing filter paired reads). Within this run, the index representation for strain AT11T was determined to 6.46%. The 697,439 paired reads were trimmed and assembled. to define the closed species genomes used for comparison analysis. Then, the potential missing genes in the genomes of interest were identified. All processes of annotation and comparison were performed using the Multi-Agent Soft- ware System DAGOBAH [8] that includes Figenix [9]. Genomic similarity was evaluated via digital DNA–DNA hybridization (dDDH) using the Genome to Genome Dis- tance Calculator (GGDC) 2.1-DSM web service (http:// ggdc.dsmz.de/ggdc.php). The species used for genomic comparison were retrieved from the 16S rDNA gene tree. Phylogenetic Analysis The following strains were selected: Blautia producta strain ATCC 27340T (ARET00000000) [7, 19, 28], Eisenbergiella tayi strain B086562T (MCGH00000000) [1, 2], Anaerostipes hadrus strain DSM 3319T (AMEY00000000) [13], Parasporobac- terium paucivorans strain DSM 15970T [20], Eubacterium ruminantium strain ATCC 17233T (GCA900167085) [3], Clostridium bolteae strain WAL 16351T (AGYH00000000) [30], and Clostridium clostridioforme strain ATCC 25,537 (GCA900113155) [12]. For each selected strain, the com- plete genome sequence was retrieved from the FTP of NCBI (National Center for Biotechnology Information). The proteome was analyzed using proteinOrtho [17]. For each couple of genomes, a similarity score was then computed. Genome Annotation and Comparison Open reading frames (ORFs) were predicted using Prodigal [10] with default parameters but the predicted ORFs were excluded if they were spanning a sequencing gap region (contain N). The predicted bacterial protein sequences were searched again the Clusters of Orthologous Groups (COG) using BLASTP with an E value of 1e−03, a coverage of 0.7, and a percent identity of 30%. If no hit was found, a search was conducted against the Nucleotide Redundant (NR) database using the same parameters. If the length of sequence was smaller than 80 amino acids, a 1e−05 E value was used. The tRNAScanSE tool [21] was used to find tRNA genes, while ribosomal RNAs were found by using RNAmmer [15]. Lipoprotein signal peptides and the num- ber of transmembrane helices was predicted using Phobius [11]. ORFans were identified if all the BLASTP performed gave no positive results with an E value smaller than 1e−03 for ORFs with a sequence size larger than 80 amino acid or an E value smaller than 1e−05 for ORFs with a sequence length smaller than 80 amino acids. Paralog genes were defined by blasting each protein gene against all protein genes of this genome. For pseudogenes, the first step was Fig. 1 Phylogenetic tree based on 16S rRNA sequence comparison highlighting the position of strain AT11T against other most closely related type strains. The scale bar repre- sents a 2% nucleotide sequence divergence Phylogenetic Analysis The spectrum generated from strain AT11T spots did not match those of Bruker and our in-house database (Sup- plementary Fig. 1) available at http://www.medit erran ee-infec tion.com/artic le.php?laref =933&titre =c-d-e. This new strain exhibited 97.76% nucleotide sequence similarity with Eisenbergiella tayi, the closest species with standing in nomenclature according to the 16S rDNA sequence analysis. Figure 1 presents the neighbor-join- ing phylogenetic tree (Fig. 1) based on 16S rRNA gene sequences and shows the relationships between strain AT11T and some related taxa. This sequence of the strain was deposited in EMBL-EBI under accession number LN881600. Phenotypic and Biochemical Characterization Strain AT11T is strictly anaerobic, its growth tempera- ture was between 28 and 45 °C, and optimal growth was observed at 37 °C. Colonies appeared light gray in color and exhibited an irregular form with a diameter between 0.5 and 1.5 mm after 72 h of culture on Columbia agar with 5% sheep blood (bioMérieux). No growth was observed above 5 g/l (10–100 g/l) salt on Schaedler agar with 5% sheep blood (bioMérieux). Cells were Gram-negative, non-motile, non-spore-forming, catalase positive, and rod shaped, measuring 1–3 µm in length and 0.4–0.5 µm wide using electron microscopy (Supplementary Fig. 2). The negativity of Gram staining was confirmed by the positive KOH test, but the strain had a positive Gram structure in electron microscopy. The characteristics of strain AT11T, according to API® gallery systems (50 CH, 20A, Zym and Rapid ID 32A), along with those of the closest species, Eisenbergiella tayi strain B086562T, are listed in Supple- mentary Table 1 and the differences between these two species are presented in Table 1. v Variable Hexadecanoic acid was the most abundant fatty acid (63%), followed by saturated and unsaturated C18 fatty acids representing approximately (33%) of total relative abundance. The fatty acid profiles of strain AT11T and the closest strain E. tayi B086562T are shown in Table 2. Accession Numbers The 16S rRNA gene sequence and whole-genome shotgun sequence of strain AT11T were deposited in EMBL-EBI under accession numbers LN881600 and OEZA00000000, respectively. The Digital Protologue database TaxonNumber for strain AT11T is TA00401. Fig. 1 Phylogenetic tree based on 16S rRNA sequence comparison highlighting the position of strain AT11T against other most closely related type strains. The scale bar repre- sents a 2% nucleotide sequence divergence Fig. 1 Phylogenetic tree based on 16S rRNA sequence comparison highlighting the position of strain AT11T against other most closely related type strains. The scale bar repre- sents a 2% nucleotide sequence divergence 3 Table 1 Differential characteristic of strain AT11T with Eisenber- giella tayi B086562T Properties Eisenbergiella massiliensis AT11T Eisenbergiella tayi B086562Ta Indole production V − Arabinose + − Arbutin + − Cellulose + − Dulcitol + − Gelatin + − Glucose + − Lactose + − Maltose V − Mannitol V − Mannose + − Raffinose + − Rhamnose + − Saccharose + − Salicin + − Sorbose + − Tagatose + − Trehalose + − Trypsin + − Xylose V − Potassium 5-cetogluconate + − Acid phosphatase + − Alkaline phosphatase V + Arginine hydrolase + − Esterase + − Esterase lipase + − Naphthol-AS-BI-phosphohy- drolase + − Tyrosine arylamidase − + α-Arabinosidase − + α-Fructosidase + − β-Glucuronidase + − Isolated from Human feces Blood Results and Discussion Table 1 Differential characteristic of strain AT11T with Eisenber- giella tayi B086562T Table 1 Differential characteristic of strain AT11T with Eisenber- giella tayi B086562T + Positive, − negative v Variable a Data for E. tayi were obtained from Amir et al. [25] Properties Eisenbergiella massiliensis AT11T Eisenbergiella tayi B086562Ta Indole production V − Arabinose + − Arbutin + − Cellulose + − Dulcitol + − Gelatin + − Glucose + − Lactose + − Maltose V − Mannitol V − Mannose + − Raffinose + − Rhamnose + − Saccharose + − Salicin + − Sorbose + − Tagatose + − Trehalose + − Trypsin + − Xylose V − Potassium 5-cetogluconate + − Acid phosphatase + − Alkaline phosphatase V + Arginine hydrolase + − Esterase + − Esterase lipase + − Naphthol-AS-BI-phosphohy- drolase + − Tyrosine arylamidase − + α-Arabinosidase − + α-Fructosidase + − β-Glucuronidase + − Isolated from Human feces Blood Genome Comparison The draft genome sequence of strain AT11T (7.11 MB) is larger in size than those of C. bolteae, B. producta, C. clostridioforme, Eubacterium ruminantium, and A. had- rus (6.38, 6.09, 5.46, 2.84, and 2.77 MB, respectively) but almost equal to that of E. tayi (7.15). Its G+C (48%) content is lower than that of C. clostridioforme and C. bolteae (49 and 49.6%, respectively), but higher than that of E. tayi, B. producta, Eubacterium ruminantium, and A. hadrus (46.3, 45.7, 37.2, and 37.2, respectively). Its gene content (6114) is higher than that of C. bolteae, B. producta, C. clostridioforme, A. hadrus, and Eubacte- rium ruminantium (5892, 5666, 5376, 2716, and 2533, respectively) but lower than that of E. tayi at 6156. The distribution of genes into COG categories was not entirely similar in all compared genomes (Fig. 3). The average genomic identity of orthologous gene sequences (AGIOS) Based on the phenotypic, chemotaxonomic, genomic, and phylogenetic characteristics, a novel bacterium iso- lated from the stool sample of a morbidly obese French woman, under the name Eisenbergiella massiliensis sp. nov., is proposed. The type strain is AT11T = DSM 100838T = CSUR P2478T. Genome Properties Two genes associated with Vancomycin (Vancomycin B-type resist- ance protein, VanW) resistance and 20 genes associated with beta-lactamase resistance were found using the RAST web server [26]. The remaining 1525 genes (24.94%) were annotated as hypothetical proteins. Genome Properties Antimicrobial agent susceptibility was tested according to the EUCAST recommendations leading to the following MIC results: 32, 0.5, 0.125, 1.6, 0.064, 0.38, 0.5, 0.5, and 0.125 µg/ml, respectively, for Amikacin, Vancomycin, Imi- penem, Ceftriaxone, Rifampicin, Benzyl penicillin, Amoxi- cillin, Minocycline, and Teicoplanin. The genome deposited in EMBL-EBI under accession num- ber OEZA00000000 (Fig. 2) is 7,114,554 bp long with 48% GC content. It is composed of 19 contigs consisting of 17 scaffolds. Of the 6176 predicted genes, 6114 were protein- coding genes and 62 were RNAs (two 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes, 56 tRNA genes). A total of 4321 genes (70.67%) were assigned a putative 4 ND Not detected a Data for E. tayi were obtained from Amir et al. [25] Fatty acid Name Strain AT11 E. tayia C16:0 Hexadecanoic acid 62.7 45.4 C18:1n9 9-Octadecenoic acid 10.3 14.8 C18:1n5 13-Octadecenoic acid 9.2 ND C18:0 Octadecanoic acid 7.8 12.8 C18:2n6 9,12-Octadecadienoic acid 4.4 1.3 C18:1n7 11-Octadecenoic acid 1.6 3.2 C17:0 Heptadecanoic acid 1.5 ND C15:0 Pentadecanoic acid < 1 < 1 C14:0 Tetradecanoic acid < 1 6.3 C16:1n7 9-Hexadecenoic acid < 1 ND C20:4n6 5,8,11,14-Eicosatetraenoic acid < 1 ND 9,10-Methylene-C16:0 2-Hexyl-cyclopropaneoctanoic acid < 1 ND Iso-C16:0 14-Methyl-pentadecanoic acid < 1 ND Iso-C15:0 13-Methyl-tetradecanoic acid < 1 ND C16: 0 2-OH 2-Hydroxyhexadecanoic acid ND 1.6 C17:2 Heptadecadienoic acid ND 11.6 C13:1 cis 12 12-Tridecanoic acid ND 2.1 Anteiso-C15:0 12-Methyl-tetradecanoic acid ND < 1 a Data for E. tayi were obtained from Amir et al. [25] values ranged from 61.7% between C. bolteae and Eubac- terium ruminantium to 90.8% between C. bolteae and C. clostridioforme among compared species without strain AT11T (Supplementary Table 2). When strain AT11T was included in the comparison, these values ranged from 62.2% with Eubacterium ruminantium to 78.4% with E. tayi (Supplementary Table 2). The dDDH values for strain AT11T ranged from 19.5% with A. hadrus to 34.4% with C. clostridioforme (Supplementary Table 3) with a prob- ability of error of ± 2%. These values are very low and below the cutoff of 70%, thus also confirming that this strain is a new species. function by COGs or by NR BLAST. 130 genes were iden- tified as ORFans (2.13%). The remaining 1525 genes (24. 94%) were annotated as hypothetical proteins. Table 2 Cellular fatty acid composition (%) of strain AT11T compared to its closest neighbor Eisenbergiella tayi strain B086562T Acknowledgements The authors thank the Xegen Company (http:// www.xegen .fr) for automating the genomic annotation process. Description of Eisenbergiella massiliensis sp. nov 3 Distribution of functional classes of predicted genes accord- ing to the Clusters of Orthologous Groups (COGs) of strain AT11T with its closest species: 1, Strain AT11T; 2, Eisenbergiella tayi strain DSM 26961T; 3, Blautia producta strain ATCC 27340T; 4, Clostrid- ium bolteae strain WAL 16351T; 5, Clostridium clostridioforme strain ATCC 25537T; 6, Parasporobacterium paucivorans strain DSM 15970T; 7, Anaerostipes hadrus strain ATCC 29173T; and 8, Eubac- terium ruminantium strain ATCC 17233T. Superscript T Type strain, ATCC American Type Culture Collection, DSM Deutsche Sammlung von Mikroorganismen, WAL Wadsworth Anaerobe Laboratory Fig. 3 Distribution of functional classes of predicted genes accord- ing to the Clusters of Orthologous Groups (COGs) of strain AT11T with its closest species: 1, Strain AT11T; 2, Eisenbergiella tayi strain DSM 26961T; 3, Blautia producta strain ATCC 27340T; 4, Clostrid- ium bolteae strain WAL 16351T; 5, Clostridium clostridioforme strain ATCC 25537T; 6, Parasporobacterium paucivorans strain DSM 15970T; 7, Anaerostipes hadrus strain ATCC 29173T; and 8, Eubac- terium ruminantium strain ATCC 17233T. Superscript T Type strain, ATCC American Type Culture Collection, DSM Deutsche Sammlung von Mikroorganismen, WAL Wadsworth Anaerobe Laboratory Funding This work was funded by Fondation Méditerranée Infection. Funding This work was funded by Fondation Méditerranée Infection. Funding This work was funded by Fondation Méditerranée Infection. 6. Drancourt M, Bollet C, Carlioz A et al (2000) 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 38:3623–3630 Compliance with Ethical Standards 7. Ezaki T, Li N, Hashimoto Y et al (1994) 16S ribosomal DNA sequences of anaerobic cocci and proposal of Ruminococcus hansenii comb. nov. and Ruminococcus productus comb. nov. Int J Syst Bacteriol 44:130–136 Conflict of interest The author declares that they have no conflicts of interest. 8. Gouret P, Paganini J, Dainat J et al (2011) Integration of evolu- tionary biology concepts for functional annotation and automa- tion of complex research in evolution: the multi-agent software system DAGOBAH. In: Pontarotti P (ed) Evolutionary biology— concepts, biodiversity, macroevolution and genome evolution. Springer, Berlin Heidelberg, pp 71–87 Description of Eisenbergiella massiliensis sp. nov Eisenbergiella massiliensis (mas.si.li.en’sis. L. fem. adj. massiliensis, of Massilia, the Latin name for Marseille). It is a strictly anaerobic bacterium which grows at a meso- thermal temperature of 37 °C. The colonies grown on Columbia agar with 5% enriched sheep blood are light gray, non-hemolytic, and irregular with a diameter of 0.5 mm. 5 Fig. 2 Graphical circular map of the genome of strain AT11T: From outside to the center: Contigs (red/gray), Clusters of Orthologous Groups (COGs) category of genes on the forward strand (three cir- cles), genes on forward strand (blue circle), genes on the reve strand (red circle), COGs category on the reverse strand (three c cles), G+C content. (Color figure online) Fig. 2 Graphical circular map of the genome of strain AT11T: From outside to the center: Contigs (red/gray), Clusters of Orthologous Groups (COGs) category of genes on the forward strand (three cir- cles), genes on forward strand (blue circle), genes on the reverse strand (red circle), COGs category on the reverse strand (three cir- cles), G+C content. (Color figure online) Cells exhibit a negative Gram-stain, are non-spore-form- ing, non-motile, catalase positive, and rod-shaped bacilli, with a size of 0.4/2 µm. Using the API Gallery systems (API® ZYM API® 50CH API® 20A and API® rapid ID 32A) in anaerobic condition, positives reactions were observed for acid phosphatase, alkaline phosphatase, ester- ase, esterase lipase, naphthol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase, α-arabinosidase, α-fucosidase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase, 6-phosphate-β-galactosidase, arbutin, D-cel- lobiose, D-glucose, D-lactose, D-lyxose, D-maltose, D-mannose, D-raffinose, D-saccharose, D-tagatose, D-trehalose, dulcitol, D-xylose, L-arabinose, L-rhamnose, L-sorbose, potassium 5-cetogluconate, and salicin. Urease and indole are not pro- duced, gelatin was not liquefied and nitrate was not reduced, although esculin was hydrolyzed. The major cellular fatty acids detected were C16:0 (62.7%) and C18:1n9 (10.3%). Its genome, consisting of one chromosome, is 7,114,554 bp in length with 48% of G+C content. 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Clin Microbiol Infect 20:O255–O266 nov. and Clostridium symbiosum (Stevens) comb. nov. Int J Syst Bacteriol 26:195–204 13. Kant R, Rasinkangas P, Satokari R et al (2015) Genome sequence of the butyrate producing Anaerobic bacterium Anaerostipes had- rus PEL 85. Genome Announc 3:e00224-15 23. Meier-Kolthoff JP, Göker M, Spröer C, Klenk H-P (2013) When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol 195:413–418 14. Kong L-C, Tap J, Aron-Wisnewsky J et al (2013) Gut microbiota after gastric bypass in human obesity: increased richness and asso- ciations of bacterial genera with adipose tissue genes. Am J Clin Nutr 98:16–24 24. Million M, Maraninchi M, Henry M et al (2012) Obesity-asso- ciated gut microbiota is enriched in Lactobacillus reuteri and depleted in Biﬁdobacterium animalis and Methanobrevibacter smithii. Int J Obes 36:817–825 15. References nov., sp. nov. Appl Environ Microbiol 67:4017–4023 32. Togo AH, Khelaifia S, Lagier J-C et al (2016) Noncontiguous finished genome sequence and description of Paenibacillus ihumii sp. nov. strain AT5. New Microbes New Infect 10:142–150 33. Togo AH, Durand G, Khelaifia S et al (2017) Fournierella massil- iensis, gen. nov., sp. nov., a new human-associated member of the family Ruminococcaceae. Int J Syst Evol Microbiol 67:1393–1399 21. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964 34. Zhang H, DiBaise JK, Zuccolo A et al (2009) Human gut micro- biota in obesity and after gastric bypass. Proc Natl Acad Sci USA 106:2365–2370 22. Matuschek E, Brown DFJ, Kahlmeter G (2014) Development of the EUCAST disk diffusion antimicrobial susceptibility testing 8
https://openalex.org/W4362620463	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Resolving_the_Heterogeneous_Tumor-Centric_Cellular_Neighborhood_through_Multiplexed_Spatial_Paracrine_Interactions_in_the_Setting_of_Immune_Checkpoint_Blockade/22544142/1/files/40007565.pdf	English	null	Supplementary Figure 1 from Resolving the Heterogeneous Tumor-Centric Cellular Neighborhood through Multiplexed, Spatial Paracrine Interactions in the Setting of Immune Checkpoint Blockade	null	2,023	cc-by	84	Supplementary Figure 1. Phenotypic Cell Classification Decision Tree. Markers from panel design assigned to each cell phenotype for cell classification Figure created with Biorender.com Supplementary Figure 1. Phenotypic Cell Classification Decision Tree. Markers from panel design assigned to each cell phenotype for cell classification Figure created with Biorender.com Supplementary Figure 1. Phenotypic Cell Classification Decision Tree. Markers from panel design assigned to each cell phenotype for cell classification Figure created with Biorender.com assigned to each cell phenotype for cell classification Figure created with Biorender.com
https://openalex.org/W3118128317	http://www.thieme-connect.de/products/ejournals/pdf/10.1055/s-0040-1715088.pdf	English	null	Endovascular Graft Infection by Neisseria meningitidis: A Rare but Fatal Infection	Aorta	2,020	cc-by	2,200	Article published online: 2020-12-23 Article published online: 2020-12-23 Article published online: 2020-12-23 THIEME Case Report 148 Case Report 148 Introduction Infection of endovascular prosthesis is a rare but challenging complication that requires a tailored approach to remove the infected graft and restore the arterial circulation, combined with long-term antibiotic therapy. Despite surgical repair, morbidity and mortality remain high.1–3 Preoperative workup consisted of a transthoracic echo- cardiogram that revealed good biventricular function; a CT coronary angiogram that did not identify signiﬁcant coro- nary stenosis and a positron emission tomography-CT (PET- CT) that conﬁrmed intense avidity in the infrarenal peri- aortic tissues (►Fig. 3). Abstract Endovascular graft infection is a rare but challenging complication that requires a tailored approach to remove the infected graft and restore the arterial circulation combined with long-term antibiotic therapy. We present a case surgically treated with explant of the graft and reconstruction of the thoracoabdominal aorta. Microbiological investigation revealed growth of Neisseria meningitidis, which is extremely rare in this location, and to our knowledge, has not been previously published in the literature. y ►infrarenal aneurysm ►EVAR ►infection ►thoracoabdominal ►meningococcal disease A multidisciplinary team (MDT) decided that the best approach was open surgery to remove the infected prosthe- sis and primarily repair the thoracoabdominal aorta (Craw- ford’s level-IV extent). Copyright © 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 760-0888. Endovascular Graft Infection by Neisseria meningitidis: A Rare but Fatal Infection Ana Lopez-Marco, PhD1 Satya Das, MD2 Robert Seraﬁno-Wani, MD2 Sadasivam Benjamin Adams, MD1 Aung Oo, MD1 Ana Lopez-Marco, PhD1 Satya Das, MD2 Robert Seraﬁno-Wani, MD2 Sadasivam Selvakumar, MD3 Benjamin Adams, MD1 Aung Oo, MD1 Das, MD2 Robert Seraﬁno-Wani, MD2 Sadasivam Selvakumar, MD3 1 Address for correspondence Ana Lopez-Marco, PhD, Department of Cardiothoracic Surgery, Barts Heart Centre, St. Bartholomew’s Hospital, West Smithﬁeld, London, EC1A 7BE, United Kingdom (e-mail: ana.lopez-marco@nhs.net). 1Department of Cardiac Surgery, Barts Heart Centre, St Bartholomew’s Hospital, London, United Kingdom 2Department of Microbiology, St. Bartholomew’s Hospital, London, United Kingdom 3Department of Vascular Surgery, Lister Hospital, Stevenage, United Kingdom AORTA 2020;8:148–151. Case Presentation We present a 69-year-old male who underwent endovascu- lar aneurysm repair (EVAR) of his infrarenal aortic aneurysm with a modular graft (aortic bifurcated body and two iliac limbs, TriVascular Ovation Prime, and Ovation iX Iliac, Tri- Vascular Inc., Santa Rosa, CA). He was then prepared for urgent aortic surgery. Intra- operative monitoring consisted of right radial and right femoral arterial lines, venous central line, near-infrared spectrometry for cerebral, paraspinal, and lower limb satu- rations, and motor-evoked potentials. A spinal drainage system was inserted for spinal protection. After 18 months, he developed ﬂu-like symptoms with persistent elevated systemic C-reactive protein (CRP; >120 - mg/L). Blood cultures were negative. Amoxicillin/clavulanic acid and steroids controlled his symptoms; however, com- puted tomography (CT) revealed a new ﬂuid and gaseous perigraft collection extending into the left iliac fossa con- ﬁrming the diagnosis of infected EVAR (►Figs. 1 and 2). A left thoracophrenolaparotomy was performed through the seventh left intercostal space toward the median abdomi- nal line. The diaphragm was divided circumferentially and the abdominal aorta approached via retroperitoneal space. The descending thoracic aorta (DTA) was mobilized, leav- ing the visceral segment undisturbed to avoid complications. received January 24, 2020 accepted after revision June 11, 2020 received January 24, 2020 accepted after revision June 11, 2020 DOI https://doi.org/ 10.1055/s-0040-1715088. ISSN 2325-4637. Open Thoracoabdominal Aortic Surgery after Infected Endovascular Repair Lopez-Marco et al. 149 Fig. 3 Axial view of the positron emission tomography-computed tomography showing a high uptake extending into the left iliac fossa. Fig. 1 Axial view of the preoperative contrasted computed tomog- raphy of the aorta demonstrating a Type-II endoleak and a periaortic gas collection (highlighted by yellow arrows). Fig. 1 Axial view of the preoperative contrasted computed tomog- raphy of the aorta demonstrating a Type-II endoleak and a periaortic gas collection (highlighted by yellow arrows). Fig. 3 Axial view of the positron emission tomography-computed tomography showing a high uptake extending into the left iliac fossa. Fig. 4 Modular components of the prosthesis used for the endo- vascular aneurysm repair after the surgical explantation. Fig. 2 Coronal view of the preoperative contrasted computed tomogra- phy of the aorta, demonstrating the presence of a periaortic gas collection extending into the left iliac fossa (highlighted by yellow arrows). Fig. Acknowledgments None. Acknowledgments None. If there is no immediate danger to the patient’s life, conser- vative treatment can be attempted with antibiotics guided by sensitivity of the causative organism when available; surgery should be attempted ﬁrst inyoung and ﬁt patients and in those unlikely to resolve conservatively (i.e., extensive perigraft purulence, pseudoaneurysm, or suspected aortoenteric ﬁstu- la). Surgical treatment consists of removal of the infected graft followed by revascularization techniques using synthetic, autologous, or cryopreserved allografts either for in situ reconstruction or extra-anatomical bypass techniques. Open Thoracoabdominal Aortic Surgery after Infected Endovascular Repair Lopez-Marco et al. Open Thoracoabdominal Aortic Surgery after Infected Endovascular Repair Lopez-Ma When the infrarenal aorta mobilization started, the pa- tient showed signs of systemic inﬂammatory response and sepsis (SIRS), requiring increasing ﬂuid replacement therapy that generated a shift of ﬂuids to the third space and progressive swelling of the intraperitoneal contents. The thoracolaparotomy wound was left open with the cavity packed with surgical swabs. Neisseria meningitidis appears as gram-negative cocci, oriented in pairs. Neisseria spp. inhabit the nasopharynx as commensals and are transmitted by close exposure through aerosolization of respiratory droplets or direct contact with secretions with an incubation period between 1 to 14 days.7 With appropriate host humoral immune response, the invasive disease is prevented. However, with suboptimal immunity, invasion and spread will occur. Meningococcus utilizes a spectrum of virulence factors to evade the host- immune responses: capsule, immunoglobulin-A proteases, transferrin binding proteins, and surface blebs containing lipopolysaccharide which functions as an endotoxin and promotes cascade of proinﬂammatory cytokines (tumor necrosis factor-α, interleukin [IL]-I, IL-6, and IL-8) that lead to endothelial damage, capillary leak, procoagulant state and microthrombi formation. Postoperative period was marked by profound hypotension and vasodilation secondary to SIRS. The patient developed pro- gressive multiorgan failure and died in the early morning hours. Cultures of the infected graft and evacuated pus collection revealed a profuse growth of Neisseria meningitidis from three different samples. Postoperative period was marked by profound hypotension and vasodilation secondary to SIRS. The patient developed pro- gressive multiorgan failure and died in the early morning hours. Cultures of the infected graft and evacuated pus collection revealed a profuse growth of Neisseria meningitidis from three different samples. AORTA Vol. 8 No. 5/2020 Conﬂict of Interest These cases are best discussed on an MDT formed by vascular and cardiovascular surgeons, radiologists, and spe- cialists of infectious diseases to ﬁnd a procedure that com- bines the best morbidity, mortality, and durability.1–4 Case Presentation 2 Coronal view of the preoperative contrasted computed tomogra- phy of the aorta, demonstrating the presence of a periaortic gas collection extending into the left iliac fossa (highlighted by yellow arrows). The left inferior pulmonary vein was cannulated with a 28-F cannula in preparation for left heart bypass. The left femoral artery was not used due to extensive calciﬁcation. The distal DTA was clamped, and the aorta was opened longitudinally along the visceral segment; distal perfusion to the limbs was established via two separate 13-F balloon- tipped cannula placed into the iliacs. Fig. 4 Modular components of the prosthesis used for the endo- vascular aneurysm repair after the surgical explantation. Reimplantation of the visceral vessels was performed with continuous 4/0 Prolene in the following sequence: celiac axis, SMA, right renal, and left renal. Removal of the main body of the EVAR (►Fig. 4) was then performed and visceral protection established by adminis- tering cold blood through the celiac axis and the superior mesenteric artery (SMA) with two separate 13-F balloon- tipped cannulae and 1 L of Custodiol solution with two separate 10-F balloon-tipped cannula into the renal arteries. A 20-mm Rifampicin-soaked four-branched graft was anastomosed to the distal DTA at the level of the diaphragm with a two-layered buttressed 3/0 Prolene. Removal of the main body of the EVAR (►Fig. 4) was then performed and visceral protection established by adminis- tering cold blood through the celiac axis and the superior mesenteric artery (SMA) with two separate 13-F balloon- tipped cannulae and 1 L of Custodiol solution with two separate 10-F balloon-tipped cannula into the renal arteries. The remaining modules of the EVAR were then fully removed (►Fig. 4), as well as the inﬂammatory tissue and clots within the aneurysmal sac and the left iliac fossa abscess. A 30-mm straight Dacron graft was anastomosed to the suprailiac aortic segment with the same buttressed tech- nique used for the proximal anastomosis and ﬁnally, the two Dacron grafts were anastomosed together. A 20-mm Rifampicin-soaked four-branched graft was anastomosed to the distal DTA at the level of the diaphragm with a two-layered buttressed 3/0 Prolene. AORTA Vol. 8 No. 5/2020 150 Funding None. There is no standard treatment and a tailored approach should always be offered. The ultimate goal is to remove the infected graft, but this is not always feasible due to general ﬁtness or comorbidities. Conﬂict of Interest The authors declare no conﬂict of interest related to this article. Discussion Graft infections after endovascular abdominal aneurysm re- pair are rare (incidence < 1%); however, they are extremely challenging to manage, are associated with signiﬁcant mor- bidity and mortality, and are potentially lethal if untreated.1–3 Meningococcal infection varies from nonlocalized febrile illness to meningitis and/or septicaemia.8 We believe that our patient had the initial bacteremia with the ﬂu-like symptoms and the graft infected by the presence of the meningococci in the blood stream. Anti- biotics were effective to treat the systemic but not the graft infection. The foreign material of the graft and the thrombus within the aneurysm sac provide a nidus where microorganisms can grow. Predominant microorganisms are gram positive (Staph- ylococci and Streptococci), although gram-negative enteric organisms and polymicrobial infections are not rare.1–4 Most likely, the SIRS during surgery was consequence of Neisseria meningitidis release into the blood stream during the EVAR and perigraft collection manipulation leading to the effects described earlier. Diagnosis is complex and requires a high index of suspi- cion, based on a combination of clinical symptoms, imaging and microbiological investigations (negative cultures in up to 33% of the cases).1–4 To conclude, we report a rare case of infrarenal EVAR infection by Neisseria meningitidis. Despite a well-planned surgery, the patient could not overcome the perioperative SIRS following the removal of the infected prosthesis and died in the early hours after the operation. Angio-CT is the gold-standard imaging technique, looking for perigraft gas/ﬂuid collections, soft-tissue attenuation, pseudoaneurysms, or ﬁstulous formations. PET-CT is useful for low-grade infections, showing high avidity within the infected area.1–4 7 Pollard AJ, Finn A. Neisseria meningitidis. In: Long SS, Pickering LK, Prober CG, eds. Principles and Practice of Pediatric Infectious Diseases. Philadelphia, PA2012:730–741 5 Schaefers JF, Donas KP, Panuccio G, et al. Outcomes of surgical explantation of infected aortic grafts after endovascular and open abdominal aneurysm repair. Eur J Vasc Endovasc Surg 2019;57(01): 130–136 6 Chaikof EL, Dalman RL, Eskandari MK, et al. The Society for Vascular Surgery practice guidelines on the care of patients with an abdomi- nal aortic aneurysm. J Vasc Surg 2018;67(01):2–77.e2 8 van Deuren M, Brandtzaeg P, van der Meer JW. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev 2000;13(01): 144–166 Infection Collaboration (MAGIC). Eur J Vasc Endovasc Surg 2016; 52(06):758–763 References 1 Setacci C, Chisci E, Setacci F, et al. How to diagnose and manage infected endografts after endovascular aneurysm repair. Aorta (Stamford) 2014;2(06):255–264 2 Wilson WR, Bower TC, Creager MA, et al. Vascular graft infection, mycotic aneurysms and endovascular infections. A scientiﬁc statement from the American Heart Association. Circulation 2016;134:412–460 3 Argyriou C, Georgiadis GS, Lazarides MK, Georgakarakos E, Anto- niou GA. Endograft infection after endovascular abdominal aortic aneurysm repair: a systematic review and meta-analysis. J Endo- vasc Ther 2017;24(05):688–697 Long-term antibiotic therapy and monitoring of CRP are usually required.1–4 Mortality after infected abdominal EVAR is still signiﬁcant despite the treatment used: 27 to 37% after surgery, 50% after endovascular, and 63% with conservative management.5,6 4 Lyons OTA, Baguneid M, Barwick TD, et al. Diagnosis of aortic graft infection: a case deﬁnition by the Management of Aortic Graft endovascular, and 63% with conservative management.5,6 7 Pollard AJ, Finn A. Neisseria meningitidis. In: Long SS, Pickering LK, Prober CG, eds. Principles and Practice of Pediatric Infectious Diseases. Philadelphia, PA2012:730–741 8 van Deuren M, Brandtzaeg P, van der Meer JW. Update on meningococcal disease with emphasis on pathogenesis and clinical management. Clin Microbiol Rev 2000;13(01): 144–166 7 Pollard AJ, Finn A. Neisseria meningitidis. In: Long SS, Pickering LK, Prober CG, eds. Principles and Practice of Pediatric Infectious Diseases. Philadelphia, PA2012:730–741 Open Thoracoabdominal Aortic Surgery after Infected Endovascular Repair Lopez-Marco et al. Open Thoracoabdominal Aortic Surgery after Infected Endovascular Repair Lopez-Marco et al. 151 Infection Collaboration (MAGIC). Eur J Vasc Endovasc Surg 2016; 52(06):758–763 6 Chaikof EL, Dalman RL, Eskandari MK, et al. The Society for Vascular Surgery practice guidelines on the care of patients with an abdomi- nal aortic aneurysm. J Vasc Surg 2018;67(01):2–77.e2 AORTA Vol. 8 No. 5/2020
https://openalex.org/W2492038593	https://europepmc.org/articles/pmc5288197?pdf=render	English	null	Radiation driven epithelial-mesenchymal transition is mediated by Notch signaling in breast cancer	Oncotarget	2,016	cc-by	7,753	Abstract Epithelial to mesenchymal transition (EMT) is developmental process associated with cancer metastasis. Here, we found that breast carcinoma cells adopt epithelial- to-mesenchymal transition (EMT) in response to fractionated-radiation. Importantly, we show that Notch signaling is highly activated in fractionally-irradiated tumors as compared to non-irradiated tumors that are accompanied by an EMT. Moreover, we uncovered the mechanism of Notch-driven EMT, in which Notch enhanced EMT through IL-6/JAK/STAT3 signaling axis in mammary tumor cells. Collectively, we present converging evidence from our studies that Notch2 is a critical mediator of radiation- induced EMT and responsible for induced malignant tumor growth. Radiation driven epithelial-mesenchymal transition is mediated by Notch signaling in breast cancer p Rae-Kwon Kim1,, Neha Kaushik1,, Yongjoon Suh1, Ki-Chun Yoo1, Yan-Hong Cui1, Min-Jung Kim2, Hae-June Lee3, In-Gyu Kim4, Su-Jae Lee1 Rae-Kwon Kim1,, Neha Kaushik1,, Yongjoon Suh1, Ki-Chun Yoo1, Yan-Hong Cui1, Min-Jung Kim2, Hae-June Lee3, In-Gyu Kim4, Su-Jae Lee1 1Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, Korea 2Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, Korea Institute of Radiological and Medical Sciences, Seoul, Korea 2Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Radiological and Medical Sciences, Seoul, Korea 3Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, Korea Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul, Korea 4Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon, Korea 4Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, Daejeon, Korea These authors contributed equally to this work These authors contributed equally to this work Correspondence to: Su-Jae Lee, email: sj0420@hanyang.ac.kr p , j y g Keywords: radiation, EMT, Notch signaling, interleukin-6, breast cancer Received: May 23, 2016 Accepted: July 13, 2016 Published: July 23, 2016 Keywords: radiation, EMT, Notch signaling, interleukin-6, breast cancer y , , g g, , Received: May 23, 2016 Accepted: July 13, 2016 Published: July 23, 2016 Published: July 23, 2016 www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ Introduction To confirm this phenomenon, the invasiveness of fractionated-radiation exposed MCF7 cells was visualized and quantified into a collagen-based matrix. Consistent with the results of Transwell boyden chamber invasion assays, the invasiveness was highly enhanced in MCF7 cells after fractionated radiation at day 9 (Figure 1F). To test the involvement of SLUG in radiation-induced EMT further, MCF7 breast cancer cells were transfected with small interfering RNA (siRNA) targeting SLUG prior to a radiation treatment. Of note, the knock-down of SLUG effectively decreased the radiation-induced migratory and invasive properties of both MCF7 and SKBR3 breast cancer cells (Supplementary Figure S1B and S1B). Importantly, the downregulation of E-cadherin protein levels was retained and the accumulation of N-cadherin and vimentin was abolished with the knockdown of SLUG (Supplementary Figure S1D) in MCF7 cells treated with fractionated radiation. In addition, immunostaining data revealed that E-cadherin was further restored while N-cadherin and vimentin levels were diminished in radiation treated MCF7 cells (Supplementary Figure S1E). Taken together, these results suggest that radiation causes breast cancer cells to acquire migratory and invasive properties by inducing SLUG and thereby triggering the EMT program. repressor to an activator by recruiting cofactors. However, the role of Notch signaling in radiation-induced EMT remains largely unknown. i Independent of these findings, some cytokines, secreted by cells within tumor microenvironment, is critical to stimulate EMT [19]. Among all of these, IL-6 can promote tumorigenicity, angiogenesis, and metastasis [20, 21]. In addition, IL-6 has been shown to be a direct regulator of the self-renewal of breast cancer stem cells as mediated by the IL-6 receptor/GP130 complex through STAT3 activation [22]. An elevated level of IL-6 mediated Jagged1/Notch signaling was also proposed to promote breast cancer bone metastasis [23]. These studies suggest a functional role of IL-6 with regard to malignant progression and metastasis. Still, mechanisms of contributory role of IL-6 in facilitating the progression from a pre-malignant state to a state of metastasis remain obscure. The challenge to establish how fractionated radiation contributes to the EMT acquiring acquisition of malignant phenotypes following exposure doses in cancer patients remains unexplored. In this study, we investigated that Notch signaling is critical for fractionated radiation- induced EMT. Mechanistically, we uncover an innovative mechanism of the regulation of Notch2 via the JAK/ STAT3 signaling pathway and subsequently-induced tumor malignancy in response to the radiation. Introduction morphological modifications from an epithelial cobblestone- like type to an elongated mesenchymal fibroblast type including decreased expression in epithelial markers such as E-cadherin with increased expression of mesenchymal markers such as N-cadherin and vimentin [9, 10]. It is widely accepted that EMT plays a key role in the radio- resistance phenomenon and has been broadly explored in several types of tumors such as gliomas and breast, and lung cancer [11–13]. Recently, it is well-believed that EMT is also triggered by extracellular stimuli such as hypoxia and radiation [14]. Most cancer deaths from breast cancer result from tumor recurrence following treatment of the primary tumor. Ionizing radiation (IR) is the preferred treatment strategy for curing cancers at advanced stages including breast cancer. However, the emergence of radio-resistance leads to the failure of radiotherapy and subsequently increases mortality frequently in patients [1]. These consequences suggest that a subcellular fraction of radiation resistant tumor cells with potent tumorigenic activity is critical for re-growth [2–4]. To overcome these problems, determining the underlying molecular mechanisms associated with radiation-induced epithelial- to-mesenchymal transition (EMT) will be helpful for breast cancer relapse predictions and would greatly improve the therapeutic approaches for this disease [5– 7]. Undertaking this aim, we focused on epithelial- to-mesenchymal transition (EMT) phenomenon in breast tumor cells. Accumulating evidences suggests that Notch signaling involved in many physiological processes including call fate control during neurogenesis, cancer stem cell maintenance, EMT, and differentiation [15–17]. Notch signaling can be activated through four transmembrane Notch receptors (Notch 1–4) that can bind with Notch family ligands (Jagged-1, Jagged-2, Delta- like 1, 3, and 4) [18]. Following ligand binding, Notch receptors undergo a series of proteolytic cleavages and the released Notch intracellular domain (NICD) translocate to the nucleus where it interacts with the DNA binding protein CSL (C protein binding factor 1/Suppressor of Hairless/Lag-1), transforming it from a transcriptional The process of EMT is crucial in metastatic dissemination and has been the subject of intense investigations [8]. EMT is controlled by multifaceted pathways in which differentiated epithelial cells undergo www.impactjournals.com/oncotarget Oncotarget 53430 levels were up-regulated in response to the fractionated radiation in MCF7 cells (Figure 1D). Concomitantly, when the protein levels of EMT transcription factors such as SNAIL, SLUG, ZEB1 and TWIST were investigated, irradiation was found to cause a strong increase of SLUG; however, other EMT transcription factors were not altered by irradiation in MCF7 breast cancer cells (Figure 1E). Introduction Furthermore, we find that radiation-induced IL-6 secretion serves as a major contributor to Notch-induced EMT in breast cancer cells in response to the fractionated radiation. The expression of Notch ligands is required for radiation-induced Notch activation leading to EMT As the Jagged-1 mediated activation of Notch signaling is important during the induction of EMT [27], we subsequently sought to determine whether Notch ligands are expressed in breast cancer cells due to fractionated radiation. Since the Notch2 receptor is recognized in irradiated breast cancer cells and the invasive and migratory properties of MCF7 breast cancer p is recognized in irradiated breast cancer cells and the Figure 1: Fractionated radiation promotes invasiveness of breast cancer cells through EMT. (A and B) Migration and invasion assay of MCF7 and SKBR3 breast cancer cells in transwells after fractionated irradiation (2 Gy × 3), respectively. (C) Immunocytochemistry for EMT markers such as E-cadherin and Vimentin in MCF7 cancer cells after irradiation. (D) Protein expression of EMT markers such as E-cadherin, N-cadherin and Vimentin in MCF7 and SKBR3 breast cancer cells after irradiation (E) Protein expression of EMT transcription factors such as SNAIL, SLUG, ZEB1 and TWIST in MCF7 and SKBR3 breast cancer cells after irradiation. (F) Invasion of GFP-transduced MCF7 spheroid cells treated in collagen-based matrix 3D culture system following irradiation. A0 represents the area of sphere at day 0 while At is the area at day 9. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05, and p < 0.01. Figure 1: Fractionated radiation promotes invasiveness of breast cancer cells through EMT. (A and B) Migration and invasion assay of MCF7 and SKBR3 breast cancer cells in transwells after fractionated irradiation (2 Gy × 3), respectively. (C) Immunocytochemistry for EMT markers such as E-cadherin and Vimentin in MCF7 cancer cells after irradiation. (D) Protein expression of EMT markers such as E-cadherin, N-cadherin and Vimentin in MCF7 and SKBR3 breast cancer cells after irradiation (E) Protein expression of EMT transcription factors such as SNAIL, SLUG, ZEB1 and TWIST in MCF7 and SKBR3 breast cancer cells after irradiation. (F) Invasion of GFP-transduced MCF7 spheroid cells treated in collagen-based matrix 3D culture system following irradiation. A0 represents the area of sphere at day 0 while At is the area at day 9. β-actin was used as a loading control. Error bars Figure 1: Fractionated radiation promotes invasiveness of breast cancer cells through EMT. (A and B) Migration and invasion assay of MCF7 and SKBR3 breast cancer cells in transwells after fractionated irradiation (2 Gy × 3), respectively. Radiation promotes the induction of mesenchymal traits associated with EMT in breast cancer cells Recently, the Notch signaling pathway has widely accepted as a vital regulator during the induction of EMT. It has been suggested as a direct target of SLUG via regulating the SLUG promoter through the CSL interaction, resulting in the up-regulation of SLUG in endothelial cells [24–26]. In line with these studies, to understand the invasiveness of cells in response to the fractionated radiation, we subsequently examined the mRNA expression levels of the Notch family. Interestingly, Notch2 mRNA levels were markedly increased in MCF7 cells after radiation, while other Notch family components such as Notch1, Notch3 and Notch4 remained unchanged (Figure 2A). Also, the activation of Notch2 (NICD2) is noticeably increased through cleavage followed by fractionated radiation (Figure 2B). In addition, to extend our observations, MCF7 cells were transfected with siRNA targeting Notch2. Down-regulation of Notch2 significantly blocked migration and invasion in MCF7 cells in response to radiation treatment (Figure 2C). Moreover, immunostaining data revealed that E-cadherin To explore the possible harmful effect of IR, we tested whether IR causes breast cancer cells to acquire mesenchymal traits. Elevated cell migration is one of the characteristic features of cells undergoing EMT. Accordingly, we initially examined the migration and invasion in MCF7 and SKBR3 cells. To this end, both cancer cells were exposed to fractionated doses of radiation (2 Gy × 3; 2 Gy for consistent 3 days). Notably, migration and invasion were significantly increased in both cells after fractionated radiation (Figure 1A and 1B). However, the cell growth rate was significantly decreased by the fractionated radiation in both cell lines with post incubation time (Supplementary Figure S1A), suggesting that radiation is able to induce EMT while also inhibiting cell growth. Loss of E-cadherin and an increase in vimentin exhibited morphological modifications in MCF7 cells as compared to un-irradiated cells after fractionated radiation (Figure 1C). In addition, E-cadherin protein levels were downregulated and N-cadherin and vimentin protein www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 53431 was restored and the N-cadherin was abolished with the knock-down of Notch2 in radiation-treated MCF7 cells (Figure 2D). Accordingly, protein levels of E-cadherin were up-regulated and accumulation of N-cadherin and vimentin was abolished after knock-down of Notch2 in irradiated MCF7 breast cancer cells (Figure 2E). It is worth mentioning here that the knock-down of Notch2 effectively down-regulates SLUG protein levels in irradiated MCF7 cells; however other transcription factors such as SNAIL and TWIST remain unchanged (Figure 2F). Radiation promotes the induction of mesenchymal traits associated with EMT in breast cancer cells Furthermore, the down-regulation of Notch2 decreased the clonogenic survival rate in irradiated MCF7 and SKBR3 cells (Supplementary Figure S2A and S2B). To confirm the involvement of Notch in fractionated radiation-induced EMT in breast cancer cells, we blocked Notch signaling using a gamma secretase inhibitor (GSI). The inhibition of Notch2 activation attenuated the radiation-induced invasive and migratory properties of MCF7 breast cancer cells (Figure 2G) and recovered the E-cadherin protein levels with the down-regulation of N-cadherin, vimentin and NICD2 (Figure 2H). These changes also mitigate radiation-induced SLUG induction in irradiated MCF7 cells (Figure 2I). Collectively, these findings suggest that Notch2 regulates radiation-induced EMT responses via the NICD2 domain in breast cancer cells. The expression of Notch ligands is required for radiation-induced Notch activation leading to EMT (C) Immunocytochemistry for EMT markers such as E-cadherin and Vimentin in MCF7 cancer cells after irradiation. (D) Protein expression of EMT markers such as E-cadherin, N-cadherin and Vimentin in MCF7 and SKBR3 breast cancer cells after irradiation (E) Protein expression of EMT transcription factors such as SNAIL, SLUG, ZEB1 and TWIST in MCF7 and SKBR3 breast cancer cells after irradiation. (F) Invasion of GFP-transduced MCF7 spheroid cells treated in collagen-based matrix 3D culture system following irradiation. A0 represents the area of sphere at day 0 while At is the area at day 9. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05, and *p < 0.01. www.impactjournals.com/oncotarget (E) Western blot for EMT markers such as E-cadherin, N-cadherin and Vimentin in MCF7 cells that are transfected with siRNA targeting Notch2 prior to irradiation. (F) Western blots for EMT transcription factors such as SNAIL, SLUG and TWIST in MCF7 cells that are transfected with siRNA targeting Notch2 and irradiated afterwards. (G) Migration and invasion assay in transwells after irradiation of MCF7 that are treated with gamma-secretase inhibitor, well known Notch signaling inhibitor (GSI, 20 µM). (H and I) Western blot for EMT markers and regulators after fractionated irradiation in MCF7 cells that are treated with pretreated with GSI. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05, and *p < 0.01. intracellular signaling is transduced by Notch ligands. By analyzing multiple Notch ligands, we found that Jagged1 and DLL4 mRNA were increased significantly in irradiated MCF7 cells as compared to un-irradiated cells, whereas the smaller changes noted in Jagged2, and DLL1 transcripts did not reach statistical significance (Figure 3A). Additionally, elevated Jagged1 and DLL4 mRNA levels resulted in increased accumulation of protein levels in irradiated MCF7 cells as compared with un-irradiated cells (Figure 3B). Immunocytochemistry also exposed higher expression levels of Jagged1 and DLL4 in MCF7 cells after fractionated radiation (Figure 3C). Due to the strong accumulation of Jagged1 and DLL4 in response to radiation, we decided to analyze the effects of Jagged1 and DLL4 inhibition in irradiated MCF7. Here we observed that NICD2 was significantly down-regulated after the knock-down of these ligands; however, NICD1 remained unchanged in irradiated MCF7 cells (Figure 3D). Interestingly, the knock-down of Jagged1 and DLL4 also blocked Notch-induced invasion and migration along with increased E-cadherin and decreased N-cadherin and vimentin protein levels in irradiated MCF7 cancer cells (Figure 3E and 3F). Restoration of pericellular E-cadherin together with attenuation of N-cadherin indicates that the expressions of Jagged1 and DLL4 are crucial for radiation activated Notch signaling for the induction of EMT (Figure 3G). investigated whether STAT3 has significance in radiation- induced Notch2 activation. To this end, when MCF7 breast cancer cells were treated with a STAT3 inhibitor or DMSO and radiated afterwards, we observed that the inactivation of STAT3 sharply attenuated the radiation- induced Notch2 (NICD2), Jagged1 and DLL4 mRNA expression and protein levels (Figure 4C and 4D). www.impactjournals.com/oncotarget Consistent with these results, interestingly the down- regulation of STAT3 also decreased the invasiveness and motility (Supplementary Figure S3B) and the expression of the epithelial marker such as E-cadherin was reappeared while the mesenchymal markers such as N-cadherin and vimentin were lost in irradiated MCF7 breast cancer cells (Supplementary Figure S3C and S3D). Because JAK (Janus kinases) is well-known regulator of STAT, we were curious to know whether the radiation-induced activation of STAT3 regulated by JAK could induce EMT through the Notch signaling pathway. Treatment with a JAK inhibitor effectively abrogates the radiation-induced invasive and migratory properties in irradiated MCF7 cells (Supplementary Figure S3E). In parallel, the inhibition of JAK mitigated the protein levels of N-cadherin and vimentin, as well as protein levels of SLUG, as induced by fractionated radiation (Supplementary Figure S3F). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 53432 i tj l / t t Figure 2: Fractionated radiation-induced Notch signaling promotes EMT in breast cancer cells. (A) qRT PCR analysis fo mRNA levels of Notch family members (Notch1-4) in MCF7 breast cancer cell after fractionated irradiation. (B) Protein expression of Notch family such as Notch1 (NICD1) and Notch2 (NICD2) in MCF7 after fractionated irradiation. (C)) Migration and invasion assay in transwell Figure 2: Fractionated radiation-induced Notch signaling promotes EMT in breast cancer cells. (A) qRT PCR analysis for mRNA levels of Notch family members (Notch1-4) in MCF7 breast cancer cell after fractionated irradiation. (B) Protein expression of Notch family such as Notch1 (NICD1) and Notch2 (NICD2) in MCF7 after fractionated irradiation. (C)) Migration and invasion assay in transwells Figure 2: Fractionated radiation-induced Notch signaling promotes EMT in breast canc Figure 2: Fractionated radiation-induced Notch signaling promotes EMT in breast cancer cells. (A) qRT PCR analysis for mRNA levels of Notch family members (Notch1-4) in MCF7 breast cancer cell after fractionated irradiation. (B) Protein expression of Notch family such as Notch1 (NICD1) and Notch2 (NICD2) in MCF7 after fractionated irradiation. (C)) Migration and invasion assay in transwells www.impactjournals.com/oncotarget Oncotarget 53433 after irradiation of MCF7 that are transfected with siRNA targeting Notch2. (D) Immunocytochemistry for EMT markers (E-cadherin and N-cadherin) after fractionated irradiation of MCF7 that are treated with siRNA targeting Notch2. (E) Western blot for EMT markers such as E-cadherin, N-cadherin and Vimentin in MCF7 cells that are transfected with siRNA targeting Notch2 prior to irradiation. (F) Western blots for EMT transcription factors such as SNAIL, SLUG and TWIST in MCF7 cells that are transfected with siRNA targeting Notch2 and irradiated afterwards. (G) Migration and invasion assay in transwells after irradiation of MCF7 that are treated with gamma-secretase inhibitor, well known Notch signaling inhibitor (GSI, 20 µM). (H and I) Western blot for EMT markers and regulators after fractionated irradiation in MCF7 cells that are treated with pretreated with GSI. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05, and *p < 0.01. after irradiation of MCF7 that are transfected with siRNA targeting Notch2. (D) Immunocytochemistry for EMT markers (E-cadherin and N-cadherin) after fractionated irradiation of MCF7 that are treated with siRNA targeting Notch2. Radiation upregulates Notch induced EMT through the activation of STAT3 signaling Because the signal transduction of IL-6 is involved in the activation of JAK leading to the activation of transcription factor STAT3 [28], we questioned if a certain secretion factor secreted by radiation could induce EMT. To this end, MCF7 breast cancer cells were irradiated at 2 Gy × 3 as previously described and other MCF7 cells were treated with a conditioned medium that has a secretion factors induced by MCF7 after irradiation (Figure 5A). Both migration and invasion levels were significantly increased in both treated conditions in response to radiation (Figure 5B). Remarkably, the expression levels of mesenchymal marker proteins were increased after treatment with radiation irradiated culture medium in MCF7 cells (Figure 5C). Loss of E-cadherin again confirms that some secretion factors were induced in culture medium by MCF7 cells in response of radiation (Figure 5D). Moreover, by analyzing multiple ILs, we found that the expression levels of IL-6 and IL-8 To gain further insight into the molecular mechanisms affecting the regulation of EMT, we sought to identify transcription factors whose expression regulates EMT through the radiation-induced Notch signaling pathway in breast cancer cells. To this end, first we examined the expression levels of the transcription factors such as STAT3, NF-κB and Fra-1 that are frequently associated with EMT signaling in breast cancer cells after fractionated irradiation. Notably, the activation of STAT3 was significantly induced by fractionated radiation in MCF7 cells, whereas the activation levels of NF-κB and Fra-1 remained unchanged (Figure 4A). In agreement with these results, the knock-down of STAT3 effectively attenuated radiation-induced Notch signaling (Figure 4B, Supplementary Figure S3A). Given that irradiation promoted the activation of STAT3, next we www.impactjournals.com/oncotarget Oncotarget 53434 Figure 3: Fractionated radiation-induced Notch ligands promote EMT in breast cancer cells. (A) qRT PCR analysis for mRNA levels of Notch ligands in MCF7 breast cancer cell after fractionated irradiation. (B) Western blot and (C) immunocytochemistry for Notch ligands such as Jagged1 and DLL4 in MCF7 breast cancer cells after irradiation. (D) Protein expression for Notch2 activation in irradiated MCF7 cells that are transfected with siRNA targeting Jagged1 and DLL4. (E) Migration and invasion assays in transwells after irradiation of MCF7 that are transfected with siRNA targeting Jagged1 and DLL4. (F) Western blot and (G) immunocytochemistry for EMT markers after fractionated irradiation of MCF7 cells transfected with siRNA targeting Jagged1 and DLL4. β-actin was used as a loading control. Radiation upregulates Notch induced EMT through the activation of STAT3 signaling Error bars represent mean ± S.D. of triplicate samples. p < 0.05, p < 0.01 and p < 0.001. Figure 3: Fractionated radiation induced Notch ligands promote EMT in breast cancer cells (A) qRT PCR analysis for Figure 3: Fractionated radiation-induced Notch ligands promote EMT in breast cancer cells. (A) qRT PCR analysis for mRNA levels of Notch ligands in MCF7 breast cancer cell after fractionated irradiation. (B) Western blot and (C) immunocytochemistry for Notch ligands such as Jagged1 and DLL4 in MCF7 breast cancer cells after irradiation. (D) Protein expression for Notch2 activation in irradiated MCF7 cells that are transfected with siRNA targeting Jagged1 and DLL4. (E) Migration and invasion assays in transwells after irradiation of MCF7 that are transfected with siRNA targeting Jagged1 and DLL4. (F) Western blot and (G) immunocytochemistry for EMT markers after fractionated irradiation of MCF7 cells transfected with siRNA targeting Jagged1 and DLL4. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05, p < 0.01 and p < 0.001. www.impactjournals.com/oncotarget Oncotarget 53435 knock-down of IL-6 and IL-8 in irradiated MCF7 breast cancer cells (Figure 5H). Next, to confirm the role of IL-6 signaling in radiation-induced EMT, its activity was inhibited in irradiated MCF7 cells by a neutralizing antibody. As expected, treatment with the IL-6 neutralizing antibody effectively decreased radiation-induced invasion and migration in MCF7 cells (Supplementary Figure S5A). Consistent with these results, the expression of the epithelial marker E-cadherin was also increased and the mesenchymal markers N-cadherin and vimentin were decreased by fractionated radiation after treatment with the IL-6 neutralizing antibody in MCF7 cells. Remarkably, Slug protein levels were also down-regulated by the IL-6 neutralizing antibody in MCF7 cells in response to radiation (Supplementary Figure S5B). Immunostaining analyses of the increased E-cadherin and diminished transcripts were markedly increased in MCF7 cells by radiation among all cytokines tested (Figure 5E). Owing to the robust induction of IL-6 and IL-8 in irradiated MCF7 cells, we sought to determine if IL-6 and IL-8 play significant roles in radiation-induced Notch2 signaling expression. As expected, the knock-down of IL-6 decreased the radiation-induced Notch2, Jagged1 and DLL4 protein levels; however, IL-8 partially inhibited Notch2, Jagged1 and DLL4 protein levels (Figure 5F). Indeed, Jagged1, DLL4 and Notch2 transcripts were significantly down-regulated by the IL-6 and IL-8 knock- down in irradiated MCF7 cells (Figure 5G). Radiation upregulates Notch induced EMT through the activation of STAT3 signaling In contrast, EMT was induced more effectively by recombinant IL-6 protein than IL-8 in MCF7 cells (Supplementary Figure S4). Meanwhile, the activation of STAT3 did not show increase due to fractionated radiation by the Oncota get 53436 www impactjournals com/oncotarget Figure 4: Fractionated radiation up-regulates Notch signaling through activation of STAT3. (A) Western blot analysis for STAT3, NF-κB and Fra-1 phosphorylation status rafter irradiation of MCF7 breast cancer cells. (B) qRT-PCR for Notch2, Jagged-1 and DLL4 mRNA levels after irradiation of MCF7 cells that are transfected with siRNA targeting STAT3, NF-κB and Fra-1. (C) qRT-PCR for Notch2, Jagged1 and DLL4 mRNA levels after irradiation of MCF7 cells that are treated with STAT3 inhibitor (2 µM). (D) Western blot for NICD2, Jagged1 and DLL4 after fractionated irradiation (2 Gy × 3) of MCF7 that are treated with STAT3 inhibitor (2 µM). Error bars represent mean ± S.D. of triplicate samples. p < 0.05, p < 0.01 and p < 0.001. Figure 4: Fractionated radiation up-regulates Notch signaling through activation of STAT3. (A) Western blot analysis for STAT3, NF-κB and Fra-1 phosphorylation status rafter irradiation of MCF7 breast cancer cells. (B) qRT-PCR for Notch2, Jagged-1 and DLL4 mRNA levels after irradiation of MCF7 cells that are transfected with siRNA targeting STAT3, NF-κB and Fra-1. (C) qRT-PCR for Notch2, Jagged1 and DLL4 mRNA levels after irradiation of MCF7 cells that are treated with STAT3 inhibitor (2 µM). (D) Western blot for NICD2, Jagged1 and DLL4 after fractionated irradiation (2 Gy × 3) of MCF7 that are treated with STAT3 inhibitor (2 µM). Error bars represent mean ± S.D. of triplicate samples. p < 0.05, p < 0.01 and p < 0.001. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 53436 Oncotarget 53437 www.impactjournals.com/oncotarget Figure 5: Radiation-induced IL6 secretion mediates activation of Notch signaling. (A) Schematic model illustrating conditioned medium treated MCF7 breast cancer cells. MCF7 cells were irradiated 2 Gy × 3 as mentioned earlier. Then cell’s medium was further harvested and treated to another MCF7 cells. (B) Migration and invasion assay in transwells after irradiated cultured medium those are compared with Figure 5: Radiation-induced IL6 secretion mediates activation of Notch signaling. (A) Schematic model illustrating conditioned medium treated MCF7 breast cancer cells. MCF7 cells were irradiated 2 Gy × 3 as mentioned earlier. Then cell’s medium was further harvested and treated to another MCF7 cells. (B) Migration and invasion assay in transwells after irradiated cultured medium those are compared with www.impactjournals.com/oncotarget Oncotarget 53437 directly irradiated MCF7 cells. (C) Western blot and (D) immunocytochemistry for EMT markers after fractionated irradiation of MCF7 that are treated with irradiated cultured medium. (E) qRT PCR analysis for mRNA levels of cytokines family in MCF7 cells after fractionated irradiation. (F) Western blot for NICD2, Jagged1 and DLL4 after fractionated irradiation of MCF7 that are transfected with siRNA targeting IL-6 and IL-8. (G) qRT-PCR for Notch2, Jagged-1 and DLL4 mRNA levels after irradiation of MCF7 cells that are transfected with siRNA targeting IL-6 and IL-8. (H) Western blot for activation of STAT3 after fractionated irradiation of MCF7 that are transfected with siRNA targeting IL-6 and IL-8. β-actin was used as a loading control. Error bars represent mean ± S.D. of triplicate samples. p < 0.05 and *p < 0.01. N-cadherin levels confirm that IL-6 upregulates radiation- induced EMT responses in cancer cells (Supplementary Figure S5C). These observations taken together suggest that IL-6 strongly mediates Notch-induced EMT through the JAK/STAT3 signaling axis in breast cancer cells. malignancy along with increased mesenchymal markers, indicating that Notch2 is necessary and sufficient for the loss of E-cadherin and increased N-cadherin and vimentin levels. Finally, we defined the molecular basis of radiation- induced EMT in breast cancer cells, finding that it results from the fact that Notch2 upregulation is accompanied by IL-6-dependent STAT3 activation. These findings provide a mechanistic link between Notch2 induction and IL-6 secretion for the progression of tumor malignancy in mammary tumor cells.i Radiation-induced Notch promotes EMT in mice The epithelial to mesenchymal transformation, along with the increased invasiveness and motility of breast cancer cells in vitro, prompted us to analyze the effects of fractionated radiation (2 Gy × 5) in vivo (Figure 6A). Two-three days after an orthotropic injection of 4T1 breast tumor cells in mice, irradiation was performed. Histological staining indicated that tumor cells were disseminating from their margins in irradiated tissues as compared to non-irradiated cases (Figure 6B). These findings indicated that cancer cells gain migratory properties and going to metastasize after radiation. In vitro data also revealed that 4T1 mouse breast tumor cells showed higher rate of migration and invasion along with decreased E–cadherin and increased N-cadherin and vimentin levels after fractionated radiation (Supplementary Figure S6A and S6B). Additionally, noticeable losses of E-cadherin expression levels and increased N-cadherin, vimentin levels were detected in irradiated tumors (Figure 6C). Remarkably, Notch2 was also overexpressed in irradiated tumors compared to non- irradiated cases (Figure 6D). Therefore, we conclude that radiation-induced Notch activation can increase the EMT in breast cancer cells in vivo. Importantly, our findings reveal that Notch2 inhibition effectively altered the expression of mesenchymal markers that requires SLUG activity. The SNAIL family members including SLUG encode zinc finger–containing transcriptional activators that promote EMT during tumor development, in part by regulating the expression of E-cadherin junctional proteins [32]. In line with this notion, a recent report demonstrates that Notch controls expression of SLUG and that abrogation of Notch signaling prevents tumor growth and metastasis in an in vivo tumor model [33]. In contrast to previous studies, we find here that the transcriptional activator SLUG, but not SNAIL, participates in the Notch-activated EMT pathway [26]. Remarkably, SLUG knockdown restored E-cadherin expression in breast cancer cells while also decreasing mesenchymal markers in response to radiation. Consistent with this, we observed that a γ-secretase inhibitor treatment reversed these pro-metastatic functions of Notch2 by disrupting the Notch pathway in associated breast cancer cells [34]. These findings broaden our understanding of radiation regulated EMT processes through Notch pathways and open possible new avenues for cancer therapeutics improvement via inhibition of Notch-mediated tumor metastasis. Discussion Recently EMT has been considered to have malignant features in tumor cells, causing these cells to gain a tendency towards metastatic dissemination, resistance to various chemotherapies, and relapse. Several reports now stated that radiation is commonly considered as one of the well-known inducer of EMT [29–31]. Hence, understanding the molecular mechanisms acting with regard to radiation towards EMT is necessary to generate novel therapeutic procedures. i We also demonstrated that elevated levels of IL-6 can be detected in the breast cancer cells after radiation. Higher levels of IL-6 have a generally been reported to be correlated with a more advanced stage of disease and a worse outcome [35]. Of note, we observed that fractionated radiation increased the secretion of IL-6 in the tumor microenvironment through the activation of the JAK/STAT3/Notch2 signaling pathway as shown in a schematic model (Figure 6E). Collectively, our findings suggest that Notch2 could be a therapeutic target of efforts to overcome radiotherapy induced relapse in the breast cancer treatment. Hence, treatment of Notch inhibitors, prior to radiotherapy, in tumors at risk for metastasizing may be a fruitful approach in the direction of improving cancer therapeutic outcomes. In the present study, we identified Notch signaling as a critical regulator of mammary tumor progression by ionizing radiation. Notch expression is significantly up- regulated in irradiated tumors arising in mice bearing mammary tumors relative to non-irradiated tumors. Interestingly, Notch2 induction accelerated tumor www.impactjournals.com/oncotarget Oncotarget 53438 Oncotarget 53439 www impactjournals com/oncotarget Figure 6: Radiation-induced Notch promotes EMT in mice. (A) Schematic representation of experimental scheme for treating in vivo mice model by fractionated radiation (2 Gy/day for five consistent days). (B) Hematoxylin and eosin (H&E) staining of breast tumor tissue in mice 24 hour after irradiation as described above. (C) Immunohistochemistry for EMT markers such as E-cadherin, N-cadherin and vimentin in breast tumor tissues of mice after irradiation. (D) Immunohistochemistry for expression of Notch2 in breast cancer tissues of mice after irradiation with 10 Gy (2 Gy × 5). (E) Schematic model that fractionated radiation promotes EMT through Notch signaling in breast cancer cells. Importantly, irradiation-activated IL-6 increases the Notch-activity via activating JAK/ STAT3 for EMT. Figure 6: Radiation-induced Notch promotes EMT in mice. (A) Schematic representation of experimental scheme for treating in vivo mice model by fractionated radiation (2 Gy/day for five consistent days). Boyden chamber invasion and migration assays Matrigel invasion assays were conducted using 24- well Transwell inserts (Corning Inc., Tewksbury, MA, USA) pre-coated with 10 mg/ml growth-factor-reduced Matrigel (BD Biosciences) at 37 °C. All breast cancer cells (2 × 104) suspended in 200 μl of serum-free medium were seeded into the upper chamber with the lower well filled with 0.8 ml of a growth medium. After incubation for 48 hr (hours), migrated cells were fixed and stained with a Diff-Quick kit (Fisher) and photographed and invasiveness was calculated as described in our previous report [36]. A migration assay was also performed in Transwell inserts containing similar membranes without the Matrigel coating. Chemical reagents and antibodies Monoclonal antibodies to N-cadherin (610920) and E-cadherin (612130) were purchased from BD Transduction Laboratory (Seoul, Korea). Polyclonal antibodies to STAT3 (sc-482), NF-κB (p65) (sc- 109), Vimentin (sc-5565), SLUG (sc-10436) and TWIST (sc- 15393) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The polyclonal antibody to ZEB1 (HPA027524) was purchased from Sigma (St Louis, MO, USA). The polyclonal antibodies to Snail (3879), p-STAT3 (Y705) (9131), p-STAT3 (S727) (9134), Fra1 (5281), p-Fra1 (3880) and Notch1 (2421) were obtained from Cell Signaling Technology (Beverly, MA, USA). Polyclonal antibodies to anti-IL-6 (ab6672), Notch2 (ab72803), Jagged1 (ab109536) and DLL4 (ab7280) were purchased from Abcam (Cambridge, UK. Monoclonal antibody to β-actin and 4, 6-diamidino- 2-phenylindole (DAPI) were purchased from Sigma (St Louis, MO, USA). The chemical inhibitors including Gamma secretase inhibitor (565770), STAT3 inhibitor (573097) and the JAK inhibitor (420099) were bought from Calbiochem (San Diego, CA, USA). Recombinant human CXCL8/IL-8 protein was purchased from R & D systems and recombinant protein IL-6 was bought from EMD Millipore. Western blot analysis Cell lysates were prepared by incubation with a lysis buffer (40 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Then proteins were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). Primary antibodies were used to detect the relevant protein, and β-actin was used as loading control. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and proteins were visualized using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL), according to the manufacturer’s protocol. Secondary antibodies, anti-mouse IgG-HRP, anti- goat IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz biotechnology (CA, USA). Irradiation Cancer cells were exposed to radiation using a 137Cs γ-ray source (Atomic Energy of Canada, Ltd, Mississauga, Canada) at a dose rate of 3.81 Gy/min. Further analysis such as migration, invasion assay, western blot and immunocytochemical analysis were done at 48 hours after fractionated irradiation (2 Gy × 3; 2 Gy per day for 3 days). Cell culture The human breast epithelial cell line MCF7, SKBR3 and the mouse breast epithelial cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA). All cell cultures were maintained in a humidified 5% CO2 atmosphere at 37°C. The hormone receptor status of the MCF7 cells is estrogen receptor positive (ER+), progesterone positive (PR+) and HER2 negative (HER2-) while that of SKBR3 is ER- PR- HER2+. MCF7, SKBR3 and 4T1 cells were grown in the minimum Eagle’s medium, RPMI medium and DMEM medium respectively. All of these media were supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). All cell culture products were purchased from GIBCO (Seoul, Korea). The gamma- secretase inhibitor (GSI) was used in cancer cells at a concentration of 20 μM; the JAK inhibitor was used at 10 μM, and the STAT3 inhibitor was used at 2 μM. Clonogenic survival assay MCF7 and SKBR3 cells were seeded into 60 mm cell culture dish at concentrations of 300–400 cells per plate after the knockdown of si-Notch2 and radiation afterwards with dose rate of 1 Gy-4 Gy. The cells were incubated for 8–10 days to allow for colony formation. The colonies composed of more than 50 cells were counted. Survival fraction was calculated as (mean colonies counted. Each assay was performed in triplicates. Transfection Cells were transfected with siRNA duplexes (40 nM) by using Lipofectamine 2000 (Invitrogen), following the procedure recommended by the manufacturer. Irradiation was performed 48 hr after transfection. All siRNA were purchased from Genolution Pharmaceuticals, Inc (Seoul, Korea). Discussion (B) Hematoxylin and eosin (H&E) staining of breast tumor tissue in mice 24 hour after irradiation as described above. (C) Immunohistochemistry for EMT markers such as E-cadherin, N-cadherin and vimentin in breast tumor tissues of mice after irradiation. (D) Immunohistochemistry for expression of Notch2 in breast cancer tissues of mice after irradiation with 10 Gy (2 Gy × 5). (E) Schematic model that fractionated radiation promotes EMT through Notch signaling in breast cancer cells. Importantly, irradiation-activated IL-6 increases the Notch-activity via activating JAK/ STAT3 for EMT. www.impactjournals.com/oncotarget Oncotarget 53439 www.impactjournals.com/oncotarget The authors declare no conflicts of interest. The authors declare no conflicts of interest. Immunocytochemistry Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in phosphate- buffered saline (PBS). Following fixation, the cells were incubated at 4°C overnight with mouse polyclonal anti-human E-cadherin (1:200) N-cadherin (1:200), anti-Jagged1 (1:200), DLL4 (1:200), Rabbit polyclonal anti-human anti-vimentin (1:200), and goat polyclonal anti-human anti-SLUG (1:200) primary antibody in PBS with 1% bovine serum albumin and 0.1% Triton X-100. Immunostaining of the proteins was visualized using Alexa Fluor 488-conjugated anti-rabbit and anti-mouse or anti-goat secondary antibodies (Molecular Probes, Seoul, Korea). Nuclei were counterstained with DAPI (Sigma- aldrich). Immunostaining was observed with an Olympus IX71 fluorescence microscope (Olympus, Seoul, Korea). Statistical analysis All experimental data are reported as means; error bars represent the standard deviation (SD) of at least three independent tests. Statistical analyses were performed using non-parametric Student’s t-tests to assess the significance levels. Results were considered as significant if p < 0.05, p < 0.01, *p < 0.001. Immunohistochemistry Athymic balb/c nude mice were euthanized and tissues were harvested and fixed in formalin for the www.impactjournals.com/oncotarget Oncotarget 53440 preparation of paraffin sections. Paraffin-embedded tissue sections were deparaffinized in xylene, 95, 90, and 70% ethanol, followed by phosphate buffered saline (PBS). Epitopes were unmasked with 20 mg/ml proteinase K in PBS with 0.1% Triton X-100. Sections were stained with hematoxylin and eosin (H & E) or immunostained overnight at 4°C with the Notch2 (1:100), E-cadherin (1:100), N-cadherin (1:100), vimentin antibody (1:100) and IL-6 antibody (1:100). After washing in PBS, a 1:200 dilution of biotinylated goat anti-rabbit IgG or anti-mouse IgG antibody in a blocking solution was applied to the sections and incubated for 30 min. After washing in PBS, the ABC reagent (ABC Peroxidase Standard Staining Kit-Thermo Fisher Scientific) was applied to the sections and incubated for 30 min. After washing in PBS, color reaction was performed with 3, 30-diaminobenzidine (Vector Laboratories) and the slides were washed with PBS. After counter-staining with hematoxylin and clearing with a graded ethanol series and xylene, the sections were mounted with Canada balsam. Observations and photography were conducted using a microscope (Olympus) equipped with a DP71 digital imaging system (Olympus). (5 weeks of age; Orient). Irradiation (2 Gy × 5; 2 Gy/day for 5 days) was then done five times every day. Tumor sizes were measured with a caliper (calculated volume = shortest diameter2 × longest diameter/2) at three day intervals. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Center for Laboratory Animal Sciences, the Medical Research Coordinating Center, and the HYU industry- University Cooperation Foundation. References 1. Bussink J, van der Kogel AJ, Kaanders JH. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer. Lancet Oncol. 2008; 9:288–96. 1. Bussink J, van der Kogel AJ, Kaanders JH. Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and neck cancer. Lancet Oncol. 2008; 9:288–96. 2. Bernier J, Hall EJ, Giaccia A. Radiation oncology: a century of achievements. Nat Rev Cancer. 2004; 4:737–47. 3. Puchner MJ, Giese A. Tamoxifen-resistant glioma-cell sub- populations are characterized by increased migration and proliferation. Int J Cancer. 2000; 86:468–73. 3. Puchner MJ, Giese A. 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https://openalex.org/W4362486321	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S5_from_Functional_Toll-like_Receptor_4_Overexpression_in_Papillary_Thyroid_Cancer_by_MAPK_ERK_Induced_ETS1_Transcriptional_Activity/22513162/1/files/39975190.pdf	French	null	Supplementary Figure S7 from Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK–Induced ETS1 Transcriptional Activity	null	2,023	cc-by	132	SUPPLEMENTARY FIGURE 5 SUPPLEMENTARY FIGURE 5 FIGURE S5. MAPK/ERK signaling inhibition repressed BRAFV600E‐induced TLR4 expression at the plasma membrane. Immunofluorescence analysis under nonpermeabilized conditions showing TLR4 protein expression levels at the plasma membrane in doxycycline (Dox)‐treated PC/BRAFV600E cells in the presence of vehicle or the chemical inhibitors PLX4032 or U0126. * P<0.001 vs. vehicle‐treated cells, ### P<0.001 vs. same condition in absence of inhibitor (one‐way ANOVA, Newman‐Keuls test). FIGURE S5. MAPK/ERK signaling inhibition repressed BRAFV600E‐induced TLR4 expression at the plasma membrane. Immunofluorescence analysis under nonpermeabilized conditions showing TLR4 protein expression levels at the plasma membrane in doxycycline (Dox)‐treated PC/BRAFV600E cells in the presence of vehicle or the chemical inhibitors PLX4032 or U0126. * P<0.001 vs. vehicle‐treated cells, ### P<0.001 vs. same condition in absence of inhibitor (one‐way ANOVA, Newman‐Keuls test). Newman‐Keuls test).
W2281676653.txt	https://www.cdc.gov/pcd/issues/2016/pdf/15_0505.pdf	en		Waterpipe Tobacco Smoking and Susceptibility to Cigarette Smoking Among Young Adults in the United States, 2012–2013	Preventing chronic disease	2,016	public-domain	4,684	PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, Volume 13, E24 AND POLICY FEBRUARY 2016 ORIGINAL RESEARCH Waterpipe Tobacco Smoking and Susceptibility to Cigarette Smoking Among Young Adults in the United States, 2012–2013 Ramzi G. Salloum, PhD; M. Rifat Haider, MHE; Tracey E. Barnett, PhD; Yi Guo, PhD; Kayla R. Getz, MPH; James F. Thrasher, PhD; Wasim Maziak, MD, PhD Suggested citation for this article: Salloum RG, Haider MR, Barnett TE, Guo Y, Getz KR, Thrasher JF, et al. Waterpipe Tobacco Smoking and Susceptibility to Cigarette Smoking Among Young Adults in the United States, 2012–2013. Prev Chronic Dis 2016;13:150505. DOI: http://dx.doi.org/10.5888/pcd13.150505. PEER REVIEWED Abstract Results Of 2,528 young adults who had never established cigarette smoking, 15.7% (n = 398) reported being waterpipe smokers (every day or some days [n = 97; 3.8%] or rarely [n = 301; 11.9%]); 44.2% (176/398) of waterpipe smokers reported being susceptible to cigarette smoking. Those who smoked waterpipe rarely were 2.3 times as susceptible to cigarette smoking as those who were not current waterpipe smokers (OR = 2.3; 95% CI, 1.6–3.4). Conclusion Introduction Waterpipe tobacco smoking, also known as hookah and shisha, has surged in popularity among young people in the United States. Waterpipe is also increasingly becoming the first tobacco product that young people try. Given the limited access to and limited portability of waterpipes, waterpipe smokers who become more nicotine dependent over time may be more likely to turn to cigarettes. This study examined the relationship between waterpipe tobacco smoking and susceptibility to cigarette smoking among young adults in the United States. Methods Using data from the 2012–2013 National Adult Tobacco Survey, a nationally representative sample of US adults, we reported rates of current waterpipe smoking and susceptibility to cigarette smoking by demographic characteristics and by use of other tobacco products among survey participants aged 18 to 24 years. Multivariable logistic regression was used to examine the relationship between current waterpipe smoking and susceptibility to cigarette smoking, defined as the lack of a firm intention not to smoke soon or within the next year. Current waterpipe smoking is associated with susceptibility to cigarette smoking among young adults in the United States. Longitudinal studies are needed to demonstrate causality between waterpipe smoking and initiation of cigarette smoking. Introduction Waterpipe tobacco smoking, also known as hookah and shisha, has surged in popularity among young people in the United States (1,2) and worldwide (3). In the 2012–2013 National Adult Tobacco Survey (NATS), among all age groups who reported using tobacco, 18.2% of adults aged 18 to 24 reported waterpipe smoking (every day, some days, or rarely); this prevalence was the highest prevalence for any age category and for any tobacco product other than cigarettes (4). Waterpipe is also increasingly becoming the first tobacco product that young people try (5). Young people may be attracted to waterpipe smoking because of its social allure (6) and the perception that it damages health less than cigarette smoking (7). Given the limited access to and limited portability of waterpipes, nicotine-dependent smokers may turn to cigarettes (8). Understanding the association between waterpipe smoking and susceptibility to cigarette smoking is the first The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2016/15_0505.htm • Centers for Disease Control and Prevention 1 PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 step in exploring whether the appeal of waterpipes among young people could lead to initiation of cigarette smoking. Whereas waterpipe smoking is primarily an intermittent method of using tobacco (3), its disproportionate popularity among young people compared with older adults is particularly relevant because nearly all adults who become daily cigarette smokers initiate smoking by age 26 (9). Susceptibility to smoking is defined as the lack of a firm decision against smoking, and it is a strong predictor of regular or established smoking among young people (10–12). Given the increasing prevalence of waterpipe smoking among young people in the United States, there is a need to understand whether it contributes to cigarette smoking susceptibility. A recent study observed subsequent cigarette smoking among waterpipe smokers at baseline (13); however, to our knowledge, this relationship has not been examined in a nationally representative US sample. Our objectives in this study were to describe the characteristics of young adults in the United States who are current waterpipe smokers and to determine whether current waterpipe smokers are more likely to report being susceptible to cigarette smoking than those who are not waterpipe smokers. Methods Data were analyzed from the 2012–2013 NATS, a stratified, nationally representative telephone survey of 60,192 noninstitutionalized civilian US adults aged 18 years or older (4). The sampling design of NATS relies on independent samples drawn from households in the 50 US states and District of Columbia. The response rate for the 2012–2013 wave was 44.9% (landline frame, 47.2%; cellular frame, 36.3%). The analytic sample was limited to the 2,528 young adult (aged 18–24 years) respondents who had never established cigarette smoking behavior. Respondents were considered to be never-established smokers if they responded negatively to the question, “Have you smoked at least 100 cigarettes in your entire life?” and responded with “not at all” to the question, “Do you now smoke cigarettes every day, some days, or not at all?” Current smokers of waterpipe tobacco were identified by using the following question: “The next question asks you about smoking tobacco in a hookah. A hookah is a type of water pipe. . . . Do you now smoke tobacco in a hookah every day, some days, rarely or not at all?” Participants who responded with either “every day” or “some days” were defined as current waterpipe smokers. Participants who selected “rarely” were defined as intermittent waterpipe smokers. Those who chose “not at all” were defined as nonsmokers of waterpipe. The survey asked adults aged 18 to 29 the following 2 questions on susceptibility to future cigarette smoking: “Do you think you will smoke a cigarette soon?” and “Do you think you will smoke a cigarette in the next year?” Response options were “definitely yes,” “probably yes,” “probably not,” and “definitely not.” Those who responded with “definitely not” to both questions were considered not susceptible to cigarette smoking, whereas all other participants were considered susceptible. This classification is based on research showing that the smoking susceptibility measure predicts subsequent smoking behavior (10–12). We conducted a sensitivity analysis, which classified “definitely yes” and “probably yes” as being susceptible to smoking cigarettes and “probably not” and “definitely not” as being not susceptible to smoking. The NATS questionnaire did not include a question that is often used in assessing susceptibility to cigarette smoking (“If one of your best friends were to offer you a cigarette, would you smoke it?”), and thus our measure of susceptibility was constructed by using only 2 questions. Associations between the current use of other tobacco products and susceptibility to cigarette smoking were also assessed. The tobacco product categories were the following: 1) cigars, cigarillos, and filtered little cigars; 2) smokeless tobacco, including chewing tobacco, dip, snuff, and snus; and 3) e-cigarettes. To establish current use, respondents were asked if they now used the product “every day,” “some days,” “rarely,” or “not at all.” Participants who responded with “every day,” “some days,” or “rarely” were considered current users of the tobacco product type. The association between harm perception and susceptibility to cigarette smoking was also assessed by using the following question: “How harmful do you think cigarette smoking is to a person’s health?” The response options were “not at all harmful,” “moderately harmful,” and “very harmful.” Finally, respondents were asked the following question to assess prior experimentation with cigarettes, “Have you ever tried cigarette smoking, even one or two puffs?” Those who responded affirmatively were deemed to have experimented with cigarettes at some point in their lifetime. The following demographic characteristics of survey respondents were assessed: sex, age in years (18–21 or 22–24), race/ethnicity (non-Hispanic white, non-Hispanic black, non-Hispanic Asian, Hispanic, and non-Hispanic other), educational attainment (no high school diploma, high school graduate or general educational development [GED], some college or associate degree, bachelor’s degree or higher), and annual household income (<$20,000, $20,000–$49,999, $50,000–$99,999, ≥$100,000). The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 2 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2016/15_0505.htm PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 Statistical analyses Data were analyzed during August 2015 using Stata 14.0 (StataCorp LP) and weighted to adjust for the differential probability of selection and response. Landline data were weighted according to the selection probability of the telephone number, the number of adults in each household, and the number of telephone numbers per household. Cell telephones were assumed to be used exclusively by the respondent and were weighted only according to the selection probability of the telephone number. Final weights were adjusted for undercoverage by sex, age, race/ethnicity, educational attainment, and telephone type (ie, landline or cellular) (14). Demographic characteristics and rates of tobacco use were summarized by using weighted percentages and confidence intervals (CIs). Rao–Scott χ2 tests were used to detect significant differences between estimates that included corrections for survey design effects. Bivariate analyses were conducted to identify significant differences by current waterpipe smoking status. Multivariable logit regression was used to examine the relationship between respondent characteristics and susceptibility to cigarette smoking. Sensitivity analysis was used in the adjusted regression model to test the interaction of sex with current waterpipe smoking on susceptibility to cigarette smoking. This study was deemed exempt by the University of Florida institutional review board because it relied on publicly available data. Results Among respondents who had never established cigarette smoking (n = 2,528), 97 (3.8%) were current waterpipe smokers, and 301 (11.9%) were intermittent waterpipe smokers (Table 1). Among adults aged 18 to 24 years, most current waterpipe smokers were men (64.0%), aged 18 to 21 years (71.7%), had only a high school diploma or GED (67.7%), and had never tried cigarettes (74.5%). In this same age group, only 50.6% of intermittent waterpipe smokers were men. In addition, most intermittent waterpipe smokers were aged 18 to 21 years old (63.4%), non-Hispanic white (55.4%), had only a high school diploma or GED (70.6%), and had tried cigarettes (66.6%). Overall, among young adults who had never established cigarette smoking, 19.7% (95% CI, 17.9%–21.7%) of waterpipe smokers reported being susceptible to smoking cigarettes (Table 2). Almost half (48.0%; 95% CI, 29.4%–67.2%) of current waterpipe smokers and 42.8% (95% CI, 36.2%–49.7%) of intermittent waterpipe smokers reported being susceptible to cigarette smoking, whereas 16.2% (95% CI, 14.4%–18.3%) of those who were not current waterpipe smokers reported being susceptible. In the adjusted logistic regression model, intermittent waterpipe smokers were 2.3 times as susceptible to cigarette smoking as those who were not current waterpipe smokers (OR = 2.3; 95% CI, 1.6–3.4). We observed no significant associations between current use of other tobacco products and susceptibility to cigarette smoking. In this model, men were more likely than women (OR = 1.6; 95% CI, 1.2–2.3) and young adults aged 18 to 21 years were more likely than those aged 22 to 24 years (OR = 1.9; 95% CI, 1.3–2.7) to report susceptibility to cigarette smoking. Young adults who perceived cigarettes as moderately harmful were significantly more likely (OR, 11.2; 95% CI, 1.2–93.6) than those who perceived them as not harmful to report susceptibility to cigarette smoking. Finally, respondents who had experimented with cigarettes at some point in their lifetime were more likely (OR, 5.0; 95% CI, 3.6–6.8) to report susceptibility to cigarette smoking than those who had never experimented with cigarettes. In the adjusted model, we found no significant differences in susceptibility to cigarette smoking by race/ethnicity, educational attainment, or annual household income. The additional regression model testing the effect of sex on the relationship between current waterpipe smoking and susceptibility to cigarette smoking did not find a significant interaction effect between sex and current waterpipe smoking. In the sensitivity analysis, classifying only those who reported “definitely yes” or “probably yes” responses as susceptible to cigarette smoking resulted in a similar adjusted effect for current waterpipe smoking on susceptibility to cigarette smoking. Most control variables had similar effects in this model as they did in the original regression model. Discussion Our study, using data from the 2012–2013 NATS, showed that among young adults who had never established cigarette smoking, 2 of 5 waterpipe smokers reported being susceptible to smoking cigarettes. Another study using the same data found that waterpipe smoking — primarily intermittent use — was the secondmost prevalent (18.2%) tobacco use method (after cigarette smoking) among young adults in the United States (4). Our study is the first to examine the relationship between current waterpipe smoking and the susceptibility to cigarette smoking in a nationally representative sample of young adults in the United States. Because longitudinal data sets that simultaneously track current waterpipe smoking and the initiation of cigarette smoking are lacking, an association between waterpipe smoking and the initiation of cigarette smoking is not clearly defined for young adult cohorts in the United States. Recent results from a longitudinal cohort study of adolescents in Jordan showed that waterpipe The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2016/15_0505.htm • Centers for Disease Control and Prevention 3 PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 smoking at baseline led to initiation of cigarette smoking at follow-up (8). Another study that examined cigarette smoking susceptibility among waterpipe-smoking young people in the Middle East (15) reported that the odds of susceptibility to cigarette smoking were more than double for those who smoked waterpipe in the previous month than for those who did not. Until such data are available in the United States, the association between waterpipe smoking and the initiation of cigarette smoking will not be clearly defined. Nevertheless, our findings suggest that young adults who smoke waterpipe tobacco are at risk for cigarette smoking initiation because they did not express a firm intention to refrain from smoking cigarettes in the future. Strengths of this study are the use of a large, nationally representative sample and the first-time assessment of the relationship between current waterpipe smoking and susceptibility to cigarette smoking. Limitations include the use of self-reported data, which are subject to recall bias. Second, because our data were cross-sectional, no inferences can be made about causality. Third, variables that may affect susceptibility to waterpipe tobacco smoking, such as whether parents, spouses, or others use tobacco, were not included in the NATS questionnaire. Fourth, although the measurement of susceptibility has predictive validity among adolescents (10–12), validity that we assume carries over to young adults, future research is required to test this assumption. To our knowledge, ours is the first US study to assess the relationship between current waterpipe smoking and susceptibility to cigarette smoking among young adults who had never established cigarette smoking. Although our study did not establish a causeand-effect relationship between waterpipe smoking and cigarette smoking initiation, such a relationship is plausible, and longitudinal studies are needed to investigate its possibility. Given the high prevalence of waterpipe smoking among young people in the United States, it is important to assess both its direct and indirect consequences. Acknowledgments Dr Ramzi Salloum and Ms Kayla Getz are supported by the Florida Department of Health (James and Esther King Biomedical Research Program) grant 4KB16. Dr Wasim Maziak is funded by the National Institute on Drug Abuse (NIDA), grant R01 DA035160. The authors thank the Centers for Disease Control and Prevention for use of the National Adult Tobacco Survey. The findings and conclusions in this report are solely those of the authors. Author Information Corresponding Author: Ramzi G. Salloum, PhD, Department of Health Outcomes and Policy, University of Florida College of Medicine, PO Box 100177, Gainesville, FL 32610. Telephone: 352-294-4997. Email: rsalloum@ufl.edu. Author Affiliations: M. Rifat Haider, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina, and Jahangirnagar University, Dhaka, Bangladesh; Tracey E. Barnett, College of Public Health and Health Professions and College of Medicine, University of Florida, Gainesville, Florida; Yi Guo, Kayla R. Getz, College of Medicine, University of Florida, Gainesville, Florida; James F. Thrasher, Arnold School of Public Health, University of South Carolina, Columbia, South Carolina; Wasim Maziak, Stempel College of Public Health and Social Work, Florida International University, Miami, Florida, and Syrian Center for Tobacco Studies, Aleppo, Syria. References 1. Salloum RG, Osman A, Maziak W, Thrasher JF. How popular is waterpipe tobacco smoking? Findings from internet search queries. Tob Control 2015;24(5):509–13. 2. Salloum RG, Thrasher JF, Kates FR, Maziak W. Water pipe tobacco smoking in the United States: findings from the National Adult Tobacco Survey. Prev Med 2015;71:88–93. 3. 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Validation of susceptibility as a predictor of which adolescents take up smoking in the United States. Health Psychol 1996; 15(5):355–61. 11. Unger JB, Johnson CA, Stoddard JL, Nezami E, Chou CP. Identification of adolescents at risk for smoking initiation: validation of a measure of susceptibility. Addict Behav 1997; 22(1):81–91. 12. Jackson C. Cognitive susceptibility to smoking and initiation of smoking during childhood: a longitudinal study. Prev Med 1998;27(1):129–34. 13. Soneji S, Sargent JD, Tanski SE, Primack BA. Associations between initial water pipe tobacco smoking and snus use and subsequent cigarette smoking: results from a longitudinal study of US adolescents and young adults. JAMA Pediatr 2015; 169(2):129–36. 14. National Adult Tobacco Survey (NATS). Atlanta (GA): Centers for Disease Control and Prevention; 2015. http:// www.cdc.gov/tobacco/data_statistics/surveys/nats/. Accessed October 30, 2015. 15. Veeranki SP, Alzyoud S, Kheirallah KA, Pbert L. Waterpipe use and susceptibility to cigarette smoking among neversmoking youth. Am J Prev Med 2015;49(4):502–11. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2016/15_0505.htm • Centers for Disease Control and Prevention 5 PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 Tables Table 1. Characteristics of Sample of US Adults Aged 18 to 24 Years Who Never Established Cigarette Smoking (n = 2,528), National Adult Tobacco Survey, 2012–2013a Waterpipe Smokers (n = 398) Characteristic Never Established Cigarette Smokingb (n = 2,528) Current (Every Day or Some Days) (n = 97) Intermittent (Rarely) (n = 301) Sex Female 53.2 (50.7–55.7) 36.0 (24.8–48.9) 49.4 (42.3–56.5) Male 46.8 (44.3–49.3) 64.0 (51.1–75.3) 50.6 (43.5–57.7) 18–21 62.8 (60.6–65.0) 71.7 (60.1–81.0) 63.4 (56.9–69.5) 22–24 37.2 (35.0–39.4) 28.3 (19.0–39.9) 36.6 (33.0–42.3) White, non-Hispanic 50.1 (47.8–52.4) 48.0 (36.2–60.0) 55.4 (48.7–61.9) Black, non-Hispanic 11.3 (9.8–13.0) 4.3 (1.1–15.7) 5.8 (3.4–9.6) Age, y Race/ethnicity Asian, non-Hispanic 5.5 (4.5–6.9) 7.1 (2.2–20.4) 4.3 (2.4–7.6) Hispanic 20.7 (18.9–22.8) 20.6 (12.2–32.6) 22.2 (17.0–28.4) Other, non-Hispanic 12.4 (11.0–13.9) 20.0 (12.7–30.1) 12.4 (8.9–17.0) Education No high school diploma 13.2 (11.3–15.3) 17.3 (8.4–32.3) 6.9 (3.9–11.9) High school graduate or GED 64.8 (62.5–67.2) 67.7 (54.0–78.9) 70.6 (64.1–76.2) 9.0 (7.7–10.4) 8.3 (3.9–17.5) 5.3 (3.0–9.1) 13.0 (11.8–14.4) 6.7 (3.0–14.2) 17.3 (13.3–22.3) 10.0 (8.8–11.5) 11.9 (6.2–21.6) 9.2 (5.9–13.9) 20,000–49,999 25.4 (23.4–27.5) 29.6 (20.2–41.1) 19.8 (15.1–25.6) 50,000–99,999 23.4 (21.5–25.5) 19.2 (11.3–30.6) 22.7 (17.2–29.4) ≥100,000 11.6 (10.1–13.2) 15.0 (7.6–27.7) 19.9 (15.1–25.7) Unspecified 29.5 (27.4–31.8) 24.3 (15.1–36.7) 28.4 (22.8–34.8) 1.1 (0.7–1.7) 16.4 (8.8–28.6) 2.9 (1.4–5.8) 3.7 (3.0–4.7) 26.4 (17.2–38.2) 13.3 (9.8–18.0) 95.2 (94.1–96.0) 57.2 (44.8–68.7) 83.8 (78.8–87.8) Some college or associate degree Bachelor degree or higher Annual household income, $ <20,000 Cigar/cigarillo smoking Every day or some days Rarely Not at all Smokeless tobacco usec Abbreviation: GED, general educational development. a All values are weighted percentage (95% confidence interval). b Respondents were considered to be never-established smokers if they responded negatively to the question, “Have you smoked at least 100 cigarettes in your entire life?” and responded with “not at all” to the question, “Do you now smoke cigarettes every day, some days, or not at all?” c Includes chewing tobacco, dip, snuff, and snus. (continued on next page) The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 6 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2016/15_0505.htm PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 (continued) Table 1. Characteristics of Sample of US Adults Aged 18 to 24 Years Who Never Established Cigarette Smoking (n = 2,528), National Adult Tobacco Survey, 2012–2013a Waterpipe Smokers (n = 398) Characteristic Never Established Cigarette Smokingb (n = 2,528) Current (Every Day or Some Days) (n = 97) Intermittent (Rarely) (n = 301) Every day or some days 2.3 (1.8–3.1) 11.5 (5.9–21.0) 2.1 (0.9–4.9) Rarely 0.7 (0.4–1.2) 5.3 (2.1–13.0) 1.0 (0.3–3.0) 97.0 (96.1–97.6) 83.2 (72.9–90.1) 96.9 (94.0–98.5) 0.5 (0.2–1.1) 8.5 (3.8–18.1) 0.7 (0.1–3.6) 1.4 (0.01–2.1) 27.4 (17.7–39.8) 6.7 (3.9–11.2) 98.1 (97.2–98.7) 64.1 (51.6–74.9) 92.7 (88.0–95.6) 92.8 (91.5–93.9) 64.2 (50.5–75.9) 90.8 (86.3–93.9) Moderately harmful 6.3 (5.3–7.6) 30.5 (20.0–43.7) 9.2 (6.1–13.7) Not at all harmful 0.9 (0.5–1.5) 5.3 (1.1–22.2) 0.0 (0.0–0.0) Former trier 36.9 (34.6–39.2) 25.5 (16.6–37.1) 66.6 (60.0–72.7) Never smoker 63.1 (60.8–65.4) 74.5 (62.9–83.4) 33.4 (27.3–40.0) Not at all E-cigarette use Every day or some days Rarely Not at all Harm perception for cigarettes Very harmful Experimentation with cigarettes Abbreviation: GED, general educational development. a All values are weighted percentage (95% confidence interval). b Respondents were considered to be never-established smokers if they responded negatively to the question, “Have you smoked at least 100 cigarettes in your entire life?” and responded with “not at all” to the question, “Do you now smoke cigarettes every day, some days, or not at all?” c Includes chewing tobacco, dip, snuff, and snus. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2016/15_0505.htm • Centers for Disease Control and Prevention 7 PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 Table 2. Estimates and Adjusted Logistic Model of Factors Associated With Susceptibility to Cigarette Smoking Among US Adults Aged 18 to 24 Years Who Never Established Cigarette Smokinga (n = 2,528), National Adult Tobacco Survey, 2012–2013 Susceptibility to Cigarette Smoking Characteristic Overall Weighted % (95% CI) P Valueb 19.7 (17.9–21.7) OR (95% CI) — — Waterpipe tobacco smoking Every day or some days 48.0 (29.4–67.2) Rarely 42.8 (36.2–49.7) Not at all 16.2 (14.4–18.3) 1.9 (0.5–7.2) <.001 2.3 (1.6–3.4)c 1 [Reference] Sex Female 14.1 (11.9–16.8) Male 25.6 (22.6–28.8) <.001 1 [Reference] 1.6 (1.2–2.3)c Age, y 18–21 22.3 (19.8–25.1) 22–24 15.3 (12.9–18.0) <.001 1.9 (1.3–2.7)c 1 [Reference] Race/ethnicity White, non-Hispanic 18.7 (16.2–21.6) Black, non-Hispanic 17.0 (12.2–23.1) Asian, non-Hispanic 13.7 (8.3–21.9) 1 [Reference] 0.9 (0.5–1.6) .10 1.3 (0.6–2.7) Hispanic 23.4 (19.1–28.3) 1.3 (0.9– 1.9) Other, non-Hispanic 22.6 (17.8–28.3) 1.4 (0.9–2.4) No high school diploma 23.1 (17.2–30.4) 1 [Reference] High school graduate 20.0 (17.6–22.7) Some college or associate degree 14.8 (10.2–20.9) Bachelor degree or higher 16.8 (13.3–21.0) 0.9 (0.4–1.7) <20,000 20.8 (15.2–27.9) 1 [Reference] 20,000–49,999 17.0 (13.8–20.8) 0.8 (0.5–1.3) 50,000–99,999 19.1 (15.4–23.4) ≥100,000 23.8 (18.4–30.3) 1.1 (0.6–1.9) Unspecified 20.5 (17.1–24.3) 1.1 (0.7–1.9) Education .16 0.8 (0.4–1.4) 0.7 (0.3–1.4) Annual household income, $ .36 1.0 (0.6–1.6) Cigar/cigarillo smoking Every day or some days 46.6 (26.2–68.1) <.001 1.9 (0.6–6.2) Abbreviations: CI, confidence interval; OR, odds ratio. a Respondents were considered to be never-established smokers if they responded negatively to the question, “Have you smoked at least 100 cigarettes in your entire life?” and responded with “not at all” to the question, “Do you now smoke cigarettes every day, some days, or not at all?” b Calculated by adjusted logistic regression. c Significantly different from reference group at P < .05. d Includes chewing tobacco, dip, snuff, and snus. (continued on next page) The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 8 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2016/15_0505.htm PREVENTING CHRONIC DISEASE VOLUME 13, E24 PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY FEBRUARY 2016 (continued) Table 2. Estimates and Adjusted Logistic Model of Factors Associated With Susceptibility to Cigarette Smoking Among US Adults Aged 18 to 24 Years Who Never Established Cigarette Smokinga (n = 2,528), National Adult Tobacco Survey, 2012–2013 Susceptibility to Cigarette Smoking Characteristic Weighted % (95% CI) Rarely P Valueb OR (95% CI) 44.4 (33.5–55.8) 1.4 (0.8–2.5) 18.4 (16.6–20.4) 1 [Reference] Every day or some days 37.3 (24.9–51.6) 1.2 (0.6–2.4) Rarely 42.5 (21.6–66.5) Not at all 37.3 (24.9–21.1) 1 [Reference] 36.3 (9.8–74.9) 1.1 (0.1–21.6) Not at all d Smokeless tobacco use <.001 1.4 (0.5–4.3) E-cigarette use Every day or some days Rarely 60.6 (40.5–77.7) <.001 2.0 (0.6–6.8) Not at all 20.1 (18.0–22.3) 1 [Reference] Very harmful 18.4 (16.5–20.6) 6.5 (0.8–51.4) Moderately harmful 36.7 (28.3–45.9) Harm perception for cigarettes Not at all harmful <.001 9.3 (2.0–33.4) 11.2 (1.3–93.6)c 1 [Reference] Experimentation with cigarettes Former trier Never smoker 36.1 (32.4–40.0) 10.1 (8.4–12.1) <.001 5.0 (3.6–6.8)c 1 [Reference] Abbreviations: CI, confidence interval; OR, odds ratio. a Respondents were considered to be never-established smokers if they responded negatively to the question, “Have you smoked at least 100 cigarettes in your entire life?” and responded with “not at all” to the question, “Do you now smoke cigarettes every day, some days, or not at all?” b Calculated by adjusted logistic regression. c Significantly different from reference group at P < .05. d Includes chewing tobacco, dip, snuff, and snus. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2016/15_0505.htm • Centers for Disease Control and Prevention 9
https://openalex.org/W2603224975	https://ccsenet.org/journal/index.php/ijef/article/download/66428/36428	English	null	Measuring the Efficiency of Health Services Areas in Kingdom of Saudi Arabia Using Data Envelopment Analysis (DEA): A Comparative Study between the Years 2014 and 2006	International journal of economics and finance	2,017	cc-by	9,632	International Journal of Economics and Finance; Vol. 9, No. 4; 2017 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education International Journal of Economics and Finance; Vol. 9, No. 4; 2017 ISSN 1916-971X E-ISSN 1916-9728 Published by Canadian Center of Science and Education Sanaa Mohamed Aly Helal & Haga Abdelrahman Elimam 1 Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia Correspondence: Sanaa Mohamed Aly Helal, Prof. Dr., Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia. E-mail: sanhelal@yahoo.com Sanaa Mohamed Aly Helal & Haga Abdelrahman Elimam 1 Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia Correspondence: Sanaa Mohamed Aly Helal, Prof. Dr., Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia. E-mail: sanhelal@yahoo.com Accepted: March 18, 2017 Online P URL: https://doi.org/10.5539/ijef.v9n4p172 Online Published: March 25, 2017 Received: February 15, 2017 doi:10.5539/ijef.v9n4p172 Received: February 15, 2017 doi:10.5539/ijef.v9n4p172 1.1 Problem of the Study The Data Envelopment Analysis (DEA) program is considered as one of the constructive programs that contribute to the measurement of the performance efficiency among hospitals. The specification of weaknesses are the areas in the efficiency of using available resources, proposal of optimal allocation of these resources, and increment of efficiency by reducing resources or decreasing outputs (Linna et al., 2006). Therefore, this study aimed to assess the efficiency of health services at Districts of Kingdom of Saudi Arabia, which are provided by the government hospitals (public) in the country. It was divided into 20 districts commencing with Riyadh and ending with Qunfudha (Yearbook, 2014); in addition, to measure the efficiency of the hospitals within each district. Abstract The study aimed to assess the efficiency of health services provided by the government hospitals in various districts of the Kingdom of Saudi Arabia. The number of beds at hospitals, doctors, nursing staff and paramedical categories were used as inputs for the model. The average productivity efficiency of government hospitals in the districts of the Kingdom of Saudi Arabia in 2014 was 92.3%; whereas, the average internal production efficiency of these districts in the provision of health services through their respective hospitals was 94.7%; and the average external productivity efficiency in the different cities of the districts in Kingdom of the Saudi Arabia was 97.5%. It has been found that the average overall productivity efficiency was 90.2%, concerning the relative efficiency indicators of government hospitals, which were based on the hospitals’ distribution of Saudi Arabian districts in 2006. An analysis of the indicator showed that the average production efficiency of the services provided (internally) by the districts of the Kingdom of Saudi Arabia was 94.7%, and that the average of the external production efficiency for such services was 95.4%. The Data Envelopment Analysis is a successful technique in measuring the performance efficiency of hospitals and it also assists to identify possible improvement and reduction in cost. Keywords: data envelopment analysis (DEA), general efficiency, relative efficiency Measuring the Efficiency of Health Services Areas in Kingdom of Saudi Arabia Using Data Envelopment Analysis (DEA): A Comparative Study between the Years 2014 and 2006 Sanaa Mohamed Aly Helal & Haga Abdelrahman Elimam 1 Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia Correspondence: Sanaa Mohamed Aly Helal, Prof. Dr., Faculty of Economics and Administration, University of King Abdul Aziz, Jeddah, Saudi Arabia. E-mail: sanhelal@yahoo.com 1.3 Relevant Scholarship The concern given by the Saudi government to health services has been underlined by the continuous increase in resources in sums allocated to the Ministry of Health in the country’s budget, coupled with parallel increase and the number of hospitals and beds at the Government hospitals, as illustrated in table 1. It indicated the increase in population, budget of the Ministry of Health (MOH) in relation to the country’s (Government “Gov.”) budget, government hospitals and beds at such hospitals from 2006-2014. Table 1. Annual reports of the Saudi ministry of health 2006-2014 Year Population in Millions No. of Hospitals No. of Beds Government’s (Gov.) Budget in Million SR MOH ‘s Budget in Million SR % of MOH’s Budget to Gov’s Budget 2006 23.7 120 30617 335 19.7 5.9 2007 24.2 225 31420 380 22.8 6.0 2008 24.8 231 31720 450 25.2 5.6 2009 25.4 244 33277 475 29.5 6.2 2010 27.0 249 34370 540 35.1 6.5 2011 28.4 251 34450 580 39.9 6.9 2012 29.2 259 35828 690 47.1 6.8 2013 30.0 268 38970 820 54.4 6.6 2014 30.8 270 40300 855 60.0 7.0 Table 1. Annual reports of the Saudi ministry of health 2006-2014 The results of Table 1 showed that in the period between 2006-2014, the positive rate of change in the total number of population reached 30%; and that the positive rate of change in the number of hospitals reached 24%, and the positive rate of change in the number of beds at hospitals reached 32% and finally the positive rate of change in the budget of the Saudi Ministry of Health reached 204.6%. Based on the above mentioned information, the following two questions can be represented:  Do the government hospitals (public) at the Administrative districts in the Kingdom of Saudi Arabia function in the required efficiency?  What are the procedures that can be adopted so as to address the situation of the Administrativ provides health services with low efficiency? Several studies have focused on measuring the efficiency of health services provided by government hospitals (public) via using DEA; and these studies are considered as the most attractive literature in the management of health services. 1.3 Relevant Scholarship Furthermore, they do not only prove that DEA is an effective technique for evaluating the efficiency of the organizations providing health care, but they also reflect a group of diversified problems in the management of health care provision that can be effectively handled via DEA. Additionally, a number of these studies have focused on resources and cost; whereas, others have concentrated on the comparison between the technique of DEA and other techniques. 1.3.1 Expansion of Data Envelopment Analysis Technique (DEA) 1.2 Importance of the Problem The healthcare services sector in the Kingdom of Saudi Arabia (KSA) has occupied a remarkably distinctive position among the remaining other service sectors. Furthermore, KSA has always been dedicated in considering health services sector as a key objective in all its development plans. However, it has been remarked that it was (and is still) facing challenges, which are represented in the provision of health care for every citizen from one aspect and raising the level of competency of the health care from the other. Therefore, the evaluation of health services and measurement of the hospitals’ efficiency have become necessity for ensuring the improvement of services quality and optimum utilization of resources allocated for them. Those in charge of hospital’s management confront a number of challenges that are not represented only in the provision of health services, but also in elevating the level of efficiency of these services. It happens because these services are directly linked to the health of individuals and their lives; and that the human resource is the foundation for any real economic and social development. Since human development and economic development are two similar aspects, it could 172 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org be found that the percentage of the development of societies is directly associated to the competency of these health services. be found that the percentage of the development of societies is directly associated to the competency of these health services. 1 3 R l S h l hi 1.3.3 DEA in Measuring the Efficiency of Hospitals The developed and developing countries have relied on measuring the efficiency of the hospital on DEA. Barnum, et al. (2011) used a sample comprising 87 hospitals in Chicago, and compared the development of the techniques for measuring the efficiency of these hospitals in connection for non-replaceable variables (fixed assets), and employed both the modern DEA measurement technique and traditional measuring techniques, such as ratio analysis and regression analysis. The results of the study have indicated that there is exaggeration in the efficiency of certain hospitals when it is measured via traditional measurement techniques. It makes any comparison doubtful between hospitals. Furthermore, the researchers have concluded, in a comparative study between DEA technique and econometric techniques, particularly the non-linear technique (one) using a sample of hospitals in North Carolina State in USA. It has been observed that DEA technique is capable of both specifying the shortcomings and of contributing to the elucidating the percentage of reducing the resources or maximizing the outputs of hospitals. This matter could not be determined by non-linear technique (Banker et al., 1984). Vincenzo and Rizzi (2006) applied DEA for the measurement of the efficiency of 85 hospitals (public and private) in the city of Veneto, Italy. The researchers found that minimum level of care for achieving the objectives of the hospital administrative policy makers at the hospital led to the reduction of the average of hospital’s efficiency. Likewise, the study concluded that public hospitals had low efficiency; that it attributed such reduction to the decision makers of these hospitals and the hospitals’ size, and inability of management to control the external factors. The study showed that private hospitals, whether profitable or non-profitable, have efficiency level higher than that of public hospitals. However, the study carried out by Sahin and Bulent (2011), which began the efficiency of hospitals in Turkey during the period between the years (2001-2006), used DEA technique, It has been found that the average efficiency rose up significantly after the Government has applied reformatory policies in the state-owned hospitals; whereas, the average of efficiency decreased in the same period in the private hospitals, particularly after due launching of reforms at the state-owned hospitals. 1.3.4 DEA in the Field of Healthcare DEA technique has been warmly received by researchers and practitioners at the hospitals of both public and private sectors. & Tone, 2003) and (Bahermz, 1996). & Tone, 2003) and (Bahermz, 1996). 1.3.2 DEA for Resources of Hospitals Evaluating the studies that have given much concern to resources and cost, it has been found that the resources of hospitals have been listed among the significant things in the studies carried out during the recent years. Furthermore, the focus of researchers regarding the efficiency of the hospitals has tremendously increased; either due to the desire for controlling the escalating costs (Bakar et al., 2010) or for achieving the greatest gain in spite of the limited resources (Sorkis & Talloru, 2002). The study conducted by Iswanto (2015) has focused on the importance of health financing as a necessity for ensuring higher efficiency of health services provided by hospitals in Indonesia. The study has depended on cross-sectional data of the financial statements for the three financial years of (Kimaj) Hospital, during (2011-2013). Furthermore, the cost of goods sold, operational cost, general cost, patient cost and the doctor’s fees were considered as variables for the inputs, whereas revenues, gross profit and net profit have been taken as variables for the outputs. The measurement of hospital’s performance has been measured by using DEA, which indicated that the efficiency of the hospital was 100% in 2013 as compared to 2011. 1.3.1 Expansion of Data Envelopment Analysis Technique (DEA) Data Envelopment Analysis technique (DEA) has been expanded in the late seventies of the twentieth century for measuring efficiency in different fields, including education, health, agriculture, industrial, engineering, sports and others. Farrell (1957) is considered the first one, who displayed the basic DEA technique, which was developed later into (CCR Model) in 1957 (Charnes et al., 1957) and then in 1978 (Charnes et al., 1978). This model relied on a scale fixed income, Constant Return Scale (CRS) for measuring the efficiency of Decision-Making Units (DMU). In 1984, Banker developed (CCR) model (Banker, 1984) into (BCC) model by using Variable Return Scale (VRS). However, researchers have employed curve as a measure for efficiency, as any point on this curve determines the amount of inputs or outputs (Niakas & Kontodimopoulos, 2005). In the Arab World, DEA is considered as a modern trend in the context of scientific and academic research; and that the first individual who wrote about this matter in Arabic in 1996, was Bahermz (1996), who was followed by Alhdokha and Bahermz in (1997), Azzaz in (2000), Baaikerm in (2002), Al-Shabi in (2004), and Fahmi in (2007). This technique is known as a mathematical method that uses linear programming for measuring relative efficiency of a number of administrative units (decision-making units) through specification of the optimal mixture of a group of its inputs and outputs, based on their actual performance (Joe Zhu, 2003; Cooper, Seiford, 173 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org 1.3.3 DEA in Measuring the Efficiency of Hospitals But, despite of the above, DEA has not been employed widely in the field of health care. David Sherman (1984) was the first researcher, who applied DEA at the group of teaching hospitals, and presented the aspects of shortcomings at hospitals. The study conducted by Al-Shayea (2011) was based on the measurement of the performance and efficiency of the departments at King Khalid University Hospital during the 12-month period. The results showed that only two of the nine department’s efficiency reached 100% during the period. In the same year, another study was published in Britain and included the usage of DEA technique in measuring the efficiency of public polyclinics in three English districts (Szczequra et al., 1993). Another study forecasted the non-governmental hospitals in USA with poor performance. Results indicated that efficiency was a positive sign, but it was weak in predicting the necessity of the hospital’s closure (Lynch & Ozcan, 1994). A study emphasized that DEA is considered as a constructive technology for the managers of health care to achieve an improvement in the efficiency of hospitals’ performance (Nayar & Ozcan, 2008). A similar study applied DEA technique to nine hospitals providing obstetric services. The study stressed the importance of this technique in measuring the efficiency and effectiveness. This justifies its inclusion among the 174 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org group of tools used by cost managers at Hospitals (Finkler & Wirtschafter, 1993). The study conducted by Hussein (2011) highlighted the significance of health services in general and hospitals, particularly as one of the most important indicators for social and technological advancement of people. To augment these activities, decision-makers need to know the results of their decisions and performance efficiency is considered as one of the most important decisions. The inputs of health-system are expected to yield a definite output, which usually refer to the labor and financial quantities and related capacities, whereas the inputs concerning the non-health inputs are referred by the income and reference to education levels (Kim & Kang, 2014). It has been noted by Greene (2004) that health expenditure is the primary and most dominant factor of input for the productivity evaluation of healthcare management programs. Labor related inputs have been incorporated by some studies in the DEA formulation by using international dataset (Afonso & St Aubyn, 2005; Bhat, 2005; Retzlaff-Roberts et al., 2004). 1.3.3 DEA in Measuring the Efficiency of Hospitals It is because the addition of labor related inputs may indicate the parsimonious DEA model at an organizational level. The aim of the study conducted by Laura et al. (2014) was to assess the efficiency of public health care systems in Europe, through the application of DEA technique, which is considered a non-teaching one. When DEA was applied to 30 European countries in 2010, the variables of inputs of the technique comprised a number of doctors, number of beds at hospitals and public health expenditure as a percentage of GDP; whereas, the variables of outputs included possibilities of a new born staying alive at delivery, life expectancy and infant mortality rate. The results concluded that there are a number of developed and developing countries that lie on the limits of efficiency; whereas, the vast majority of countries in the sample are incompetent. Despite the fact that many studies have underlined the importance of DEA in measuring efficiency, it has been found that many researchers are of the opinion that DEA is not a useful technique for evaluating efficiency, without the presence of additional sources (Szczepura et al., 1993). 1.4 Contribution of the Study The data envelopment analysis has been suggested by the study to manage the efficiency of the hospital management and services according to the nature of activities performed by the hospitals. The differences and diversity between several activities within the hospital can also be managed by the DEA technique. The study has contributed to measure the efficiency of the hospitals for the years 2006-2014 using the DEA technique. The study demonstrated that efficiency of the hospitals can be easily analyzed with tools and indicators developed by the econometrician, stakeholders and statisticians to measure the efficiency and human productivity. DEA is efficient enough to manage the input and output ratios, which do not comprise of all inputs and outputs. 2. Method The population included 270 public hospitals of the Ministry of Health, Kingdom of Saudi Arabia, distributed over 20 Administrative districts, in the year 1435/2014. This number was compared with 220 hospitals, distributed over the same number of Administrative districts for the year 1426/2006. Annual Health Statistical book issued by the Ministry of Health in the Kingdom of Saudi Arabia in 2006-2014 was considered as one of the data collection methods. The selection of the inputs and outputs is considered as one of the important factors in assessing the efficiency of hospital via using DEA technique, because it directly impact the results that were obtained. The study employed these variables (Yearbook of the Ministry of Health 2014-2006) and inputs for all public (government) hospitals at each Administrative District in the Kingdom of Saudi Arabia) consisted the number of beds, number of doctors, number of nurses, and number of personnel of allied medical (health) categories. Outputs for all public (government) hospitals at each Administrative District in the Kingdom of Saudi Arabia comprised the number of individuals visiting admitted patients. The number of inpatients, number of beneficiaries from the radiography service, and number of beneficiaries from laboratory tests have also been included. 2.2 BCC Model (Banker-Charnes-Cooper) Variable Return Model This particular Model was developed to overcome the disadvantages of CCR Model as presented by Charles et al. (1984). The assumption adopted by that CCR Model that returns on production was constant and does no longer apply to certain productivity unit. However, BCC model, which was amended from DEA Model, assumes that return on production was not constant. Furthermore, Charnes (1994) distinguished in this Model between technical efficiency and size efficiency (one) through the following points: Determine the technical efficiency at in a certain level of operations. Specify the possibility for availability of constant increasing or decreasing return on production.  Specify the possibility for availability of constant increasing or decreasing return on production This means that this model plays a role of regressor in the model, on the volume of production (Variable Return Scale) (Joe Zhu, 2003). The following mathematical formula in the event represented the goal to reduce the amount of input Orientation (Cooper et al., 2007). S.C 3. Results S.C 2.1 The Most Important Models of the Technique of Data Envelopment Analysis (DEA) where: n: number of administrative units, m: number of inputs, s: Number of outputs, jo: unity whose efficiency is required to be measured, θ the degree of efficiency (0≤ θ≤ 1): : weight of the unit xij: the amount of input i for unit j, yij: amount of input r for unit j entrance of the unit i j,: r the amount of the entrance of the unit j. : amount of input i for unit whose efficiency is required to be measured jo, yij of amount of input r for unit whose efficiency is required to be measured jo. Also, the values of the units under study are fixed values obtained from the past information related to the inputs and outputs resulting then, as it must be noted that the best values of the weights may change from one unit to another (Lewin et al., 1981). 2.2 BCC Model (Banker-Charnes-Cooper) Variable Return Model 2.1 The Most Important Models of the Technique of Data Envelopment Analysis (DEA) Models of the Technique of Data Envelopment Analysis (DEA) can be classified into four basic models that include CRS Model (Constant Return Scale) or CCR Model, (logarithmic) Multiplicative Model, Additive Model and BCC Variable Return Model, (Bahermz, 1996; Fahmi, 2006; Babakor, 2002). The researcher has used BCC Model and CCR Model, technical efficiency (overall efficiency), assuming constant return in relation to production (CRS). Charles, Cooper and Rhodes (Charnes et al., 1978) evolved this model, which was considered as the main model of all the above mentioned ones (Data Envelopment Analysis). Through this particular model, relative efficiency of administrative units under analysis has been evaluated; and that the sources and amount of inefficiency for each administrative unit have been determined. The relative efficiency of any unit (jo) has also been calculated for other units. It is worthy to mention, that CCR model is a model with constant return to 175 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org production model Constant Return to Scale (CRS), denoting that the rate of return on production increases at the same rate of increase in the factors of production. This property, which is known as the constant return to production, is considered appropriate only when all units subjected to comparison operating at the level of their optimal sizes (overall efficiency). But, in fact there may be a lot of obstacles that prevent the units from achieving these sizes, such as incomplete competition, funding constraints and others. CCR Model takes following formula: S.C (1) (2) (3) S.C (1) (2) (3) (1) (1) (2) (3) (2) (3) (3) where: n: number of administrative units, m: number of inputs, s: Number of outputs, jo: unity whose efficiency is required to be measured, θ the degree of efficiency (0≤ θ≤ 1): : weight of the unit xij: the amount of input i for unit j, yij: amount of input r for unit j entrance of the unit i j,: r the amount of the entrance of the unit j. 3. Results It is worth mentioning that (DEA) model measures efficiency, depending on the size of existing operations in DMU to provide services to the beneficiaries during the period of measurement. Furthermore, this model secures the possibility of the presence of constant increase or decrease in returns on the scope of effective units, which resulted from the change in the quantity of inputs to obtain efficiency (Cooper et al., 2003). Sherman and Joe (2006) classified DEA technique as the best way for reference comparison (Benchmark), due to the excellence in specifying the best counterpart units, depending on many inputs and outputs. Furthermore, the efficiency measurement with this technique does not require provision of information on the prices of inputs or outputs; and it is not stipulated that the inputs and outputs should be of the same measurement unit, but concentration should be made on decision making units. Additionally, there is no restriction in using a particular form of production functions that meet the strict justice criteria for each unit decision-making unit (Manzoni, 2009). BCC model was used among other Models or techniques of DEA for measuring hospitals’ efficiency. The researcher has employed this model, because it gives a variable return on the volume of production and that it is possible to know whether the return is constant, increasing or decreasing. Furthermore, reduction of inputs and increment of outputs have been dully chosen (Charnes, 1985; Bahermz, 1994). DEA was applied to the data of the hospitals (public) of the Ministry of Health, which are distributed over 20 Administrative Districts of the Kingdom of Saudi Arabia to measure efficiency at these hospitals. The results are indicated in the Table 2: 176 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org Table 2. 3.2 Internal Efficiency It denotes the exploitation of the hospital’s management for the available resources (inputs) for the provision of the best possible health services to the beneficiaries (outputs). 3.1 General Efficiency It denotes the level of efficiency of the administrative unit (of hospitals) in the utilization of available resources (inputs) and its ability to overcome external factors to secure the provision of the best possible service to the beneficiaries (outputs). If the general efficiency is 100%, this means that the relative efficiency has become complete. It denotes the level of efficiency of the administrative unit (of hospitals) in the utilization of available resources (inputs) and its ability to overcome external factors to secure the provision of the best possible service to the beneficiaries (outputs). If the general efficiency is 100%, this means that the relative efficiency has become complete. 3.2 Internal Efficiency 3. Results The general efficiency, in turn, is divided into internal efficiency and external efficiency (Abou El- Seoud, 2013; Nadir et al., 2013). From Table 2, the most important findings have been extracted that are reached through the application of Data Envelopment Analysis to government hospitals in the Administrative Districts of the Kingdom of Saudi Arabia for the year 2014. The table shows the general efficiency of the selected Administrative Districts that provide health services via government hospitals. The general efficiency, in turn, is divided into internal efficiency and external efficiency (Abou El- Seoud, 2013; Nadir et al., 2013). From Table 2, the most important findings have been extracted that are reached through the application of Data Envelopment Analysis to government hospitals in the Administrative Districts of the Kingdom of Saudi Arabia for the year 2014. The table shows the general efficiency of the selected Administrative Districts that provide health services via government hospitals. The general efficiency, in turn, is divided into internal efficiency and external efficiency (Abou El- Seoud, 2013; Nadir et al., 2013). 3. Results Comparison between the efficiency of health services provided by hospitals at the administrative districts of the Kingdom of Saudi Arabia between the years 2006-2014 Efficiency Indicator for the year 2006 Efficiency Indicator for the year 2014 Causes of inefficiency External Operation Efficiency Scale )%( Internal Operation Efficiency VRS )%( General Operation Efficiency CRS )%( Administrative Districts Causes of inefficiency External Operation Efficiency Scale )%( Internal Operation Efficiency VRS )%( General Operation Efficiency CRS )%( Administrative Districts EE I E G E EE I E G E --- 100 100 100 100 Medinah --- 100 100 100 Medinah --- 100 100 Qaseem --- 100 100 100 Qaseem --- 100 100 100 Hafr Al-Baten --- 100 100 100 Eastern --- 100 100 100 Aseer --- 100 100 100 Al-Ahsa --- 100 100 100 Bishah --- 100 100 100 Bishah --- 100 100 100 Ha’il --- 100 100 100 Jazan --- 100 100 100 Northern --- 100 100 100 Al-Bahah --- 100 100 100 Najran --- 100 100 100 Qurayyat --- 100 100 100 Al-Bahah --- 100 100 100 Qunfudah 100 100 100 Qunfudah IE, EE 99.6 98.3 97.9 Tabouk EE 96.6 100 96.6 Qurayyat EE 97.5 100 97.5 Aseer IE, EE 97.7 97.7 95.5 Al-Ahsa EE 96.7 100 96.7 Makkah EE 92.5 100 92.5 Eastern EE 91.5 100 91.5 Ta’if EE 90.2 100 90.2 Jazan IE, EE 88.9 98.2 87.4 Ha’il IE, EE 88.9 95.4 84.8 Tabouk EE 85.9 100 85.9 Riyadh EE 78.8 100 78.8 Makkah IE, EE 90.8 92.4 83.8 Northern IE, EE 96.7 74.2 71.8 Al-Jouf IE, EE 99.9 80.6 80.5 Hafr Al-Baten EE 70 100 70 Riyadh IE, EE 99.6 78.8 78.4 Najran IE, EE 97.8 69.4 67.9 Ta’if IE, EE 99.4 75.2 74.8 Jeddah IE, EE 98.4 57.6 56.6 Jeddah IE, EE 99.9 71 70.9 Al-Jouf IE, EE 95.4 94.7 90.2 Average IE, EE 97.5 94.7 92.3 Average Table 2. Comparison between the efficiency of health services provided by hospitals at the administrative districts of the Kingdom of Saudi Arabia between the years 2006-2014 From Table 2, the most important findings have been extracted that are reached through the application of Data Envelopment Analysis to government hospitals in the Administrative Districts of the Kingdom of Saudi Arabia for the year 2014. The table shows the general efficiency of the selected Administrative Districts that provide health services via government hospitals. 3.3 External Efficiency t signifies the efficiency of the hospital’s management in overcoming the external factors, which ffect the level of service provided by them to (outputs). By analyzing the results of the table’s relative efficiency of the hospitals in the Administrative Districts, there were many remarked points. The average of the general productive efficiency throughout the Administrative Districts of the Kingdom of Saudi Arabia in general is (92.3%). This means that the government hospitals in the selected districts must be able to provide the same level of output (number of individuals visiting inpatients, the number of laboratory tests, number of beneficiaries benefiting from radiography service) or more (if their objective is to reduce inputs together with maintaining the same level of outputs). However, with the usage of only (92.3%) or less of the current inputs (the number of doctors, the number of nurses, the number of allied 177 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org health personnel), the selected hospital has been efficient. In other words, they must reduce the inputs with a percentage of (7.7%) together with maintaining the current amount of outputs to show that they were efficient. But if the goal of the hospitals is to maximize outputs while maintaining the same amount of inputs, this means that government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia must increase services by (8.3%), to reach the complete general efficiency. This represents the inverse of the efficiency indicator in this case (Al-Ahmadi, 2009). health personnel), the selected hospital has been efficient. In other words, they must reduce the inputs with a percentage of (7.7%) together with maintaining the current amount of outputs to show that they were efficient. But if the goal of the hospitals is to maximize outputs while maintaining the same amount of inputs, this means that government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia must increase services by (8.3%), to reach the complete general efficiency. This represents the inverse of the efficiency indicator in this case (Al-Ahmadi, 2009). When the selected indicator has broken down, it become conspicuous that the average of internal operation efficiency (IE) for the government’s hospitals across the Administrative Districts of the Kingdom of Saudi Arabia is generally (94.7%). 3.4 Relative Efficiency Indicators of Government’S Hospitals in 2006 3.4 Relative Efficiency Indicators of Government’S Hospitals in 2006 The average of the General productive efficiency (GE) throughout the Administrative districts of the Kingdom of Saudi Arabia in general is (90.2%). This means that the government hospitals in the selected districts must be able to provide the same level of output (number of individuals visiting inpatients, the number of laboratory tests, number of beneficiaries benefiting from radiography service) or more (if their objective is to reduce inputs together with maintaining the same level of outputs). However, with the usage of only (90.2%) or less of the current inputs (the number of doctors, the number of nurses, the number of allied health personnel), the selected hospital could be efficient. In other words, they must reduce the inputs with a percentage of (9.2%) together with maintaining the current amount of outputs so that they were being efficient. But if the goal of the hospitals is to maximize outputs while maintaining the same amount of inputs, this means that government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia must increase the services by (8.3%) and increase their services by (10.9%) to reach the complete general efficiency. This represents the inverse of the efficiency indicator in this case (Talal, 2009). When the selected indicator broken down, it become obvious that the average of internal operation efficiency (IE) for the government’s hospitals across the Administrative Districts of the Kingdom of Saudi Arabia is generally (94.7%), and the average of External productive efficiency (EE) of the government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia is generally (95.54%). This means that the inability of the government’s hospitals reach the complete general efficiency across the Administrative Districts of the Kingdom of Saudi Arabia. It is evident from the above table that the groups of government hospitals at the Administrative Districts that failed to achieve the relative efficiency can be divided into two categories:  The government hospitals, that attribute their failure to achieve relative efficiency to the weakness of their management in overcoming the environmental or external factors as well as to the weaknesses in the internal management processes, and these hospitals are located in Tabuk, Hail, Riyadh, the northern border, Hafr Al-Batin, Najran, Jeddah, Al-Jawf. 3.4 Relative Efficiency Indicators of Government’S Hospitals in 2006  The government hospitals, that attribute their failure to achieve relative efficiency to the weakness of their management in overcoming the environmental or external factors as well as to the weaknesses in the internal management processes, and these hospitals are located in Tabuk, Hail, Riyadh, the northern border, Hafr Al-Batin, Najran, Jeddah, Al-Jawf.  The group of government hospitals, that attribute their failure to achieve relative efficiency to the weakness of their management in overcoming the environmental or external factors, and these hospitals are located in: Asir, Makkah, Taif, and Riyadh. 3.3 External Efficiency The average of EE (External Operation efficiency (EE) of the government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia is generally (97.5%). This means that, the inability of the government’s hospitals to reach the complete general efficiency across the Administrative Districts of the Kingdom of Saudi Arabia, is basically due to the weakness of the efficiency level for managing internal operations more than weakness of the efficiency level for managing and overcoming of the external environmental factors. The number of hospitals, that attribute inefficiency to the weakness management level in the general overcoming of the external environmental factors and the weakness of the level of efficiency in managing internal operations, was eleven. Whereas, it has been found that only seven hospitals attribute inefficiency to the general weakness of the level of efficiency in managing internal operations. 3.5 The Options Available to Inefficient Hospitals for Improving Their Efficiency There are many useful information provided by Data Envelopment Analysis. In addition to determining inefficient units (those who failed to achieve complete general relative efficiency), this analysis technique determines the amount of inefficiency at inefficient units and the proposed amounts for each of them to become efficient. Whether the goal is to maximize the outputs (output maximization) or to reduce the inputs (input minimization); in order to improve efficiency so that incompetent hospitals become really competent. The column labeled “actual values” indicates the value of used inputs and the values of produced output, which was actually produced at inefficient hospital in Jeddah Province. However, the column labeled (targeted) values 178 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org indicates the target and the values of the amounts of inputs and outputs that should be produced by inefficient hospitals. This study has applied the options available for improving the efficiency of inefficient government hospitals at Jeddah Province so that they are become efficient. indicates the target and the values of the amounts of inputs and outputs that should be produced by inefficient hospitals. This study has applied the options available for improving the efficiency of inefficient government hospitals at Jeddah Province so that they are become efficient. 3.6 The Options Available to Inefficient Hospitals in Jeddah Province for Improving Their Efficiency First Option: Reduction in inputs and obtaining the same outputs: Through the application of DEA with the usage of input minimization, the results in the following Table 3 are as follows: 3.6 The Options Available to Inefficient Hospitals in Jeddah Province for Improving Their Efficiency First Option: Reduction in inputs and obtaining the same outputs: Through the application of DEA with the usage of input minimization, the results in the following Table 3 are as follows: Table 3. 3.5 The Options Available to Inefficient Hospitals for Improving Their Efficiency There was reduction in the number of doctors in 2014 compared with 2006 with a percentage of 25.0% There was reduction in the number of nurses in 2014 compared with 2006, with a percentage of 15.0% - There was reduction in the number of allied health personnel in 2014 compared with 2006, with a percentage of 17.4%. - There was increase of the number of inpatients in 2014 compared with 2006 with a percentage of 10.6% after this reduction in inputs. 3.5 The Options Available to Inefficient Hospitals for Improving Their Efficiency Level of reduction in inputs and obtaining the same outputs at inefficient hospital in Jeddah Province 2006 2014 Category % Slack Optimization Target Value Actual Value % Slack Optimization Target Value Actual Value Number -43.4 -1302 1701 3003 -25.2 -755 2238 2993 Beds Input -50.6 -755 737 1492 -25.6 -584 1694 2278 Doctors -43.4 -1134 1481 2615 -28.4 -1464 3686 5150 Nurses -42.6 -679 825 1594 -25.2 -1099 3256 4355 Allied health personnel 3.11 19781.5 655822.5 636041 765941 765941 Individuals visiting patients Output 10.5 7726 81210 73484 20.6 18717 109614 90897 Inpatients 0 0 7193032 7193032 12367572 12367572 patients benefiting from laboratory tests 0 0 253457 253457 440589 440589 Patients benefiting from radiography Medinah Aseer, Al-Bahah Madinah, AlQaseem, Eastern District, Jazan, Al-Bahah, Madinah Madinah Jazan ,Al-Baha, Al-Qunfudah, Referral Units in Jeddah Province Out-Patients Department Visits for the number of beneficiaries of radiography service. vel of reduction in inputs and obtaining the same outputs at inefficient hospital in Jeddah Province From the results of the above table 3, following conclusion can be made: From the results of the above table 3, following conclusion can be made:  In order to reach the complete efficiency, like their referral hospitals, government hospitals in the Province of Jeddah region in 2014 should reduce the number of beds to 755 or by (25.2%) and reduce the number of doctors to 584, i.e. (25.6%), and reduce the number of nurses to 1464 i.e. (28.4%), and reduce the number of allied health personnel 1099 i.e. (25.2%). However, there is room for increasing the number of inpatients to 18,717 patients i.e. (20.6%) after this reduction in inputs.  Likewise, the same option at all inefficient government hospitals can be applied in other Provinces, and which were indicated in table 2; and the desired improvement referral units can be achieved as applied in inefficient government hospitals at Jeddah Province  When the efficiency in the performance of government hospitals for 2014 and 2006 was compared, its aspects can be summarized via the results of the selected table through the usage of DEA in the following points: - There was reduction in the number of beds at hospitals for 2014 compared to 2006 with a percentage o 18.2%. 3.7 Second Option: An Increase in Outputs at the Same Inputs 3.7 Second Option: An Increase in Outputs at the Same Inputs The following table 4 shows the increases that must be obtained as the outputs at the same number of inputs, through the application of DEA with the use of outputs output maximization: The following table 4 shows the increases that must be obtained as the outputs at the same number of inputs, through the application of DEA with the use of outputs output maximization: 179 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org Table 4. Levels of required increases in the outputs at the same inputs for inefficient hospitals in Jeddah Province 2006 2014 Category % Slack Optimization Target Value Actual Value % Slack Optimization Target Value Actual Value Number of 0 0 0 3003 0 0 2238 2993 Beds Input 13 -191 1301 1492 0 -12 2266 2278 Doctors 0 0 2615 2615 0 -220 4930 5150 Nurses 8.6 -137 1457.2 1594 0 0 4355 4355 Allied health personnel 82 521943 1157984 636041 34 258474 1024415 765941 individuals visiting inpatients Output 95 56266 143392 73484 61 55707 146604 90897 Inpatients 77 5507680 12700712 7193032 34 4173555 16541127 12367572 patients benefiting from laboratory tests 77 194071 447528 253457 34 148681 589270 440589 patients benefiting from radiography Medinah, Aseer, Al-Bahah Madinah, AlQaseem, Eastern District, Jazan, Al-Bahah, Madinah Madinah Jazan ,Al-Baha, Al-Qunfudah, Referral Units in Jeddah Province When the results of the Table 4 were summarized on the health services provided by the hospitals in Jeddah Province, it has been found that the selected table illustrates how to reach the current inputs with the complete efficiency, like referral hospitals. The government hospitals in the city of Jeddah should increase the reception of patients at different clinics at such hospitals with a percentage of (34%), increase the number of inpatients by (61%); and that they increase the number of patients benefiting from radiography service by (34%), and increase the number of laboratory tests at their respective primary health care centers by (34%). After these increases in outputs, it can also reduce the number of doctors to 12 with a percentage of (0.5%) and finally reduce the number of allied health personnel working at respective primary health care centers to (220) i.e. at a percentage of 4%. 3.7 Second Option: An Increase in Outputs at the Same Inputs  Similarly, the same option to all inefficient government hospitals can be applied in other Administrative Districts; and that reach the desired level of improvement and referral units as applied in inefficient government hospitals at Jeddah Province.  When the efficiency in the performance of government (public) hospitals in 2014 with that in 2006 is compared, this can be summed up through the results of the table via using the DEA program (technique) in the following points:  When the efficiency in the performance of government (public) hospitals in 2014 with that in 2006 is compared, this can be summed up through the results of the table via using the DEA program (technique) in the following points: - There was an improvement in the number of individuals visiting clinics in 2014 compared with that in 2006 by 48%. - There was an improvement in the number of individuals visiting clinics in 2014 compared with that in 2006 by 48%. - There was an improvement in the number of patients in 2014 compared with that in 2006 by 34%. - There was an improvement in the number of those benefited from the laboratory tests in 2014 compared with that in 2006 by 43%. - There was an improvement in the number of those benefited from radiography service in 2014 compared with that in 2006 by 43%. 4. Discussion The average general productivity efficiency of government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia in 2014, was generally 92.3% in comparison with that of 90.2% for the same hospitals in 2006. The groups of government hospitals at the Administrative Districts that failed to achieve the relative efficiency can be divided into two categories: The average general productivity efficiency of government hospitals across the Administrative Districts of the Kingdom of Saudi Arabia in 2014, was generally 92.3% in comparison with that of 90.2% for the same hospitals in 2006. The groups of government hospitals at the Administrative Districts that failed to achieve the relative efficiency can be divided into two categories:  The government hospitals, that attribute their failure to achieve relative efficiency to the weakness of their management in overcoming the environmental are located in Tabuk, Hail, Riyadh, the northern border, Hafr Al-Batin, Najran, Jeddah, Al-Jawf.  The group of government hospitals that attribute their failure to achieve relative efficiency to the weakness of their management in overcoming the environmental or external factors, and these hospitals are located in: Asir, Makkah, Taif, and Riyadh. The referral for the general hospitals of Jeddah Province in 2014 included those located at the Provinces of The referral for the general hospitals of Jeddah Province in 2014 included those located at the Pr 180 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org Medina, Jizan, Al-Baha and Qunfudah 2014. Whereas, referral hospitals for the same Jeddah’s general hospitals in 2006 comprised those situated in the Provinces of Medina, Asir and Baha. The average general productive efficiency for government hospitals of Jeddah Province was 74.8% in 2014, compared with that of 56.6% in 2006 for the same hospitals. Furthermore, it turns out that the average internal productive efficiency of government hospitals of Jeddah Province was (75.2%) in 2014 compared with 57.6% in 2006 for the same hospitals. Likewise, average of external productive efficiency for government of Jeddah Province was 99.4% in 2014, compared with that of (98.4%) in 2006 for the same hospitals. 4. Discussion This means that the inability of the government hospitals to reach the complete general efficiency across the Administrative Districts of the Kingdom of Saudi Arabia, was basically due to the weakness of the efficiency level for managing internal operations more than weakness of the efficiency level for managing for the general overcoming of the external environmental factors. The study indicated that there are two options for inefficient hospitals at Jeddah Province to reach efficiency; the first option is via reductions in inputs and obtaining of outputs and second option is via increasing outputs at the same levels of outputs. With application of these two options. The comparison of the performance efficiency of inefficient government (public) hospitals via reduction of resources (inputs) and obtaining of the same services (outputs) in 2014 from 2006 can be summarized. There was a reduction in the number of beds by 18.2%, and a reduction in the number of doctors by 25%, as the reduction in the number of nurses by 15%, reduction in allied health service personnel by 17.4% and finally an increase in the number of inpatients in 2014 from 2006 by 10.1% after this reduction in inputs. The comparison of the performance efficiency of inefficient government (public) hospitals in 2014 from 2006 can be summarized. There was a remarkable improvement in the number of individuals visiting patients by 48% for the improvement in the number of inpatients by 34%. In addition to the previously mentioned improvement in the number of those benefited from laboratory tests by 43%, and finally an improvement in the number of those benefiting from radiography service improved by 43%. Regardless of the knowledge about the effectiveness in healthcare delivery, the healthcare performance conceptualization has remained imperfect. There is inefficient healthcare system, which is often determined by the unsatisfactory and inadequate measures of uncertain objectives and misidentified service activities (Kim & Kang, 2014). Successive studies have proclaimed that the processes of health care delivery appear to be less vigorous and less routinized as compared to other areas and it remains highly sensitive in terms of different environmental settings (Spinks & Hollingsworth, 2009; Hollingsworth, 2008). DEA allows the productivity assessments of every country and its healthcare system through comparing a relatively technical efficiency to its peer group. The DEA technique has certain drawbacks, it produces outcomes that are specifically sensitive to the error measurements. 4. Discussion If an organization or hospital can become an outlier that more prominently distorts the frontier shape and reduces the effectiveness of scores nearby organizations or other hospitals. DEA has another drawback that it only evaluates the efficiency relative to the particular sample of best practices (Cooper et al., 2006). This study presented that Data Envelopment Analysis (DEA) technique (Model, which is successful in measuring performance efficiency of hospitals. Moreover, DEA allows analysis of various aspects of shortcomings and helps to identify possible improvements and specify the possible reduction in cost. Relative Efficiency Scale is considered a good indicator for evaluation and comparison of the performance of government hospitals in Administrative Districts of the Kingdom of Saudi Arabia. By using the technique DEA for measuring the degree of efficiency and specification of causes of inefficiency, their sources and , as well as by applying the selected technique to 20 Administrative Districts in the framework of comparison of hospitals performance between 2014 and 2006, The following results have been obtained: Based on the results, the study presents some recommendations as follows: Based on the results, the study presents some recommendations as follows:  Encourage the studies for the determination of the causes that for the lack of government hospitals from achieving relative efficiency with a view to secure optimal utilization of available resources, via comparing the levels (inputs/outputs) of these hospital with the levels (inputs/outputs) of the referral hospitals.  Encourage the studies for the determination of the causes that for the lack of government hospitals from achieving relative efficiency with a view to secure optimal utilization of available resources, via comparing the levels (inputs/outputs) of these hospital with the levels (inputs/outputs) of the referral hospitals.  Urge to carry out periodical (annually or biennially) studies on performance measurement at the level of primary health care centers and government hospitals regular studies (annual or biennial). It must highlight the comparative performance between public hospitals in 2014 and in 2006 so as to ensure of the good utilization of their resources and that they enjoy high efficiency.  Provide detailed information to researchers about the resources and outputs of hospitals to conduct such an  Provide detailed information to researchers about the resources and outputs of hospitals to conduct such an 181 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org important study. References AbouEl-Seoud, M. (2013). Measuring efficiency of reformed public hospitals in Saudi Arabia: An application of data envelopment analysis. Int J Econ Manag Sci, 2(9), 44-53. AbouEl-Seoud, M. (2013). Measuring efficiency of reformed public hospitals in Saudi Arabia: An application of data envelopment analysis. Int J Econ Manag Sci, 2(9), 44-53. Afonso, A., & St Aubyn, M. (2005). Non-parametric approaches to education and health efficiency in OECD countries. 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Discussion  Direct to conduct a study on measuring the impact of external factors on efficiency rates, which include the impact of the hospital’s site (densely populated/thinly populated area), the nature of the city where the hospital is located, the demographic composition of the city, the proportion of residents in the city to the citizens etc.  Direct to conduct a study on measuring the impact of external factors on efficiency rates, which include the impact of the hospital’s site (densely populated/thinly populated area), the nature of the city where the hospital is located, the demographic composition of the city, the proportion of residents in the city to the citizens etc.  Request to redistribute health resources, the most significant of which is manpower, at government hospitals with low productivity over hospitals and primary health care centers with high productive efficiency.  Request to redistribute health resources, the most significant of which is manpower, at government hospitals with low productivity over hospitals and primary health care centers with high productive efficiency. Acknowledgments The authors are very thankful to all the associated personnel in any reference that contributed in/for the purpose of this research. Further, this research holds no conflict of interest and is not funded through any source. References The authors are very thankful to all the associated personnel in any reference that contributed in/for the purpose of this research. Further, this research holds no conflict of interest and is not funded through any source. References D., & Wirtschafter, D. D. (1993). Cost-effectiveness and data envelopment analysis. Health Care Management Review, 18(3), 81-88. https://doi.org/10.1097/00004010-199301830-00010 Greene, W. (2004). Distinguishing between heterogeneity and inefficiency: Stochastic frontier analysis of the World Health Organization's panel data on national health care systems. 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University Ca’Foscari of Venice, Dept. of Economics Research Paper Series, (13/06). https://doi.org/10.2139/ssrn.946885 Retzlaff-Roberts, D., Chang, C. F., & Rubin, R. M. (2004). Technical efficiency in the use of health care resources: A comparison of OECD countries. Health Policy, 69(1), 55-72. https://doi.org/10.1016/j.healthpol.2003.12.002 Sahin, M., & Bulent, S. (2011). Analyzing the Efficiencies of Hospitals: An Data Envelopment Analysis. Journal of Global Strategic Management, (10). https://doi.org/10.20460/JGSM.2011515804 Sarkis, J., & Talluri, S. (2002). Efficiency measurement of hospitals: Issues and extensions. International Journal of Operations & Production Management, 22(3), 306-313. https://doi.org/10.1108/01443570210417605 Sherman, H. (1984). Hospital Efficiency Measurement and Evaluation, Empirical Test of aNew Technique. Medical Care, 22(10), 922-939. https://doi.org/10.1097/00005650-198410000-00005 Sherman, H. D., & Zhu, J. (2006). Service productivity management: Improving service performance using data envelopment analysis (DEA). Springer Science & Business Media. Spinks, J., & Hollingsworth, B. (2009). Cross-country comparisons of technical efficiency of health production: A demonstration of pitfalls. Applied Economics, 41(4), 417-427. https://doi.org/10.1080/00036840701604354 183 International Journal of Economics and Finance Vol. 9, No. 4; 2017 ijef.ccsenet.org Szczepura, A., Davies, C., Fletcher, J., & Boussofiane, A. (1993). Efficiency and effectiveness in general practice. Journal of Management in Medicine. https://doi.org/10.1108/eb060565 Szczepura, A., Davies, C., Fletcher, J., & Boussofiane, A. (1993). Efficiency and effectiveness in general practice. Journal of Management in Medicine. https://doi.org/10.1108/eb060565 Copyrights Copyright for this article is retained by the author(s), with first publication rights granted to the journal Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). 184
https://openalex.org/W4382284345	https://www.researchsquare.com/article/rs-3071456/latest.pdf	English	null	Change of deep subduction seismicity after a large megathrust earthquake	Research Square (Research Square)	2,023	cc-by	11,638	Change of deep subduction seismicity after a large megathrust earthquake Authors: Blandine Gardonio1,3, David Marsan2, Thomas Bodin1, Anne Socquet2, Stéphanie Durand1, Mathilde Radiguet2, Yanick Ricard1,Alexandre Schubnel3 Authors: Blandine Gardonio1,3, David Marsan2, Thomas Bodin1, Anne Socquet2, Stéphanie Durand1, Mathilde Radiguet2, Yanick Ricard1,Alexandre Schubnel3 Keywords: Posted Date: June 27th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: There is NO Competing Interest. Version of Record: A version of this preprint was published at Nature Communications on January 2nd, 2024. See the published version at https://doi.org/10.1038/s41467-023-43935-3. Affiliations: 1Univ Lyon 1, ENSL, CNRS, LGL-TPE, F-69622, Villeurbanne, France 2Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, IRD, Univ. Gustave Eiffel, ISTerre, 38000 Grenoble, France 3Laboratoire de Géologie, Département de Géosciences, École Normale Supérieure, CNRS UMR 8538, PSL Research University, Paris, France. 3Laboratoire de Géologie, Département de Géosciences, École Normale Supérieure, CNRS UMR 8538, PSL Research University, Paris, France. *Correspondence to: blandine.gardonio@univ-lyon1.fr 1 1 Subduction zones are home to the world’s largest and deepest earthquakes. Recently, large- scale interactions between shallow (0-60 km) and intermediate (80-150km) seismicity have been evidenced before megathrust earthquakes along with large-scale changes in surface motion. Large-scale deformation transients following major earthquakes have also been observed possibly due to a change in slab pull or to a bending/unbending of the plates, which suggests the existence of interactions between the deep and shallow parts of the slab. In this study, we analyze the spatio-temporal variations of the Japanese seismicity before and after the 2011/3/11 Mw9.0 Tohoku-oki earthquake. We observe that the rate of the intermediate to deep (150-450km) seismicity underwent a deceleration of 55% south of the rupture zone and an acceleration of 30% north of it, coherent with the GPS surface displacements. This shows for the very first time how a megathrust earthquake can affect the stress state of the slab over a 2500 km lateral-range and a large depth range, demonstrating that earthquakes interact at a much greater scale than the surrounding rupture zone usually considered. Our vision of subduction zones must consequently be renewed as a whole, from shallow to large depths. 2 2 Recent studies suggest that large subduction earthquakes can affect the plate stress regime by showing either a synchronicity of the shallow and intermediate seismicity before large megathrust earthquakes1,2,3,4, a slab wide deformation transient concomitant with deep and shallow earthquake swarms before a large deep-focus earthquake5 or a gravity field change6,7. The observation of large- scale surface displacements reversals, several months before megathrust earthquakes also suggests the existence of deep precursors8. However, the observations of such large-scale interactions are rare and their mechanisms, and spatio-temporal extent remain poorly understood. To tackle this riddle, one solution is to analyze of the seismic productivity as a proxy for the slab stress conditions after a megathrust earthquake9,10. Observed variations in rate of intermediate and deep seismicity Our aim is to investigate the temporal and spatial evolution of the background seismicity, as a proxy for changes in loading rates. We work on 21 years of earthquake activity located in the subduction zone of the Pacific slab, recorded by the JMA (Japanese Meteorological Agency) catalogue from 2000 to 2021. To avoid a signal mostly affected by aftershocks and foreshocks sequences, we removed dependent earthquakes following the method of Marsan et al. (2017)33 (see Methods, Fig. S1 to S4). Keeping only the independent background seismicity34 evidences temporal and spatial changes in loading rates (Fig. S5). 144 reference points are defined over a depth-range of 80km to 640km (colored dots in Fig. 1). For each point, we select the 200 closest earthquakes with magnitude ≥ 3.5 which corresponds to the magnitude of completeness at these depths. Note that there are fewer earthquakes in the Japan Sea so the distance between the reference points and the related earthquakes reaches 600km in this region whereas it is 300km for the other points (Fig. S6a). For every set of earthquakes, we investigate the temporal evolution of the seismicity rate given by λ = N/Δt, where N is the number of events occurring in the time period Δt (see Methods). We compute the relative change of seismicity rate λ at the time of the Tohoku-oki earthquake as (λa–λb)/λb, where λa and λb are the seismicity rates after and before (over 11 years) the Tohoku-oki earthquake. The effect of the Tohoku-oki earthquake on the seismicity rate at depth is far from being homogeneous over the Japanese islands and we observe three main areas of opposite behaviors (Fig. 1a). Underneath the island of Hokkaido, the seismic rate increases, up to 30% between 150 and 450km depth. This is confirmed by the stack of all the time series at latitude > 42°N (Fig. S6b). The maximum change is found at 200km at 45°N latitude (Fig. 2a and b, red curve). We detect no significant effect of the Tohoku-oki earthquake on the deep seismicity (Fig. 1a). The stack of the time series of reference points lying between 35°N and 42°N shows no change of the seismic rate at the time of the Tohoku-oki earthquake (Fig. S6c). Observed variations in rate of intermediate and deep seismicity The southern part of the Japanese island arc, from 26° to 35°N latitude, shows a decrease of the seismicity rate from -55 to - Our aim is to investigate the temporal and spatial evolution of the background seismicity, as a proxy for changes in loading rates. We work on 21 years of earthquake activity located in the subduction zone of the Pacific slab, recorded by the JMA (Japanese Meteorological Agency) catalogue from 2000 to 2021. To avoid a signal mostly affected by aftershocks and foreshocks sequences, we removed dependent earthquakes following the method of Marsan et al. (2017)33 (see Methods, Fig. S1 to S4). Keeping only the independent background seismicity34 evidences temporal and spatial changes in loading rates (Fig. S5). 144 reference points are defined over a depth-range of 80km to 640km (colored dots in Fig. 1). For each point, we select the 200 closest earthquakes with magnitude ≥ 3.5 which corresponds to the magnitude of completeness at these depths. Note that there are fewer earthquakes in the Japan Sea so the distance between the reference points and the related earthquakes reaches 600km in this region whereas it is 300km for the other points (Fig. S6a). For every set of earthquakes, we investigate the temporal evolution of the seismicity rate given by λ = N/Δt, where N is the number of events occurring in the time period Δt (see Methods). We compute the relative change of seismicity rate λ at the time of the Tohoku-oki earthquake as (λa–λb)/λb, where λa and λb are the seismicity rates after and before (over 11 years) the Tohoku-oki earthquake. The effect of the Tohoku-oki earthquake on the seismicity rate at depth is far from being homogeneous over the Japanese islands and we observe three main areas of opposite behaviors (Fig. 1a). Underneath the island of Hokkaido, the seismic rate increases, up to 30% between 150 and 450km depth. This is confirmed by the stack of all the time series at latitude > 42°N (Fig. S6b). The maximum change is found at 200km at 45°N latitude (Fig. 2a and b, red curve). We detect no significant effect of the Tohoku-oki earthquake on the deep seismicity (Fig. 1a). The stack of the time series of reference points lying between 35°N and 42°N shows no change of the seismic rate at the time of the Tohoku-oki earthquake (Fig. S6c). Affiliations: In that respect, the 2011/3/11 Mw9.0 Tohoku-oki earthquake is an ideal case study, since it is the best recorded megathrust earthquake of the instrumental period of seismology11-16 and it occurred within one of the best-monitored regions of the world, Japan. Several studies conducted after the Tohoku-oki earthquake evidenced a preseismic phase1,6,17-22. After the earthquake, there was a shift from predominant compression to tension in the forearc region off the coast23-27. The offshore GPS-Acoustic stations located above the main coseismic slip area have shown large landward post-seismic displacement due to an early viscoelastic relaxation of the oceanic mantle below the subducting plate28,29. Furthermore, segments adjacent to the Tohoku-oki earthquake experienced a landward increase of surface velocity that could either be due to the acceleration of the subducting plate30,31 or to a viscoelastic response32. The Tohoku-oki earthquake is thus characterized by both a large-scale preparatory phase and a large-scale post-seismic response. Here, we investigate the intermediate and deep seismicity (from 150 to 450km depth and even 680km in the Izu-Bonin area) of the entire Pacific slab, over the 2,500 km long Japanese island arc. The spatio-temporal evolution of the seismicity rate λ (the number of earthquakes per unit of time) is analyzed using a probabilistic approach. Our study provides new observations on the consequences a large mega-thrust earthquake on intermediate and deep seismicity, and hence on the 3 3 Observed variations in rate of intermediate and deep seismicity The southern part of the Japanese island arc from 26° to 35°N latitude shows a decrease of the seismicity rate from -55 to - 4 10% with an average of -30% (Fig. 1a and S6d). The maximum change of -55% is found at 34°N latitude and 280km depth (Fig. 2a and b, blue curve). By increasing the length of the analyzed period, the maximum change underneath Hokkaido and the negative change in the south attenuate with time until 2017 (Fig. 1b). The attenuation in the south continues until 2021, while there is a down-dip migration of the acceleration pattern in the north (Fig. 1c). Statistical significance of the variation in the seismicity rate In order to test the robustness of the observations, we perform various statistical tests on the onset of the seismicity rate change, focusing on the 2000/3/11-2013/3/11 period when we see the most significant changes due to the Tohoku-oki earthquake. We use different statistical approaches (see Methods) assuming that the rate of seismicity follows a non-homogeneous Poisson process. First, we analyze the probability for a change in seismicity rate, assuming that it occurs at the time of the Tohoku-oki earthquake. To that aim, we follow the formalism of Marsan and Wyss (2011)35. The probability density function (pdf) of the rate change r is shown in Fig. 2c. A rate change r less than or greater than 1 means a decrease or increase in seismicity rate, respectively. At 34°N latitude, the probability density for a rate change after the Tohoku-oki earthquake is maximum at rmax=0.45 meaning that it is most likely to have undergone a decrease of seismicity rate in that region at that time (Fig. 2c, blue curve). Conversely, an increase of the seismicity rate under the island of Hokkaido is confirmed with rmax=1.27 (Fig. 2c, red curve). It is possible to track this evolution by examining the pdf of the seismicity rate, λ before and after the Tohoku-oki earthquake, separately (Fig. S7). We compute the standard deviation of r, σ, for a change at every grid point (Fig. S8). Plotting rm, rmax-σ (Fig. S8 left) or rmax+σ (Fig. S8 right) leads to the same conclusions as we still see values close to 1 under Hokkaido for rmax-σ and a decrease of the earthquake rate around 34°N latitude for rmax+σ. The high probability of having a change in seismicity rate at the time of the Tohoku-oki earthquake is thus confirmed whether we see an increase or a decrease of λ. We then focus on the probability of this change occurring at any other time. We again compute the pdf of r 5 5 using the same dataset but now exploring different times of change (Fig. S9 and S10). While rmax, i.e. the value that maximizes the pdf, is stable between 2000 and Tohoku-oki, there is a significant change of slope of the maximum at the time of the Tohoku-oki earthquake (black lines in Fig. S10) implying that the regime changes at this precise moment only, both at 34°N and 44°N latitude. Statistical significance of the variation in the seismicity rate More specifically, rmax>1 between 2000 and the Tohoku-oki earthquake at 44°N latitude and rmax<1 at 34°N, showing the increase/decrease, respectively, of the seismicity rate at the time of the megathrust earthquake. Finally, we estimate the probability of having a change in the seismicity rate at any given time for the whole region by stacking the pdf of seismicity rate change calculated at each reference point (see Methods and Fig. 3). This probability shows a significant change at the time of the Tohoku-oki earthquake (Fig. 3a) indicating that a change in seismicity rate is most likely at the time of the Tohoku-oki earthquake, although its likelihood varies with latitude (Fig. 3b). Seeing no effect of the Tohoku-oki earthquake on the seismicity rate at the northern (above 45°N latitude) and southern (below 28°N latitude) tips confirms the spatial delimitation of our observations. Thus, the Tohoku-oki earthquake affected the stress state of the slab from the Izu- Bonin to the Hokkaido islands with a dichotomy between north (acceleration) and south (deceleration). We further clustered the 144 reference points according to their rmax values at the time of Tohoku-oki earthquake applying the k-means clustering method (an iterative, data- partitioning algorithm that assigns n observations to exactly one of k clusters) considering 4 clusters (Fig. S11a). For each cluster thus formed, we display in Fig. S11b-e the mean of the pdf computed at each time step for all the points in the cluster. The first cluster is located underneath Hokkaido (Fig. S11b) and its rmax value is maximum at the time of the mega-thrust (Fig. S11b). It corresponds to the area where the seismicity rate increases after the Tohoku-oki earthquake. Cluster 2 corresponds to locations where the seismicity rate is constant (rmax=1, Fig. S11c). Cluster 3 gathers the southern points where there is a decrease of the seismicity rate after the megathrust earthquake. Its rmax value is indeed minimal at the time of the Tohoku-oki earthquake. The same applies to 6 6 cluster 4 with a smaller decrease, rmax being close to 1. Note that all the grid points in the northern and southern tips belong to either cluster 2 or 4 which are the ones showing the mildest effect of the Tohoku-oki earthquake, hence outlining indicating the spatial limit of the Tohoku-oki effect. Statistical significance of the variation in the seismicity rate This confirms the dichotomy between the northern and southern parts of the Pacific slab and the extent of the Tohoku-oki effect on the intermediate and deep seismicity. After the Tohoku-oki earthquake, many small magnitude earthquakes went undetected, especially at great depths, masked by the high productivity of larger, shallower, aftershocks. To address this issue, we analyze again the JMA catalog taking a magnitude of completeness of 4.5 and considering the 70 closest earthquakes to each reference points (Fig. S12). We still see the positive and negative changes underneath Hokkaido and at 34°N latitude, respectively. Note that the seismicity rate change in the Izu-Bonin at depth is still negative but less statistically significant. We finally carry out the same exercise (taking Mc = 4.5 and the 70 closest events) using the USGS catalog and observe a strong increase underneath Hokkaido and a less visible decrease at 34°N latitude (Fig. S13). Possible causes of changes in the seismicity rate We have shown that the changes of seismicity rate are statistically significant on the entire Japanese Pacific subduction zone between 150 and 450 km depth, over a spatial extent spanning 2500km from 26° to 48° of latitude. Interestingly, the maximum and the minimum rate changes are both at major bends of the Pacific subducting slab36 (Fig. 1). Furthermore, the dip is larger in the Southern bend, around 34°N latitude, where the maximum negative variation was recorded, than underneath Hokkaido, where the maximum positive variation was recorded. The analysis of the Coulomb stress transfer on the area of the rupture zone of the Tohoku-oki earthquake, i.e. at a smaller depth than this study (0-150km), highlights a cessation of the thrust earthquakes in the rupture zone that might last for centuries9,10. To get a first order estimate of the elastic loading/unloading on the subduction interface, we compute the Coulomb stress change for two receiver points located on the slip 7 7 interface at depth, where we observe the maximum acceleration and deceleration of the seismicity rate (orange and yellow stars in Fig. 4a). To do so, we use COULOMB3.337,38 and impose a 50m of slip on one dislocation located on the rupture zone of the Tohoku-oki earthquake. The receiver points are located according to the interface geometry36 and we vary the rake angle from -180 and 180° for comparison with the rake values of faults in the surrounding area (Fig. 4b). A positive Coulomb stress change, i.e. a loading of the fault, is found for most dominant rake values at 44°N latitude, while a negative Coulomb stress change, an unloading, is found for most of the characteristics rakes at 34°N latitude coherent with an increase/decrease of the seismicity rate at these latitudes. Our GPS data analysis (Fig. 1) confirms the enhanced landward motion that follows the Tohoku-oki earthquake observed in the Hokkaido region28,29, that induces an anticlockwise rotation of the velocity change and an enhanced trenchward motion in Honshu. This landward GPS velocity change in Hokkaido is located right above the maximum positive change of the seismicity rate observed at 44°N latitude. Furthermore, we see a possible second, larger-scale, clockwise rotation of the displacement in the south, at 34°N latitude, where we observe the maximum negative change. Possible causes of changes in the seismicity rate Therefore, there is a clear spatial correlation between the surface displacements and the changes in the seismicity rate which places constraints on the location of the changes at great depth that GPS alone cannot resolve. Note that a Mw7.6 aftershock of the Tohoku-oki earthquake, which occurred offshore the Boso peninsula, and its subsequent postseismic slip could be responsible for part of this velocity change39. Such rotation patterns have different characteristics according to their timing in the seismic cycle40 and different mechanical models could explain the enhanced landward motion seen in Hokkaido : an apparent increase in coupling in regions adjacent to the megathrust30,31; or a viscoelastic relaxation of both oceanic and continental plates caused by coseismic and postseismic fault slip28,29,41 that results in gear-train rotation patterns32. Because the deep seismic response after the Tohoku-oki earthquake is both extremely fast and extensive a rapid and large scale stress transfer along the slab is required and could be facilitated by Because the deep seismic response after the Tohoku-oki earthquake is both extremely fast and extensive, a rapid and large scale stress transfer along the slab is required and could be facilitated by 8 a low-viscosity channel, similar to the one modelled down to 135km to explain the post-seismic deformation following the Maule earthquake42. As the variation in seismicity rate disappears slowly with time, this low viscosity is likely a transient feature, that can be modeled as a Burgers rheology combining Maxwell and Kelvin moduli commonly invoked in visco-elastic post-seismic relaxation models, including after Tohoku-oki earthquake26,43,44. The Tohoku-oki earthquake has large-scale consequences on the very-deep (150-450km and deeper in the Izu-Bonin area) seismicity of the Pacific plate. We observe significant changes in deep seismicity rate together with changes in surface displacement rates over a 2500 km lateral-range and up to 450km depth, demonstrating that interactions between shallow and deeper parts of the slab exist at every stage of the seismic cycle. This questions our understanding and our vision of subduction zones that must be considered as a whole. In the future, taking greater depths into account in 3D rheological models and seismicity studies will bring more insights to the stress state of the slab and its continuity as a geodynamical object. References : References : 1. Bouchon, M. et al. Potential slab deformation and plunge prior to the Tohoku-oki, Iquique and Maule earthquakes. Nature Geosci 9, 380–383 (2016). 1. Bouchon, M. et al. Potential slab deformation and plunge prior to the Tohoku-oki, Iquique and Maule earthquakes. Nature Geosci 9, 380–383 (2016). 2. Jara, J., Socquet, A., Marsan, D., & Bouchon, M. (2017). Long‐term interactions between intermediate depth and shallow seismicity in North Chile subduction zone. Geophysical Research Letters, 44(18), 9283-9292. 3. Bouchon, M., Marsan, D., Jara, J., Socquet, A., Campillo, M., & Perfettini, H. (2018). Suspected deep interaction and triggering between giant earthquakes in the Chilean subduction zone. Geophysical Research Letters, 45(11), 5454-5460. 4. 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Wang, K., Sun, T., Brown, L., Hino, R., Tomita, F., Kido, M., ... & Fujiwara, T. (2018). Learning from crustal deformation associated with the M9 2011 Tohoku-oki-oki earthquake. Geosphere, 14(2), 552-571. 44. Freed, A. M., Hashima, A., Becker, T. W., Okaya, D. A., Sato, H., & Hatanaka, Y. (2017). Resolving depth-dependent subduction zone viscosity and afterslip from postseismic displacements following the 2011 Tohoku-oki-oki, Japan earthquake. Earth and Planetary Science Letters, 459, 279-290. 13 Acknowledgments: Earthquake data were kindly provided by the Japan Meteorological Agency in cooperation with the Ministry of Education, Culture, Sports, Science and Technology. We acknowledge the help of the National research Institute for Earth science and Disaster Prevention, Tsukuba, for making available their waveform data. The authors would like to thank Cécile Lasserre, Pascal Bernard, Claudio Satriano, Hélène Lyon-Caen and Marianne Métois for insightful discussions. Funding: We acknowledge support from the European Research Council (grant number 681346-REALISM and 865963 DEEP-trigger). Author contributions: BG conceived the study. BG, DM, TB, SD, YR developed the statistical methods, AS and MR analysed the GPS data. All authors participated to the writing of the manuscript. Competing interests: Authors declare no competing interests. Data and materials availability: All data is available in the main text or the supplementary materials. 1) Declustering We perform declustering of the earthquake dataset so to remove aftershocks that would otherwise contaminate the analyzed changes in seismicity rates. This is done based on the method already described in Marsan et al. (2017), which uses an epidemic-type model of seismicity with a spatially heterogeneous background rate. We here provide the details of the method. Earthquake occurrences are modeled as a number of earthquakes per unit time and unit area λ(x, y, t), defined as the sum of two contributions : λ (x , y ,t )=μ (x, y )+ν (x , y ,t) in which ν accounts for triggering by previous earthquakes, and μ is the activity that would occur in the absence of any such interactions, i.e., the background rate, which we here assume to depend on position (x, y) but not on time. Any earthquake with index i occurring at {xi, yi, ti}, with magnitude mi, triggers aftershocks with rate : in which ν accounts for triggering by previous earthquakes, and μ is the activity that would occur in the absence of any such interactions, i.e., the background rate, which we here assume to depend on position (x, y) but not on time. Any earthquake with index i occurring at {xi, yi, ti}, with magnitude mi, triggers aftershocks with rate : ν (x , y ,t )=∑ i/ti< t Ke α mi (t+c−ti) p . γ −1 2π . Li γ −1 ((x−xi) 2+( y −yi) 2+Li 2) γ+1 2 ν (x , y ,t )=∑ i/ti< t Ke α mi (t+c−ti) p . γ −1 2π . Li γ −1 ((x−xi) 2+( y −yi) 2+Li 2) γ+1 2 hence the product of the Omori-Utsu law with a power-law spatial density. Parameter values are fixed for all parameters based on either previous analyses or independent knowledge, except for K which depends on completeness magnitude and the regional level of seismicity, and that therefore cannot be fixed a priori (we describe below how the value of K is determined). Materials and Methods 14 14 14 1) Declustering We set α to 2.0, which is a typical median value when considering previous studies, p = 1 which corresponds to the simple Omori’s law, c = 10−4 days (the exact c value has no impact on our study as long as it is sufficiently less than several minutes), γ = 2.0 which lies in the interval already proposed by Felzer and Brodsky (2006) and Marsan and Lengliné (2010). The aftershock zone radius is defined as Li = 0.2×100.5(mi−2.5) (in km), which has the same 100.5m scaling as in Utsu and Seki (1955) and Van der Elst and Shaw (2015). This radius is 4 times larger than the rupture radius of a 30 bar stress drop earthquake (Eshelby 1957). hence the product of the Omori-Utsu law with a power-law spatial density. Parameter values are fixed for all parameters based on either previous analyses or independent knowledge, except for K which depends on completeness magnitude and the regional level of seismicity, and that therefore cannot be fixed a priori (we describe below how the value of K is determined). We set α to 2.0, which is a typical median value when considering previous studies, p = 1 which corresponds to the simple Omori’s law, c = 10−4 days (the exact c value has no impact on our study as long as it is sufficiently less than several minutes), γ = 2.0 which lies in the interval already proposed by Felzer and Brodsky (2006) and Marsan and Lengliné (2010). The aftershock zone radius is defined as Li = 0.2×100.5(mi−2.5) (in km), which has the same 100.5m scaling as in Utsu and Seki (1955) and Van der Elst and Shaw (2015). This radius is 4 times larger than the rupture radius of a 30 bar stress drop earthquake (Eshelby 1957). The values of ν(xi, yi, ti) / K are computed once and for all, making the rest of the computation fast with an Expectation-Maximization method. The probability that earthquake i is a background 15 15 earthquake is ωi = μ(xi,yi) / λ(xi,yi,ti) . We start by taking arbitrary (but non-zero) values of ωi, e.g., ωi = 0.5, and smooth these values to obtain the a priori background rate: μ (x, y )=∑ i ωie −√(x−xi) 2+(x−xi) 2/l. 1 2 πl 2 where ℓ is a smoothing parameter taken equal to 50 km. 1) Declustering Parameter K is then computed as: K= ∑ i 1−ω1 ∑ i Fi K= ∑ i 1−ω1 ∑ i Fi where Fi = eαmi (ln(te + c − ti) − ln c), with te the ending date of the studied period. This corresponds to the Maximum Likelihood Estimate of K knowing ωi, for p=1 as assumed here. Given μ(x, y) and K, the a posteriori probabilities ωi can then be computed, and the procedure is thus iterated until all values eventually converge to their final estimates. The latter do not depend on the initial, arbitrary choice of ωi. To demonstrate how the declustering affect the data, we show in Figures S1, S2 and S3 the earthquake catalog for all magnitude≥2.5 earthquakes before and after declustering, for two selected periods and (extended) zones. The declustering method yields probabilities of being a background earthquake for each event as the final product. We here randomly draw background earthquakes based on these probabilities – for the purpose of simplifying the visual aspect of the plots. Figure S1 and S3 demonstrate that visually evident aftershock clusters are indeed removed efficiently, which is the case for all aftershock sequences except the remarkable case of the 2011 Tohoku-oki mainshock itself (Figure S2). Here the aftershock sequence is only attenuated but is still very visible in the declustered catalog. This is mainly due to our assumption that aftershock triggering is controlled by the epicentral distance between the mainshock and its aftershocks ; this assumption is clearly not valid for an extended source like a M9 mainshockwhen performing an analysis at the regional scale as we do, hence the poor behavior of the method for this particular mainshock. Figure S4 shows the selected deep earthquake dataset analyzed in this study, before and after declustering. This subset is not processed separately from the rest of the catalog: we decluster the catalog as a whole, and afterward extract the selected (deep) subset out of the overall declustered 16 catalog. While 57 % of all earthquakes are found to be aftershocks, this reduces to 38 % when considering only the deep selected earthquakes. This drop is expected as intermediate and deep activity is less prone to large aftershock triggering (Hainzl et al., 2019 ; Sippl et al., 2019). 1) Declustering Figure E offers a complementary view to Figure S5 by plotting the inter-event time vs date before and after declustering, showing that the aftershock sequences have efficiently been removed. 2) Statistical analysis of the earthquake catalogue Δta 1+ Na Δt b 1+ Nb r Na ( Δtb+r Δ ta) 2+N a+Nb Third statistical analysis: We want to compute the probability for the position of the change point to be at a given time t0. If N events occur at times d = {t1,t2,…,tN} and the rate λ(t) is variable with time, following [Rasmussen, 2018], the probability to observe those events at these times is p (d∨λ (t ))=∏ i=1 N λ (ti)exp(−∫λ (t )dt ) p (d∨λ (t ))=∏ i=1 N λ (ti)exp(−∫λ (t )dt ) where the integral is computed from the start to the end of the time interval under consideration. where the integral is computed from the start to the end of the time interval under consideration. Our model λ(t) = λb if t < to and λ(t) = λa if t > to with to the change point. The probability to have a change in the earthquake rate at t0 given the observations is where the integral is computed from the start to the end of the time interval under consideration. Our model λ(t) = λb if t < to and λ(t) = λa if t > to with to the change point. 2) Statistical analysis of the earthquake catalogue In this study, we make the assumption that the period of interest is divided into 2 periods with durations [tb , ta], representing the period of time before and after the time of change, respectively, and characterized by constant seismicity rates [λb, λa]. In each of these periods, the rate of seismicity is assumed to follow a Poisson process, i.e, the probability to observe N events in a period given the rate λ is respectively, and characterized by constant seismicity rates [λb, λa]. In each of these periods, the rate of seismicity is assumed to follow a Poisson process, i.e, the probability to observe N events in a period given the rate λ is p (N∨λ )= (λ Δt ) N exp (−λ Δt ) N ! p (N∨λ )= (λ Δt ) N exp (−λ Δt ) N ! First statistical analysis : we impose the time of change between the two periods at the Tohoku-oki earthquake occurrence and given observed Na and Nb, and given ta and tb , we look for the values of [λb λa] that maximize p (Nb\|λb) and p (Na\|λa), which yields : λb= Nb/tb and λa= Na/ta. Second statistical analysis: for a given time of change between the two periods, the probability to observe a rate of λb= Nb/tb is : p (λb )= Δt b( λb Δt b) Nbexp (−λb Δtb) Nb! Following Marsan and Wyss (2011), we compute the probability that the rate is increased by more than a given ratio r: P( λa λb >r)=∫ 0 ∞ d λb p ( λb)∫ rλb ∞ d λa p ( λa) The pdf of r is then p (r )=−dP dr . Together with equation (1) this leads to : The pdf of r is then p (r )=−dP dr . Together with equation (1) this leads to : The pdf of r is then p (r )=−dP dr . Together with equation (1) this leads to : p (r )= Δt a 1+Na Δt b 1+Nb Na! Nb! r Na∫ 0 ∞ d λ λ 1+N a+Nbe −λ(Δtb+r Δt a). 17 Under the integral, we recognize the Gamma function Γ (Na+Nb+2) which is the continuous prolongation of the factorial (i.e., n! = Γ (n+1)), therefore p (r )= (Na+Nb+1)! Na! N b! 2) Statistical analysis of the earthquake catalogue 18 Since ( 1 e) Na+Nb is a constant, it does not depend on to, and lumping together all the constants in a new Since ( 1 e) Na+Nb is a constant, it does not depend on to, and lumping together all the constants in a new Since ( 1 e) Na+Nb is a constant, it does not depend on to, and lumping together all the constants in a new factor, we finally obtain p (t o∨d )= k √Nb Na N b N bN a Na Δt b N b+1 Δt a N a+1 p (t o∨d )= k √Nb Na N b N bN a Na Δt b N b+1 Δt a N a+1 used to compute Fig. 3. used to compute Fig. 3. 2) Statistical analysis of the earthquake catalogue The probability to have a change in the earthquake rate at t0 given the observations is p (t o∨d )=∫∫p (t o, λb, λa∨d )d λbd λa t o∨d )=∫∫p (t o, λb, λa∨d )d λbd λa Using Bayes’ Theorem, this becomes : Using Bayes’ Theorem, this becomes : p (t o∨d )=∫∫p (t o, λb, λa) p (d∨λb, λa,t o)d λbd λa=p (t o) p (λb) p ( λa)∫∫p(d∨λb λa,t o)d λbd λa p (t o∨d )=∫∫p (t o, λb, λa) p (d∨λb, λa,t o)d λbd λa=p (t o) p (λb) p ( λa)∫∫p(d∨λb λa,t o)d λbd λa where p (t o) p (λb) p ( λa) is called k in the following hence where p (t o) p (λb) p ( λa) is called k in the following hence where p (t o) p (λb) p ( λa) is called k in the following hence where p (t o) p (λb) p ( λa) is called k in the following hence p (t o∨d )=k∫∫λb Nb λa Naexp(−λb Δtb−λaΔt a)d λbd λa ¿ ¿ We now perform a change of variable where u = λΔt We now perform a change of variable where u = λΔt p (t o∨d )=k 1 Δtb Nb+1∫u N bexp (−u)du. 1 Δ ta Na+1∫u N aexp (−u)du Under the two integrals, we recognize again the Gamma function. Using Stirling’s approximation : Under the two integrals, we recognize again the Gamma function. Using Stirling’s approximation : Nb!=√2 π N b( N b e ) N b we get Nb!=√2 π N b( N b e ) N b we get we get p (t o∨d )=k 1 Δtb Nb+1 Δta Na+1 √2 π N b( N b e ) Nb √2 π N a( N a e ) N a . 3) GPS data processing These GNSS position time series are then corrected from any jumps associated with documented material changes. The change of velocity before and after the Tohoku-oki earthquake are simply calculated as the difference of slope in each time series, between a linear trend before the earthquake from 2008/01/01 to 2011/03/08 and a linear trend after the earthquake from 2011/03/13 to 2013/03/10. 3) GPS data processing The data of the 1439 GEONET stations have been processed in double difference using GAMIT/GLOBK software [Herring et al., 2018]. For each day, the GEONET data were split into sub-networks of about 40 sites chosen to minimize the baseline between stations and improve the resolution of phase ambiguity. Subnetworks share 2 common sites with nearby other sub-networks to “tie” the solutions together, following a similar approach than the one presented in the framework of the PBO project Herring et al. [2016]. A reference tie network containing at least 1 station from every other sub-network is also constructed to provide additional stability to the entire network combination. For each sub-network, we reduce 24-hr measurement sessions to daily estimates of station position, choosing the ionosphere-free combination and fixing the ambiguities to integer values. We use IGS final products for the satellite orbits, satellites clocks and Earth orientation parameters (https://www.igs.org/products). Following [Herring et al., 2016], the orbit parameters are fixed to the IGS values. Ocean loading corrections are applied at each stations, using FES2004 (Finite Element Solution) [Lyard et al., 2021] ocean tidal loading. The Vienna Mapping Function (VMF1) [Bohm et al., 2006] is used to map the tropospheric delay in zenithal direction. The zenith delay is estimated every 2-hours and 1 gradient parameter is estimated per day. We apply atmospheric tidal and non-tidal loading correction, following Tregoning and van Dam [2005] 19 recommendation. The different sub-networks are then combined together into a single daily solution, in a regional stabilization approach using a Kalman filter with GLOBK software to obtain daily loosely constrained solutions. Then daily solutions are combined into a multiyear solution to derive the time series, and to express the solutions in the ITRF2014 [Altamimi et al., 2017] with a 7-parameter transformation using regional IGS sites. The daily GNSS position time series [gnss products, 2019] are available at recommendation. The different sub-networks are then combined together into a single daily solution, in a regional stabilization approach using a Kalman filter with GLOBK software to obtain daily loosely constrained solutions. Then daily solutions are combined into a multiyear solution to derive the time series, and to express the solutions in the ITRF2014 [Altamimi et al., 2017] with a 7-parameter transformation using regional IGS sites. The daily GNSS position time series [gnss products, 2019] are available at at are are https://doi.osug.fr/staging/GNSS_products/GNSS.products.Japan.html. Herring, T. A., T. I. Melbourne, M. H. Murray, M. A. Floyd, W. M. Szeliga, R. W. King, D. A. Phillips, C. M. Puskas, M. Santillan, and L. Wang (2016), Plate Boundary Observatory and related networks: GPS data analysis methods and geodetic products, Reviews of Geophysics, 54, 759–808, doi:10.1002/2016RG000529. References Altamimi, Z., Métivier, L., Rebischung, P., Rouby, H., & Collilieux, X. (2017). ITRF2014 plate motion model. Geophysical Journal International, 209(3), 1906-1912. Altamimi, Z., Métivier, L., Rebischung, P., Rouby, H., & Collilieux, X. (2017). ITRF2014 plate motion model. Geophysical Journal International, 209(3), 1906-1912. Böhm, J., Niell, A., Tregoning, P., & Schuh, H. (2006). Global Mapping Function (GMF): A new empirical mapping function based on numerical weather model data. Geophysical research letters, 33(7). Eshelby, J. D. (1957). The determination of the elastic field of an ellipsoidal inclusion, and related problems. Proceedings of the royal society of London. Series A. Mathematical and physical sciences, 241(1226), 376-396. Felzer, K. R., & Brodsky, E. E. (2006). Decay of aftershock density with distance indicates triggering by dynamic stress. Nature, 441(7094), 735-738. gnss products. (2019). Gnss position solutions in japan. ”CNRS, OSUG, ISTERRE”. https://doi.osug.fr/staging/GNSS_products/GNSS.products.Japan.html Hainzl S., C. Sippl, B. Schurr, 2019. Linear Relationship Between Aftershock Productivity and Seismic Coupling in the Northern Chile Subduction Zone, J. Geophys. Res., 124, 8726-8738 Herring, T. A., T. I. Melbourne, M. H. Murray, M. A. Floyd, W. M. Szeliga, R. W. King, D. A. Phillips, C. M. Puskas, M. Santillan, and L. Wang (2016), Plate Boundary Observatory and related networks: GPS data analysis methods and geodetic products, Reviews of Geophysics, 54, 759–808, doi:10.1002/2016RG000529. 20 Herring, T. A., R. W. King, M. A. Floyd, and S. C. McClusky (2018b), Introduction to GAMIT/GLOBK : Release 10.7, Tech. rep., Massachusetts Institute of Technology. Lyard, F. H., Allain, D. J., Cancet, M., Carrère, L., & Picot, N. (2021). FES2014 global ocean tide atlas: design and performance. Ocean Science, 17(3), 615-649. Marsan, D., & Lengliné, O. (2010). A new estimation of the decay of aftershock density with distance to the mainshock. Journal of Geophysical Research: Solid Earth, 115(B9). Marsan, D. & Wyss, M. Seismicity rate changes. (2011) doi:10.5078/CORSSA-25837590. Marsan, D. et al. Change in seismicity along the Japan trench, 1990-2011, and its relationship with seismic coupling: CHANGES IN SEISMICITY, JAPAN TRENCH. J. Geophys. Res. Solid Earth 122, 4645–4659 (2017). Rasmussen, J. G. (2018). Lecture notes: Temporal point processes and the conditional intensity function. arXiv preprint arXiv:1806.00221. Sippl C., B. Schurr, T. John, S. Hainzl, 2019. Filling the gap in a double seismic zone: Intraslab seismicity in Northern Chile, Lithos, 346, 10.1016/j.lithos.2019.105155 Tregoning, P., & van Dam, T. (2005). Atmospheric pressure loading corrections applied to GPS data at the observation level. Geophysical Research Letters, 32(22). References Utsu, T., & Seki, A. (1955). A relation between the area of the aftershock region and the radius of the sensibility circle. Zisin, 3(34), 1955. van der Elst, N. J., & Shaw, B. E. (2015). Larger aftershocks happen farther away: Nonseparability of magnitude and spatial distributions of aftershocks. Geophysical Research Letters, 42(14), 5771- 5778.+ 21 Fig. 1. Relative change in the rate of intermediate and deep seismicity of the Pacific plate taking the Tohoku-oki earthquake as time of change. The data span a) from 2000/03/11 (yy/mm/day) to 2013/03/11; b) from 2000/03/11 to 2017/03/11 period, using the 260 closest events to sample the same area c) from 2000/03/11 to 2021/03/11 period using the 320 closest events to sample the same area. Negative values (blue) indicate a decrease of seismicity rate, positive values (red) indicate an increase of seismicity rate. Every 20km isodepth contours of the Pacific plate are shown from 80 to 400 km34. Black arrows give the log scaled GPS post-seismic displacement. The orange and grey areas show the contours of the co and post-seismic slips12. Fig. 1. Relative change in the rate of intermediate and deep seismicity of the Pacific plate taking the Tohoku-oki earthquake as time of change. The data span a) from 2000/03/11 (yy/mm/day) to 2013/03/11; b) from 2000/03/11 to 2017/03/11 period, using the 260 closest events to sample the same area c) from 2000/03/11 to 2021/03/11 period using the 320 closest events to sample the same area. Negative values (blue) indicate a decrease of seismicity rate, positive values (red) indicate an increase of seismicity rate. Every 20km isodepth contours of the Pacific plate are shown from 80 to 400 km34. Black arrows give the log scaled GPS post-seismic displacement. The orange and grey areas show the contours of the co and post-seismic slips12. Fig. 1. Relative change in the rate of intermediate and deep seismicity of the Pacific plate taking the Tohoku-oki earthquake as time of change The data span a) from 2000/03/11 (yy/mm/day) to Fig. 1. Relative change in the rate of intermediate and deep seismicity of the Pacific plate taking the Tohoku-oki earthquake as time of change. The data span a) from 2000/03/11 (yy/mm/day) to 2013/03/11; b) from 2000/03/11 to 2017/03/11 period, using the 260 closest events to sample the same area c) from 2000/03/11 to 2021/03/11 period using the 320 closest events to sample the same area. References Negative values (blue) indicate a decrease of seismicity rate, positive values (red) indicate an increase of seismicity rate. Every 20km isodepth contours of the Pacific plate are shown from 80 to 400 km34. Black arrows give the log scaled GPS post-seismic displacement. The orange and grey areas show the contours of the co and post-seismic slips12. 22 Fig. 2. Change of seismicity rate near latitudes 34°N and 44°N. a) Location of the earthquakes color coded with depth. They correspond to the 200 closest earthquakes of the two black stars. b) Cumulative number of earthquakes with time after declustering normalized at Tohoku-oki, for 34°N (blue) and 44°N latitude (red). Note the change of slope at the time of the Tohoku-oki earthquake, with λb the slope before and λa, the slope after the megathrust earthquake. c) probability density function (pdf) of the rate change (see Methods). A rate change r < 1, =1, >1, means decrease, no change and increase, respectively. The probability of change at Tohoku-oki is maximum with rmax =0.47 at 34°N (blue) and rmax=1.3 at 44°N latitude (red). ) b) c) b) c) a) a) Fig. 2. Change of seismicity rate near latitudes 34°N and 44°N. a) Location of the earthquakes color coded with depth. They correspond to the 200 closest earthquakes of the two black stars. b) Cumulative number of earthquakes with time after declustering normalized at Tohoku-oki, for 34°N (blue) and 44°N latitude (red). Note the change of slope at the time of the Tohoku-oki earthquake, with λb the slope before and λa, the slope after the megathrust earthquake. c) probability density function (pdf) of the rate change (see Methods). A rate change r < 1, =1, >1, means decrease, no change and increase, respectively. The probability of change at Tohoku-oki is maximum with rmax =0.47 at 34°N (blue) and rmax=1.3 at 44°N latitude (red). 23 23 Fig. 3. Probability of having a seismicity rate change at any given time, stacked over all the reference points. a) Stack of all the data with or without points with latitude < 28°. b) Probability at each reference point plotted according to its latitude (see Methods). a)a) b) a)a) b) a)a) a)a) b) Fig. 3. Probability of having a seismicity rate change at any given time, stacked over all the reference points. a) Stack of all the data with or without points with latitude < 28°. References b) Probability at each reference point plotted according to its latitude (see Methods). Fig. 3. Probability of having a seismicity rate change at any given time, stacked over all the reference points. a) Stack of all the data with or without points with latitude < 28°. b) Probability at each reference point plotted according to its latitude (see Methods). 24 Fig. 4. a) Focal mechanisms of intermediate and deep earthquakes (source from NIED https://www.fnet.bosai.go.jp/). The orange and yellow stars locate the points where we compute the Coulomb stress change of b) and c). b) Blue: Coulomb stress change computed with COULOMB3 on a point located underneath Hokkaido, on the slab (lon=140.8°, lat=44.25°, depth=-204km, strike=250°, dip=26.7°, orange star in Fig. 4a) for different rake values, Orange: histogram of the rake values of all the earthquakes with latitude > 42°, c) Blue: same as b) for a point underneath 34°N latitude on the slab (lon=138.4°, lat=34.70°, depth=-181km, strike=161°, dip=39°, yellow star in Fig. 4a); Orange : same as b) for earthquakes with latitude < 35°. Here we assume a 0.4 friction coefficient. Fig. 4. a) Focal mechanisms of intermediate and deep earthquakes (source from NIED https://www.fnet.bosai.go.jp/). The orange and yellow stars locate the points where we compute the Coulomb stress change of b) and c). b) Blue: Coulomb stress change computed with COULOMB3 on a point located underneath Hokkaido, on the slab (lon=140.8°, lat=44.25°, depth=-204km, strike=250°, dip=26.7°, orange star in Fig. 4a) for different rake values, Orange: histogram of the rake values of all the earthquakes with latitude > 42°, c) Blue: same as b) for a point underneath 34°N latitude on the slab (lon=138.4°, lat=34.70°, depth=-181km, strike=161°, dip=39°, yellow star in Fig. 4a); Orange : same as b) for earthquakes with latitude < 35°. Here we assume a 0.4 friction coefficient. Fig. 4. a) Focal mechanisms of intermediate and deep earthquakes (source from NIED https://www.fnet.bosai.go.jp/). The orange and yellow stars locate the points where we compute the Coulomb stress change of b) and c). b) Blue: Coulomb stress change computed with COULOMB3 on a point located underneath Hokkaido, on the slab (lon=140.8°, lat=44.25°, depth=-204km, strike=250°, dip=26.7°, orange star in Fig. Fig. S2. Same as Fig. S1, for a time period after the 2011 Tohoku-oki-Oki megathrust earthquake. References 4a) for different rake values, Orange: histogram of the rake values of all the earthquakes with latitude > 42°, c) Blue: same as b) for a point underneath 34°N latitude on the slab (lon=138.4°, lat=34.70°, depth=-181km, strike=161°, dip=39°, yellow star in Fig. 4a); Orange : same as b) for earthquakes with latitude < 35°. Here we assume a 0.4 friction coefficient. 25 Fig. S1: (top) all m≥2.5 earthquakes in the JMA catalog, for the selected time period and latitude interval. The circle radius grows with the magnitude. Aftershock sequences are readily visible as clusters initiated by large shocks. (Bottom) same as top graph after declustering. The aftershock sequences are all removed. 26 Fig. S2. Same as Fig. S1, for a time period after the 2011 Tohoku-oki-Oki megathrust earthquake. 27 27 Fig. S3. Deep earthquakes selected in this study, before (top) / after (bottom) declustering Fig. S3. Deep earthquakes selected in this study, before (top) / after (bottom) declustering. 28 g. S4. Time between two successive earthquakes for the selected deep earthquakes, before (top d after (bottom) declustering. The vertical ‘streaks’ corresponding to aftershock sequences (e.g 2001, 2002, 2004) are removed by the declustering. Fig. S4. Time between two successive earthquakes for the selected deep earthquakes, before (top) and after (bottom) declustering. The vertical ‘streaks’ corresponding to aftershock sequences (e.g., in 2001, 2002, 2004) are removed by the declustering. 29 Fig. S5. Comparison of the relative seismicity rate change (in %) obtained before (left) and after (right) declustering. We see the dichotomy between North and South. After declustering, the decrease of the seismicity rate is less important in the South and the increase is more important in the North. The declustering method allows to unbias the seismicity rate change, especially at latitudes 36 and 40°. Note that we remove the area close to the Tohoku-oki and the Mw8.0 2003 Tokachi-oki rupture zones since the seismicity of the region is dominated by aftershocks even after declustering due to the very intense seismic activity (see Fig. S2). Fig. S5. Comparison of the relative seismicity rate change (in %) obtained before (left) and after (right) declustering. We see the dichotomy between North and South. After declustering, the decrease of the seismicity rate is less important in the South and the increase is more important in the North. The declustering method allows to unbias the seismicity rate change, especially at latitudes 36 and 40°. References Note that we remove the area close to the Tohoku-oki and the Mw8.0 2003 Tokachi-oki rupture zones since the seismicity of the region is dominated by aftershocks even after declustering due to the very intense seismic activity (see Fig. S2). Fig. S5. Comparison of the relative seismicity rate change (in %) obtained before (left) and after (right) declustering. We see the dichotomy between North and South. After declustering, the decrease of the seismicity rate is less important in the South and the increase is more important in the North. The declustering method allows to unbias the seismicity rate change, especially at latitudes 36 and 40°. Note that we remove the area close to the Tohoku-oki and the Mw8.0 2003 Tokachi-oki rupture zones since the seismicity of the region is dominated by aftershocks even after declustering due to the very intense seismic activity (see Fig. S2). 30 Fig. S6: Map of Japan showing the location of earthquakes studied in this paper selected with the method described in the main text (gray dots) and location of the reference points color coded with the maximum distance between the point and the farthest earthquake; b) Stack of the time series for all the reference points located at lat > 42°N earthquakes with time after selection and declustering, c) same as b for 35°N < lat < 42°N earthquakes. d) same as b for lat < 35°N earthquakes. Note that the total number of selected earthquakes is smaller for the Japan Sea than underneath Hokkaido or in the south. There is no significant change of seismic rate in this area while there is a clear increase and decrease in the North and South, respectively. Fig. S6: Map of Japan showing the location of earthquakes studied in this paper selected with the method described in the main text (gray dots) and location of the reference points color coded with the maximum distance between the point and the farthest earthquake; b) Stack of the time series for all the reference points located at lat > 42°N earthquakes with time after selection and declustering, c) same as b for 35°N < lat < 42°N earthquakes. d) same as b for lat < 35°N earthquakes. Note that the total number of selected earthquakes is smaller for the Japan Sea than underneath Hokkaido or in the south. References There is no significant change of seismic rate in this area while there is a clear increase and decrease in the North and South, respectively. 31 Fig. S7. Probability density function of the earthquake rate p(λ), in 1/day, observed in a) Hokkaido (44°N latitude), b) 34°N latitude and c) Izu-Bonin area : before, i.e. λb (2000/3/11 – 2011/3/10, blue) and after, i.e. λa (2011/3/11 – 2013/3/11, pink) the Tohoku-oki earthquake. Note that with more earthquakes before the distribution of the λ is sharper before than after the Tohoku-oki earthquake. a) b) c) a) b) c) Fig. S7. Probability density function of the earthquake rate p(λ), in 1/day, observed in a) Hokkaido (44°N latitude), b) 34°N latitude and c) Izu-Bonin area : before, i.e. λb (2000/3/11 – 2011/3/10, blue) and after, i.e. λa (2011/3/11 – 2013/3/11, pink) the Tohoku-oki earthquake. Note that with more earthquakes before the distribution of the λ is sharper before than after the Tohoku-oki earthquake. 32 32 Fig. S8. Analysis of the uncertainty on the maximum of the pdf of r. To show the uncertainty on rmax, i.e. the r value that maximizes f(r) (middle), we show the r-values rmax-σ (left) and rmax+σ (right) with the same colorbar for comparison. We see that even after removing 1σ to the maximum, there is still an increase of the seismicity rate underneath Hokkaido. On the contrary, there is still a decrease of seismicity rate at latitude 34° when adding σ. Note that the data in the middle is the same as in Fig. 1 with the difference that the scale is here not in percentage. Fig. S8. Analysis of the uncertainty on the maximum of the pdf of r. To show the uncertainty on Fig. S8. Analysis of the uncertainty on the maximum of the pdf of r. To show the uncertainty on rmax, i.e. the r value that maximizes f(r) (middle), we show the r-values rmax-σ (left) and rmax+σ (right) with the same colorbar for comparison. We see that even after removing 1σ to the maximum, there is still an increase of the seismicity rate underneath Hokkaido. On the contrary, there is still a decrease of seismicity rate at latitude 34° when adding σ. Note that the data in the middle is the same as in Fig. 1 with the difference that the scale is here not in percentage. Fig. S8. References Analysis of the uncertainty on the maximum of the pdf of r. To show the uncertainty on rmax, i.e. the r value that maximizes f(r) (middle), we show the r-values rmax-σ (left) and rmax+σ (right) with the same colorbar for comparison. We see that even after removing 1σ to the maximum, there is still an increase of the seismicity rate underneath Hokkaido. On the contrary, there is still a decrease of seismicity rate at latitude 34° when adding σ. Note that the data in the middle is the same as in Fig. 1 with the difference that the scale is here not in percentage. 33 33 9 Pdf of the seismicity rate change at each time plot computed for different times of c neath Hokkaido. a) cumulative number of earthquakes with time, b) pdf of r, c) pdf of nt times of change. The example shows that in 2006 (blue line), the increase of the seism ue to the Tohoku-oki earthquake is in the post-period (between 2006/3/11 and 2013/3/ value that maximizes the pdf (blue line) is slightly above 1 Choosing Tohoku-oki as the a) b) c) a) a) b) Fig. S9 Pdf of the seismicity rate change at each time plot computed for different times of change underneath Hokkaido. a) cumulative number of earthquakes with time, b) pdf of r, c) pdf of r for different times of change. The example shows that in 2006 (blue line), the increase of the seismicity rate due to the Tohoku-oki earthquake is in the post-period (between 2006/3/11 and 2013/3/11) so the r-value that maximizes the pdf (blue line) is slightly above 1. Choosing Tohoku-oki as the time of change, the pdf maximum is obtained for rmax=1.4. Choosing a time of change in 2012 gives a r < 1 because a part of the increase is in the pre-period (between Tohoku-oki and 2012). We see that r >1 before Tohoku-oki followed by a significative change of slope at the time of Tohoku-oki when r decreases after the megathrust earthquake. a) b) c) a) c) a) Fig. S9 Pdf of the seismicity rate change at each time plot computed for different times of change underneath Hokkaido. a) cumulative number of earthquakes with time, b) pdf of r, c) pdf of r for different times of change. References The example shows that in 2006 (blue line), the increase of the seismicity rate due to the Tohoku-oki earthquake is in the post-period (between 2006/3/11 and 2013/3/11) so the r-value that maximizes the pdf (blue line) is slightly above 1. Choosing Tohoku-oki as the time of change, the pdf maximum is obtained for rmax=1.4. Choosing a time of change in 2012 gives a r < 1 because a part of the increase is in the pre-period (between Tohoku-oki and 2012). We see that r >1 before Tohoku-oki followed by a significative change of slope at the time of Tohoku-oki when r decreases after the megathrust earthquake. Fig. S9 Pdf of the seismicity rate change at each time plot computed for different times of change underneath Hokkaido. a) cumulative number of earthquakes with time, b) pdf of r, c) pdf of r for different times of change. The example shows that in 2006 (blue line), the increase of the seismicity rate due to the Tohoku-oki earthquake is in the post-period (between 2006/3/11 and 2013/3/11) so the r-value that maximizes the pdf (blue line) is slightly above 1. Choosing Tohoku-oki as the time of change, the pdf maximum is obtained for rmax=1.4. Choosing a time of change in 2012 gives a r < 1 because a part of the increase is in the pre-period (between Tohoku-oki and 2012). We see that r >1 before Tohoku-oki followed by a significative change of slope at the time of Tohoku-oki when r decreases after the megathrust earthquake. 34 Fig. S10: pdf of the seismicity rate change at each time for 34°N latitude (left) and 44°N latitude (right) with the black line showing rmax. A wider pdf means that the level of change is less constrained, as it is the case when one of the 2 periods contains a lower number of events. Fig. S10: pdf of the seismicity rate change at each time for 34°N latitude (left) and 44°N latitude (right) with the black line showing rmax. A wider pdf means that the level of change is less constrained, as it is the case when one of the 2 periods contains a lower number of events. 35 35 Fig. S11. Location and pdf of the seismicity rate change at each time of the clusters using the k- means clustering on the rmax value at the time of the Tohoku-oki earthquake. References The number of points present in each cluster is given in brackets. b-e) the black line shows rmax and the red line shows the Tohoku-oki earthquake occurrence time. Note that points in the northern and southern tips belong to clusters 2 and 4 which undergo less effect of the Tohoku-oki earthquake indicating the spatial limit of the Tohoku-oki effect. Fig. S11. Location and pdf of the seismicity rate change at each time of the clusters using the k- Fig. S11. Location and pdf of the seismicity rate change at each time of the clusters using the k- means clustering on the rmax value at the time of the Tohoku-oki earthquake. The number of points present in each cluster is given in brackets. b-e) the black line shows rmax and the red line shows the Tohoku-oki earthquake occurrence time. Note that points in the northern and southern tips belong to clusters 2 and 4 which undergo less effect of the Tohoku-oki earthquake indicating the spatial limit of the Tohoku-oki effect. Fig. S11. Location and pdf of the seismicity rate change at each time of the clusters using the k- means clustering on the rmax value at the time of the Tohoku-oki earthquake. The number of points present in each cluster is given in brackets. b-e) the black line shows rmax and the red line shows the Tohoku-oki earthquake occurrence time. Note that points in the northern and southern tips belong to clusters 2 and 4 which undergo less effect of the Tohoku-oki earthquake indicating the spatial limit of the Tohoku-oki effect. 36 36 Fig. S12. Analysis of the seismicity from the JMA catalog at three different reference points : Hokkaido, Chubu and the Izu-Bonin. We use M≥4.5 earthquakes and the 70 closest events at each reference point and show the location (left), cumulative number (middle), probability of a change in seismicity rate (right). The acceleration and deceleration of the seismicity rate undernearth Hokkaido and Chubu, respectively, is clear while the deceleration at the Izu-Bonin is less robust. Fig. S12. Analysis of the seismicity from the JMA catalog at three different reference points : Hokkaido, Chubu and the Izu-Bonin. We use M≥4.5 earthquakes and the 70 closest events at each reference point and show the location (left), cumulative number (middle), probability of a change in seismicity rate (right). References The acceleration and deceleration of the seismicity rate undernearth Hokkaido and Chubu, respectively, is clear while the deceleration at the Izu-Bonin is less robust. 37 Fig. S13. Analysis of the seismicity from the USGS catalog at two different points : Hokkaido, Chubu and the Izu-Bonin. We select only M≥4.5 earthquakes and the 70 closest earthquakes at ea S13. Analysis of the seismicity from the USGS catalog at two different points : Hokkaid Chubu and the Izu-Bonin. We select only M≥4.5 earthquakes and the 70 closest earthquakes at each reference point and show the location (left), cumulative number (middle), probability of a change in seismicity rate (right). The acceleration and deceleration of the seismicity rate undernearth Hokkaido is clear while the deceleration underneath Chubu is less robust. Chubu and the Izu-Bonin. We select only M≥4.5 earthquakes and the 70 closest earthquakes at each reference point and show the location (left), cumulative number (middle), probability of a change in seismicity rate (right). The acceleration and deceleration of the seismicity rate undernearth Hokkaido is clear while the deceleration underneath Chubu is less robust. 38 38
https://openalex.org/W4298311726	https://www.biodiversitylibrary.org/itempdf/116539	English	null	The birds of Texas; an annotated check-list,	null	1,912	public-domain	21,360	The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. ALBERT R. MANN LIBRARY NEw YorRK STATE COLLEGES OF AGRICULTURE AND HoME ECONOMICS AT CORNELL UNIVERSITY NEw YorRK STATE COLLEGES OF AGRICULTURE AND HoME ECONOMICS The original of this book is in the Cornell University Library. The original of this book is in the Cornell University Library. GX GUY 7T¢ Sua 361667 GX GUY 7T¢ Sua 67 10 TVA SD Facet TS Tex Bayley CORNELL UNIVERSITY THE F Flower Heterinary Library a i FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 356 10 TVA SD Facet TS Tex Bayley CORNELL UNIVERSITY THE F Flower Heterinary Library a i FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 356 INTRODUCTION To the true sportsman, lover of the woods and fields, who BAYLOR UNIVERSITY BULLETIN 4 believes in obeying the laws, not only those of the State but also those of common decency and humanity and kills within the limit and then only such birds as can be considered came, I am willing to take off my hat and call brother. But for the man (?) who considers Robins, Larks and Mockingbirds fair game, I have absolutely no re- spect. ; ae believes in obeying the laws, not only those of the State but also those of common decency and humanity and kills within the limit and then only such birds as can be considered came, I am willing to take off my hat and call brother. But for the man (?) who considers Robins, Larks and Mockingbirds fair game, I have absolutely no re- spect. ; ae suffer for their sin of omission in not shot- The Agriculturists are beginning to suffer for their sin of omission in not shot- gunning the lunk-headed louts who formerly overran their fields and wooded pas- tures, slaughtering for the sake of killing, every living wild thing in sight, and then chucking the contents of their bag in the first trash box on their return home. In my day I was too late to witness the great flights of wild pigeons; but I can _remember a time when plume birds and other water fowl were myriad in the grassy lagoons of Eastern and Southern Texas, and many of these are soon to be numbered -with the Ivory-billed Woodpecker and Carolina Paroquet as birds which have recent- ly become extinct! The agency? Man’s inhumanity to man and every other mov- ing, living, breathing thing on earth. Of what benefit is a beautiful Heron, a curious Snake Bird, an ungainly Pelican?—I am asked. Of what benefit is a flower—a weeping willow—a tiny cascade—a rugged hill? Merely something natural for us . to gaze upon and reflect on the greatness and goodness of Him who created every- thing and on the insignificance of man who created nothing but selfishness and van- dalism and whose principal aim seems to be to reduce to dust, every beautiful thing before its time. As this is merely a preliminary list, I have made no attempt to append a com- plete bibliography. INTRODUCTION In compiling the following check-list of Texas birds, the writer has tried to be conservative and has only included those species which he was satisfied from authentic published record or personal knowledge, were inhabitants of the State. He regrets that the members of the United States Biological Survey were not permitted by the State authorities to continue their investigations, as they would in all prob- ability have added much to our knowledge concerning the birds of Western Texas. The writer, although he is connected with one of the largest educational institutions in Texas, is not permitted to take birds for scientific purposes; yet it is a fact that during the three years prior to the énactment of the present law, he collected only eleven specimens, six of which were migrating waterbirds, for the shooting of which he would not at any time have been liable for a fine. At the same time, out-of-State correspondents write that they constantly receive birds and eggs from Southern Texas and wonder why he has discontinued his investigations. The chances are that the most of these “Southern Texas birds” were collected along the Valley of the Lower Rio Grande in Northern Mexico and labeled up to suit the occasion. Such things have happened! I had the privilege of meeting one of the Biological Survey collectors—Mr. Arthur Howell, a gentleman in every sense the word implies and a true lover of Na- ture. He is an investigator, not a bird murderer and all of the specimens taken by him were for the purpose of studying their food habits in connection with the bolt weevil investigation and the whole number collected during his Texas stay would not amount to as much in the sum total as the vandal-like work of the aver- age small boy during the summer season. The birds of Texas, or at least the majority of them, are on the rapid road to extermination, the result of the clearing up of waste lands, the cutting down of pine forests, the draining of swamps and lagoons, and the brutality of those inhuman beasts in the guise of men who annually get the “blood lust” and go forth to slay for gain or so-called sport. INTRODUCTION I have consulted every book or paper containing a reference to _a Texas bird that was in any way accessible to me. . In its preparation I have made liberal use of the ranges of many species as ontlined in Mr. Ridgway’s monumental work on the Birds of North and Middle America. (Parts I-II-III-IV.) In the case of species recorded from the trans-Pecos country, I have as a rule used the notes of Oberholser and Bailey in preference to my own, as they were first on the ground. In most instances I have followed the arrangement used in the A. O. U. Check-List as to family divisions but in the case of the Passerine birds I have faithfully followed Ridgway as I consider him the greatest living authority on our Avi-Fauna. Iam a little doubtful of the status of some of the recently described subspecies, but if the splitters (and they are in the majority at the present time) are willing to accept these as valid races, I must bow in deference to their superior dis- crimination. For a description of the physical characteristics of. the different regions of the State, I will refer the reader to Mr. Vernon Bailey’s excellent report on the Biological THE BIRDS OF TEXAS 5 Survey of Texas (North American Fauna, No. 25, 1905, published by the Bureau of Biological Survey, Department of Agriculture, Washington, D. C.). By far the best physical map that I have seen is one by Prof. Robert Hill. It gives a splendid idea of the location and extent of the Grand and Coast Prairies and the other divi- sions in Eastern and Central Texas and indicates the line of the Staked Plains in Western Texas with marvelous accuracy. Published by the United States Geological Survey. W. W. Cooke’s “Report on Bird Migration in the Mississippi Valley,” published by the U. S. Department of Agriculture in 1888, and the same author's recent report on “The Distribution and Migration of North American Warblers,” were invaluable to me, as they aided me greatly in working out the status of many species and en- abled me to divide them into migrants and winter residents. The only possible ob- jection to the Warbler report is that it includes mention of some of Giraud’s “Texas” (?) species which should have long ago been excluded from the A. O. U. Check-List. INTRODUCTION Davie’s “Nests and Eggs of North American Birds” (fifth edition) and Bendire’s “Life Histories of North American Birds,” furnished breeding records not found in other sources. Mrs. Florence Merriam Bailey’s “Handbook of the Birds of the Western United States,” one of the best general works on the birds of the western region ever pub- lished, was consulted frequently, as were also the works of Elliot, Coues, Baird, Cassin and many others. The following papers furnished the majority of the local records aside from the author’s manuscript notes: Attwater, H. P. List of Birds Observed in the Vicinity of San Antonio, Bexar Attwater, H. P. List of Birds Observed in the Vicinity of San Antonio, Bexar County, Texas. (The Auk, IX, 1893, pp. 229-238 and 337-345.) Attwater, H. P. List of Birds Observed in the Vicinity of San Antonio, Bexar County, Texas. (The Auk, IX, 1893, pp. 229-238 and 337-345.) Beckham, C. W. Observations on the Birds of Southwestern Texas. (Proc. U. S. Nat. Mus., 1887, pp. 633-697.) : Beckham, C. W. Observations on the Birds of Southwestern Texas. (Proc. U. S. Nat. Mus., 1887, pp. 633-697.) : Beyer, Geo. E. The Avi-Fauna of Louisiana. (1889. Author’s separate.) Brown, N.C. A Reconnaissance in Southwestern Texas. (Bull. Nutt. Ornith. Club, VII, 1882, 33-42.) A Second Season in Texas. (The Auk, I, 1884, pp. 120-124). Brown, N.C. A Reconnaissance in Southwestern Texas. (Bull. Nutt. Ornith. Club, VII, 1882, 33-42.) A Second Season in Texas. (The Auk, I, 1884, pp. 120-124). Butcher, H. B. List of Birds Collected at Laredo. (Proc. Acad. Nat. Sci. Phila., Vol. XX, 1868, pp. 148-150). Butcher, H. B. List of Birds Collected at Laredo. (Proc. Acad. Nat. Sci. Phila., Vol. XX, 1868, pp. 148-150). Carroll, J. J. Notes on the Birds of Refugio County, Texas. (The Auk, XVII, 1900, pp. 337-348). BAYLOR UNIVERSITY BULLETIN 6 Chapman, F. M. On the Birds Observed near Corpus Christi, Texas, during parts of March and April, 1901. (Bull. Am. Mus. Nat. Hist., III, No. 2, 1891, pp. 315-328). Donald J. A. and More, R. L. A List of the Birds of Wise County, Texas. (The Naturalist, Austin, Texas, I, No’s. 3-4-5-6-8). Dresser, H. E. Notes on the Birds of Southern Texas. (Ibis, 1865, pp. 312-330; 466-495; 1866, pp. 23-46). Hancock, J. L. Notes and Observations on the Ornithology of Corpus Christi and Vicinity, Texas. (Bull. Ridgway Orn. Club, 1887, 2, pp. 11-23). INTRODUCTION Lloyd, William. Birds of Tom Green and Concho Counties, Texas. (The Auk, Lloyd, William. Birds of Tom Green and Concho Counties, Texas. (The Auk, IV, 1884). Merrill, J.C. Notes on the Ornithology of Southern Texas, being a List of Birds Observed in the Vicinity of Fort Brown, from February, 1876, to June 1878. (Proc. U.S. Nat. Mus., I, 1878, pp, 118-173). Oberholser, H.C. Some Notes from Western Texas. (The Auk, XIX, 1902, pp. 300-301). Sennett, G. B. Notes on the Ornithology of the Lower Rio Grande, from Obser- vations made in 1877. (Bull. U. S. Geol. Surv. Terr., IV, No. 1, 1878, pp. 1-66). Further Notes on the Ornithology of the Lower Rio Grande, from Observations made in the Spring of 1878. (Bull. U. S. Geol. Surv. V, 1879, No. 3, pp. 371-440). Singley, J. A. List of Birds Observed in Lee County, Texas. Notes on the Birds of Galveston Island. List of Birds Observed at Corpus Christi and on the Lower Rio Grande. (Report Texas Geological Survey). Also manuscript notes supplementing the Lee county paper, and many papers by Brewster, Sennett, Brown, Ragsdale and others. The writer has been a resident of Waco, Texas, since the fall of 1889, and be- lieves that he has a pretty general knowledge of the Avi-Fauna of the central sec- tion of the State, including the counties of McLennan, Bosque, Limestone, Falls, Hill, Robertson, Williamson, Burnet, Travis, Llano, and others in the district. He has col- lected material in Bee, Refugio and Matagorda counties in the southeastern coast district, in Midland on the southern plains, in Jeff Davis, Presidio and Brewster in the mountains of trans-Pecos Texas, in Armstrong and Potter in the Panhandle, and in scattered localities in other sections. The hypothetical list appended is short and deals with species sometimes re- ferred to as Texan birds but of which up to the present time the writer has no evi- THE BIRDS OF TEXAS 7 dence to show that they are really entitled to a place here. Such species as Giraud’s Flycatcher, the Red, Brasher’s and Bell's Warblers and others described by the same authority have until late years been considered stragglers to Texas but more recent explorations serve to show that many of them do not even occupy intervening territory between their natural habitats and our southern boundary. ORDER Pygopodes. Diving Birds Colymbus auritus. Horned Grebe. Gulf coast in winter (November to the latter part of April). - Colymbus nigricollis californicus. American Eared Grebe. - Colymbus nigricollis californicus. American Eared Grebe. Summer resident in the western half of the State. Recorded as breeding in Wise county by Donald and More. Southeastward in winter to the coast. Colymbus dominicus brachypterus. Least Grebe. Colymbus dominicus brachypterus. Least Grebe. Lower Rio Grande (Cameron and Hidalgo counties) resident. Podilymbus podiceps. Pied-billed Grebe. Colymbus dominicus brachypterus. Least Grebe. Lower Rio Grande (Cameron and Hidal Colymbus dominicus brachypterus. Least Grebe. Lower Rio Grande (Cameron and Hidalgo counties) resident. Podilymbus podiceps. Pied-billed Grebe. Lower Rio Grande (Cameron and Hidalgo counties) resident. odilymbus podiceps. Pied-billed Grebe. Podilymbus podiceps. Pied-billed Grebe. Resident, breeding from the northern border south to Brownsville. In the coast counties more abundant in winter. FAMILY Gaviidae. Loons Gavia imber. oon: Great Northern Diver. Migrates through the eastern half of the State, wintering on the coast. Interior records: San Angelo (Lloyd) and Waco (Strecker). The bulk of the birds return north about the 10th of April. One Waco bird was shot on April 18. In the fall Loons usually pass through Waco about the middle of November. INTRODUCTION Probably no more than three of Giraud’s “Sixteen Species of Texas (?) Birds” will ever be taken within the boundaries of the Lone Star State. dence to show that they are really entitled to a place here. Such species as Giraud’s Flycatcher, the Red, Brasher’s and Bell's Warblers and others described by the same authority have until late years been considered stragglers to Texas but more recent explorations serve to show that many of them do not even occupy intervening territory between their natural habitats and our southern boundary. Probably no more than three of Giraud’s “Sixteen Species of Texas (?) Birds” will ever be taken within the boundaries of the Lone Star State. FAMILY Laridae. Gulls and Terns. Larus argentatus. Herring Gull. Winters along the Gulf coast. Larus argentatus. Herring Gull. Winters along the Gulf coast. Larus californicus. California Gull. Larus californicus. California Gull. Singley collected two immature specimens on Galveston Island. Identified by Ridgway. BAYLOR UNIVERSITY BULLETIN 10 Larus delawarensis. Ring-billed Gull. Common during the fall and winter months, frequenting the Gulf coast and the lakes in the southern section of the State. During the migrations occasionally remaining on the small inland lakes for several days on a stretch. Specimens from Waco (October and April) in Baylor museum. Larus atricilla. Laughing Gull. Abundant resident along the coast. Breeding on Bird and Padre Islands, Matagorda Peninsula, etc. 10. Larus franklinii. Franklin Gull. Tolerably common winter resident on the Gulf coast. Regular mi- grant in spring, some years remaining on inland lakes for weeks at a time. Specimens from Waco bear dates from April 29 to May 5. 11. Larus glaucus. Glaucous Gull. Straggler from the north. Clay county, Texas, (Ragsdale, Bull. Nutt. Orn. Club, VI, 1881, p. 187.) 12. 13. Larus philadelphia. Bonaparte Gull. Winter resident on Gulf coast, not common. Xema sabinii. Sabine Gull. 13. 14. Xema sabinii. Sabine Gull. Casual visitor to the coast in winter. Gelochelidon nilotica. Gull-billed Tern. 14. 15. Gelochelidon nilotica. Gull-billed Tern. Resident along the Gulf coast. Sterna caspia. Caspian Tern. 14. 15. Gelochelidon nilotica. Gull-billed Tern. Resident along the Gulf coast. Sterna caspia. Caspian Tern. 15. Sterna caspia. Caspian Tern. Resident on the coast. Common. 16. Sterna maxima. Royal Tern. Common on the Gulf coast and on many of the lagoons and marshes from Corpus Christi southward. 17. 18. Sterna sandvichensis acuflavida. Cabot Tern. Common resident of the coast and l Sterna forsteri. Forster Tern. 17. 18. Sterna sandvichensis acuflavida. Cabot Tern. Common resident of the coast and lagoons in Southeastern Texas. Sterna forsteri. Forster Tern. 18. Sterna forsteri. Forster Tern. Abundant in the lagoons and marshes of Southern Texas. Migrates through Eastern Texas with considerable regularity in the spring, these birds of course being the northern contingent from the upper Missis- sippi Valley. 19. Sterna hirundo. Common Tern. Resident in some portions of Southern Texas. Common migrant. THE BIRDS OF TEXAS 11 20. 21. Sterna antillarum. Least Tern. Formerly abundant on the coast, breeding in all favorable localities. Now extremely rare. A bird that has suffered greatly from the bru- talities of the millinery gunner. Sterna fuliginosa. Sooty Tern. 21. FAMILY Suiidae. Gannets, Boobies, etc. FAMILY Suiidae. Gannets, Boobies, etc. 26. 27. Sula bassana. Gannet. Occasional on the Gulf coast. Sula sula. Booby. 27. Sula sula. Booby. Irregular visitant to the coast. FAMILY Rhynchopidae. Skimmers. 24, Rhynchops nigra. Black Skimmer. Common resident along the coast. FAMILY Laridae. Gulls and Terns. 22. Sterna fuliginosa. Sooty Tern. Rather rare resident along the coast. Hydrochelidon nigra surinamensis. Black Tern. 21. 22. Sterna fuliginosa. Sooty Tern. Rather rare resident along the coast. Hydrochelidon nigra surinamensis. Black Tern. 22. 23. Hydrochelidon nigra surinamensis. Black Tern. Irregular migrant. Three records from Waco in April. Anous stolidus. Noddy. 23. Anous stolidus. Noddy. Rather uncommon resident on the coast. FAMILY Anhingidae. Darters. 25. Anhinga anhinga. Anhinga. Rather common resident of the eastern and southern parts of the State. Rare summer resident in the vicinity of San Antonio. Speci- mens from Waco in the Baylor Museum (November and April.) FAMILY Fregatidae. Man-o’War Birds. 33. Fregata aquila. Man-o’War Bird. Coast region from Louisiana to the mouth of the Rio Grande River. In July, 1908, the writer observed many specimens on Matagorda Bay. FAMILY Pelecanidae. Pelicans. 31. Pelecanus erythrorhynchos. American White Pelican. Abundant migrant. Winters on the Gulf coast in great numbers. Very abundant during the migrations in Central and Western Texas. 32. Pelecanus occidentalis. Brown Pelican. Common resident on the coast, breeding on the islands and formerly on the mainland. Butcher recorded specimens from Laredo in May but as a rule the birds rarely go inland. FAMILY Phalacrocoracidae. Cormorants. 28. Phalacrocorax auritus. Double-crested Cormorant. Straggler. A specimen in the Baylor University collection was shot at Elm Lake, McLennan County, on October 15, 1911. Length 32; Tail 6.75; Bill 3.50: Tarsus 2.30. 29. Phalacrocorax auritus floridanus. Florida Cormorant. Rather common on the Gulf coast andin extreme Eastern Texas, breeding in favorable localities. BAYLOR UNIVERSITY BULLETIN 12 30. Phalacrocorax mexicanus. Mexican Cormorant. Gulf coast region, breeding abundantly in the marshes of the Brown- ville country. North in summer to Louisiana. 30. Phalacrocorax mexicanus. Mexican Cormorant. Gulf coast region, breeding abundantly in the marshes of the Brown- ville country. North in summer to Louisiana. ORDER Anseres. Lamellirostral Swimmers. ORDER Anseres. Lamellirostral Swimmers. FAMILY Anatidae. Ducks, Geese and Swans. BAYLOR UNIVERSITY BULLETIN 14 50. Marila nearctica. American Scaup Duck. This species is a rather rare straggler to the northern Gulf coast. 51. Marila affinis. Lesser Scaup Duck. 50. Marila nearctica. American Scaup Duck. This species is a rather rare straggler to the northern Gulf coast. 51. Marila affinis. Lesser Scaup Duck. 51. Marila affinis. Lesser Scaup Duck. Tolerably common winter resident of Southern and Western Texas. Occurs only as a migrant in the vicinity of Waco. 51. Marila affinis. Lesser Scaup Duck. Tolerably common winter resident of Southern and Western Texas. Occurs only as a migrant in the vicinity of Waco. 52. Marila collaris. Ring-necked Duck. 52. Marila collaris. Ring-necked Duck. Winter resident of Southern and Western Texas. Occurs only as a migrant in the northern and eastern sections of the State. 53. Clangula americana. American Golden-eye. Rare winter visitor to the Gulf coast. 54. Charitonetta albeola. Bufflehead. 53. Clangula americana. American Golden-eye. Rare winter visitor to the Gulf coast. 54. Charitonetta albeola. Bufflehead. 54. Charitonetta albeola. Bufflehead. Rather rare winter resident. Abundant during the migrations. 55. Harelda hyemalis. Old Squaw. 55. Harelda hyemalis. Old Squaw. Occasional visitor to the coast in winter. A mounted specimen in the Baylor Museum is said to have been collected near the mouth of the Rio Crande River in Cameron county. 56. Nomonyx dominicus. Masked Duck. Occasional on the Lower Rio Grande. Cooke thinks it probable that it breeds in the vicinity of Brownsville. 57. Erismatura jamaicensis. Ruddy Duck. 57. Erismatura jamaicensis. Ruddy Duck. Rare winter resident but abundant during the migrations. Cooke says that it breeds over much of the Mississippi Valley, from Texas to Minnesota, but I have no record of its breeding in this State. 58. Chen hyperborea. Lesser Snow Goose. 58. Chen hyperborea. Lesser Snow Goose. An abundant winter resident on the coast. 59. Chen hyperborea nivalis. Greater Snow Goose. 58. Chen hyperborea. Lesser Snow Goose. An abundant winter resident on the coast. 59. Chen hyperborea nivalis. Greater Snow Goose. 59. Chen hyperborea nivalis. Greater Snow Goose. This goose winters on the coast but is by p 60. Chen caerulescens. Blue Goose. 59. Chen hyperborea nivalis. Greater Snow Goose. This goose winters on the coast but is by po means common. 60. Chen caerulescens. Blue Goose. 60. Chen caerulescens. Blue Goose. Common winter resident on the coast. 60. Chen caerulescens. Blue Goose. Common winter resident on the coast. 61. FAMILY Anatidae. Ducks, Geese and Swans. 34. Merganser americanus. American Merganser. Winter resident, rather common on the coast but rare in the interior. 35. Merganser serrator. Red-breasted Merganser. Winter resident, not uncommon in the coast counties. Rare in East- central Texas (Waco). 36. Lophodytes cucullatus. Hooded Merganser. Not uncommon winter resident of the coast counties. Reported only as a migrant from San Antonio, Giddings, Waco and other stations in the interior. 37. Anas platyrhyncha. Mallard. 37. Anas platyrhyncha. Mallard. Common migrant and winter resident. 38. Anas fulvigula maculosa. Mottled Duck. 37. Anas platyrhyncha. Mallard. Common migrant and winter resident. 38. Anas fulvigula maculosa. Mottled Duck. Eastern Texas, south to the Rio Grande. Breeds abundantly in the marshes and lagoons of Neuces, San Patricio, Refugio and other south- eastern counties. May breed throughout its state range. Rare in Central Texas (Waco). THE BIRDS OF TEXAS 13 39. Anas rubripes. Black Duck. Exceedingly rare migrant. The majority of the records for this spe- cies evidently refer to the Mottled Duck. Rare migrant, possibly straggler, in the vicinity of Waco. 40. Chaulelasmus streperus. Gadwall. Abundant migrant and common winter resident. Rare in the interior in winter but abundant on the coast. 41. Mareca americana. Baldpate. Rather common winter resident. Cooke (Bird Migration in the Mis- sissippi Valley) says that it has been known to breed in Texas but I have no record of it. Nettion carolinensis. Green-winged Teal. 42. Nettion carolinensis. Green-winged Teal. Abundant migrant, not uncommon winter resident. 43. Querquedula discors. Blue-winged Teal. Rather common in winter, exceedingly abundant during the migrations. “Accidental breeding at San Antonio and Spring Lake.” (Cooke). 44. Querquedula cyanoptera. Cinnamon Teal. Trans-Pecos region and southern plains, migrating southeastward to the southern Gulf coast (San Antonio Bay, southward). Breeds in the vicinity of Midland, Midland county, rarely. Two records for Waco, probably stragglers., 45. Spatula clypeata. Shovellor. Common resident, breeding locally throughout the State. Very abundant in winter. 46. Dafila acuta. Pintail. Abundant migrant and winter resident. Aix sponsa. Wood Duck. 47. Aix sponsa. Wood Duck. Resident, breeding locally throughout the State. Formerly abundant, now exceedingly rare. 48. Marila americana. Redhead. Rare winter resident in Central and Eastern Texas; common in the southern and west-central sections of the State. 49, Marila vallisneria. Canvasback. Winter resident along the coast. “Rather common in early winter in Tom Green and Concho counties.” (Lloyd.)’ Rare in the vicinity of Waco and in Eastern Texas away from the coast. ORDER HERODIONES. Herons, Storks, Ibises, Etc. ORDER HERODIONES. Herons, Storks, Ibises, Etc. FAMILY PHOENICOPTERIDAE. Flamingoes. 69. Phoenicopterus ruber. American Flamingo. In former years a frequent visitor, probably summer resident, of the coast counties. Recorded from Western Texas (Tom Green and Con- cho counties), in 1881 and 1882 by William Lloyd. FAMILY Anatidae. Ducks, Geese and Swans. Anser albifrons gambeli. American White-fronted Goose. Common winter resident on the coast. Asa rule very few remain in the interior. 62. Branta canadensis. Canada Goose. Abundant migrant and common winter resident. 63. Branta canadensis hutchinsii. Hutchin’s Goose. 63. Branta canadensis hutchinsii. Hutchin’s Goose. Common migrant. Winters in the marshes and lagoons near the coast. THE BIRDS OF TEXAS 15 64. Branta nigricans. Black Brant. “Lloyd states that it was shot in Tom Green county in the fall of 1884” (Cooke). This species is evidently a very rare or irregular visitor, as this is the only definite record of its occurrence that I can find. 65. Dendrocygna autumnalis. Black-bellied Tree Duck. Summer resident on the Lower Rio Grande (Fort Brown, Hidalgo, etc.) remaining from April to October. 66. Dendrocygna fulva. Fulvous Tree Duck. Lower Rio Grande (Cameron and Hidalgo counties) straggling north- ward along the coast to Louisiana. Summer resident as far north as the sources of the Neuces. Lloyd recorded it as tolerably common in winter on the North Concho. 67. Olor columbianus. Whistling Swan. Winters abundantly on Galveston and Corpus Christi bays and at other points along the coast. Migrates over the entire region east of the Pecos. 68. Olor buccinator. Trumpeter Swan. Common winter resident of the coast. Lloyd recorded it as wintering in Western Texas. FAMILY PLATALEIDAE. Spoonbills. 70. Ajaia ajaja. Roseate Spoonbill. Eastern and Southern Texas, formerly breeding in abundance in the marshes and lagoons, in company with Ibises and Herons. Formerly bred near Houston (Nehrling), in recent years reported from various localities along the coast (Refugio, Bee, Neuces counties, etc.) BAYLOR UNIVERSITY BULLETIN 16 FAMILY CICONHDAE. Storks and Wood Ibises. 74. Mycteria americana, Wood Ibis. Formerly an abundant species throughout the Eastern and South- eastern sections or the State. still common in some localities. At the end of the breeding season, in July and August, these birds straggle all over Texas. Recorded from the Red river, near Texarkana by Overholser in 1662. Common at Coon Lake, Bee county in 1905. (Strecker). 75. Jabiru mycteria, Jabiru: American Wood Stork. A tropical American species. One specimen was taken near Galves- ton, the only record for the United States. FAMILY IBIDIDAE. Ihises. 71. Guara alba. White Ibis. . Gulf coast counties, breeding in the marshes and lagoons. 72. Guara rubra. Scarlet Ibis. 72. Guara rubra. Scarlet Ibis. A tropical American species formerly straggling to the Gulf coast. None have been recorded in recent years. 73. Plegadis guarauna. White-faced Glossy Ibis. An abundant summer resident of Southern Texas. Up to recent years, immense colonies nested in the lagoons.and marshes in the region be- tween Refugio. and Brownsville. FAMILY ARDEIDAE. Herons, Bitterns, Etc. 76. Betaurus lentiginosus. American Bittern. Winter resident. 77. Ixobrychus exilis. Least Bittern. Summer resident of the entire State, breeding from the northern boundary south to Brownsville. Winters chiefly near the southern Gulf coast. 78. Ardea herodias. Great Blue Heron. Abundant winter resident, especially in the coast counties. Summer resident, breeding locally throughout the State. 79. Ardea herodias wardi. Ward Heron. Coast region of Texas. A fine breeding male in the Baylor Univer- sity collection was shot near Refugio in June, 1904. THE BIRDS OF TEXAS 17 80. Herodias egretta. American Egret. Formerly an abundant summer resident of the cypress swamps of Eastern and Southeastern Texas, now exceedingly rare. Winters in the southern coast counties. 81. Egretta candidissima. Snowy Heron. 80. Herodias egretta. American Egret. Formerly an abundant summer resident of the cypress swamps of Eastern and Southeastern Texas, now exceedingly rare. Winters in the southern coast counties. 81. Egretta candidissima. Snowy Heron. 81. Egretta candidissima. Snowy Heron. Formerly an abundant species in the marshes and lagoons of South- ern and Eastern Texas, now almost exterminated. Capt. M. B. Davis, Secretary of the Texas Audubon Society, recently reported a small colony of fifteen pairs in a locality in Eastern Texas where the birds had formerly bred by the hundreds. They were not disturbed and it is reasonable to suppose that they are responsible for adding at least fifty more Snowy Egrets to our avian population. At the close of the breeding season in former years these’birds would occasionally strag- gle westward into the central portion of the State (Waco and Marlin). 82. Dichromanassa rufescens. Reddish Egret. 82. Dichromanassa rufescens. Reddish Egret. » © Gulf coast counties, formerly , abundant but, now rather uncommon. F ... Formerly reported from as far west as, San Antonio. Reported by Sennett and Hancock as breeding i in Laguna Madre and other locali- ties near Corpus Christi. Carroll reported it as rare summer resident of the coast region of Refugio county, 4 and the writer observed quite a 13 number of specimens in Matagorda county in 1908. 83. Hydranase tricolor ruficollis. Louisiana Heron. 83. Hydranase tricolor ruficollis. Louisiana Heron. r _ Rather uncommon summer resident. of the coast counties. Dresser reported its occurence at San Antonio, but it is not mentioned from that locality by Attwater. Reported breeding near Houston by Nehrling ’ and as.an,abundant breeder on the Lower Rio Grande by Merrill and Sennett. FAMILY ARDEIDAE. Herons, Bitterns, Etc. Recorded as, common along. the ‘Red River north of Tex- arkana in 1902 by Oberholser (Howell, \| Birds of Arkansas). 84. Florida caerulea. Little Blue Heron, 84. Florida caerulea. Little Blue Heron, Eastern half of the State, breeding in suitable localities, abundant. Recorded by Lloyd as a resident as far west as San Angelo. Breeds in several localities near Waco. I have seen thousands of these birds in the white phase of plumage in the marshes of Central Texas in late June and early July. 85. Butorides virescens. Green Heron. 85. Butorides virescens. Green Heron. The entire State, most abundant in the eastern half, breeding locally throughout its range. BAYLOR UNIVERSITY BULLETIN 18 86. 87. Nycticorax nycticorax naevius. Black-crowned Heron. Abundant migrant, winter resident of the coast counties, summer resi- dent ‘of some portions of Eastern, Central and Southern Texas. Nyctanassa violaceus. ‘ Yellow-crowned Night Heron. 87. Nyctanassa violaceus. ‘ Yellow-crowned Night Heron. “ Winter resident of the coast counties. Breeds in various localities in the eastern half of the State. In 1907, Mr. Howell discovered a small colony nesting at Gurley, Falls county, and together we paid it a visit: I should judge that there was about twenty pairs in the colony. We did not disturb them. Attwater records this species as a rare migrant at San Antonio. FAMILY ARAMIDAE. Courlans. 91. Aramus giganteus. Limpkin. The “crying bird” occasionally straggles to the coast of Texas. FAMILY RALLIDAE. Rails, Gallinules, Coots, Etc. FAMILY GRUIDAE. Cranes. 88. Grus americana.’ Whooping Crane. Formerly an abundant winter resident of Western and Southern Tex- as. Common duritig the migrations in the eastern and central sec- tions of the sie Now uncommon in'.winter, except in the middle west. 89. Gus canadensis. ‘Little Bawa Crane. Cooke in “Bird Migration in the Mississippi Valley,” states that he was _informed by Mr. Robert Ridgway that there was a specimen of this Crane from Texas in the National collection. Butcher records it from ‘Laredo (June! 1868). Grus mexicana. Sandhill Crane. $0. Grus mexicana. Sandhill Crane. Migrant through the eastern half of the State. This species was ‘formerly an abundant breeding bird in the lakes and marshes near ‘the coast. It is doubtful whether it can now be considered a summer resident of any portion of Texas. Winters along the southern coast. FAMILY RALLIDAE. Rails, Gallinules, Coots, Etc. $2. Rallus elegans. King Rail. Eastern half of the State, rare migrant and winter visitant. (South Concho [Lloyd], Corpus Christi [Sennett], Waco [Strecker] ). THE BIRDS OF TEXAS 19 93. Rallus crepitans saturatus. Louisiana Clapper Rail. Northern Gulf coast, probably breeding, 93. Rallus crepitans saturatus. Louisiana Clapper Rail. Northern Gulf coast, probably breeding, 94. Rallus crepitans caribaeus. Carribean Clapper Rail. A West Indian species found as far north on the Texas coast as Gal- veston, where it was first taken by Sennett. 95. Rallus virginianus. Virginia Rail. Rare migrant. Reported from San Antonio by Dresser. Two speci- mens collected at Waco by Prade are now in the writer’s private col- lection. This species is not mentioned in any of the local lists of Texas birds. 96. Coturnicops jamaicensis. Black Rail. Lloyd found this species breeding at San Angelo from March to June, 1884. Coturnicops noveboracensis. Yellow Rail. 97. Coturnicops noveboracensis. Yellow Rail. Heerman reported this species from Mitchell’s Lake, near San An- tonio (Dresser). This is the only record that J can find. 98. Porzana carolina. Sora. . Migrant and winter resident, reported as common by most observers. 99. Ionornis martinica. Purple Gallinule. This species is not a common bird in Texas. Recorded as a rare mi- grant at San Antonio and not mentoned in any of the other local lists. A rather rare summer resident of McLennan and Falls counties, where it breeds in the marshes and lagoons in May. 100. 101. Gallinula galeata. Florida Gallinule. Resident, breeding locally from the northern boundry south to Browns- ville. Winters along the coast, in the marshes. Rare in the vicinity of Waco. Fulica americana. American Coot. Pe 101. Fulica americana. American Coot. Pe Resident, breeding in favorable localities throughout the State. FAMILY Phalaropodidae. Phalaropes. 102. Steganopus tricolor. Wilson Phalarope. Migrant, apparently most abundant in spring. Rare near the Gulf coast. Rare at Waco. Abundant in Western Texas (Alpine, May 7-8.) BAYLOR UNIVERSITY BULLETIN 20 FAMILY Recurvirostridae. Avocets and Stilts. 103. Recurvirostra americana. American Avocet. Winters on the coast, migrating through the entire State. Known to breed in West-central and Western Texas (Mason and Midland counties. ) 104. Himantopus mexicanus. Black-necked Stilt. Summer resident of the coast counties (Bee, Refugio, Neuces, etc.) and of the southern plains region (Midland, Ector, counties, etc.) FAMILY Scolopacidae. Snipes, Sandpipers, etc. 105. Philohela minor. American Woodcock. Winter resident in Eastern Texas, occasionally as far west as San An- gelo. Rare at Waco. This species is said to be resident in the Big Thicket of Eastern Texas. 106. 107. Gallinago delicata. Wilson Snipe. Common winter resident. 107. Macrorhamphus griseus. Dowitcher. Reported from Corpus Christi and Hidalgo by Singley. Probably an irregular migrant. 108. Macrorhamphus griseus scolopaceus. Long-billed Dowitcher. Migrant. Winters on the Gulf coast principally south of Corpus Christi. 109. 110. Micropalama himantopus. Stilt Sandpiper. Common (?) during the fall migration at San Angelo (Lloyd), rare at Waco, rare at San Antonio. Pisobia maculata. Pectoral Sandpiper. 110. 111. Pisobia maculata. Pectoral Sandpiper. Migrant, especially abundant in spring. Pisobia fuscicollis. White-rumped Sandpiper. 110. 111. Pisobia maculata. Pectoral Sandpiper. Migrant, especially abundant in spring. Pisobia fuscicollis. White-rumped Sandpiper. 111. 112. Pisobia fuscicollis. White-rumped Sandpiper. Abundant migrant, common winter resident on the coast. Mild win- ters remains in Central Texas during the season. 112. 113. Pisobia bairdi. Baird Sandpiper. Uncommon migrant. Reported in spring at San Angelo (Lloyd) and Alpine (Strecker). Not recorded in the lists of Hancock, Chapman, Carroll or Attwater. One at Boerne (Brown). Pisobia minutilla. Least Sandpiper. 113. Pisobia minutilla. Least Sandpiper. Abundant migrant in the interior, winters in Southeastern Texas and along the Gulf coast. 113. Pisobia minutilla. Least Sandpiper. Abundant migrant in the interior, winters in Southeastern Texas and along the Gulf coast. THE BIRDS OF TEXAS 21 114. 115. Tringa canutus. Knot: Robin Snipe. Recorded from Corpus Christi by F. M. Chapman. Pelidna alpina pacifica. Red-backed Sandpiper. 115. 116. Pelidna alpina pacifica. Red-backed Sandpiper. Winter resident on the Gulf Coast. Abundant in marshes of the Lower Rio Grande as late as May 16, according to Dr. Merrill. Re- ported by the majority of the Texas observers. Apparently rare in Western Texas. Ereuntes pusillus. Semipalmated Sandpiper. 116. 117. Ereuntes pusillus. Semipalmated Sandpiper. Common migrant. Winters regularly o Ereuntes occidentalis. Western Sandpiper. 116. 117. Ereuntes pusillus. Semipalmated Sandpiper. Common migrant. Winters regularly on the coast, rarely in the interior. Ereuntes occidentalis. Western Sandpiper. 117. 118. Ereuntes occidentalis. Western Sandpiper. Migrant, reported from Tom Greene and Concho counties (Lloyd), Corpus Christi (Chapman) and the lower Rio Grande. Probably a rare winter resident around the delta of the Rio Grande. Calidris arenaria. Sanderling. 118. Calidris arenaria. Sanderling. Migrant. FAMILY Scolopacidae. Snipes, Sandpipers, etc. Common in spring along the coast, from Galveston to the mouth of the Rio Grande River. 119. 120. Limosa fedoa. Marbled Godwit. Spring and fall migrant, not uncommon in Southeastern Texas (Cor- pus Christi, etc.) Totanus melanoleucus. Greater Yellow-legs. 120. 121. Totanus melanoleucus. Greater Yellow-legs. Abundant migrant. Common winter resident on the Gulf coast. A few winter in the interior. Totanus flavipes. Yellow-legs. 121. 122. Totanus flavipes. Yellow-legs. Winter resident, most abundant in the coast counties, but remaining in favorable localities througout the state. Helodromas solitarius. Solitary Sandpiper. 122. 123. Helodromas solitarius. Solitary Sandpiper. Common migrant. Catoptrophorus semipalmata inornata. Wester 122. 123. Helodromas solitarius. Solitary Sandpiper. Common migrant. Catoptrophorus semipalmata inornata. Western Willet. 123. Catoptrophorus semipalmata inornata. Western Willet. Coast country, resident, more abundant in winter. Reported near Houston by Nehrling. Breeds in Refugio county (eggs in Baylor Museum from St. Marys), common at Corpus Christi (Hancock). Bartramia longicauda. Bartramian Sandpiper. 124. Bartramia longicauda. Bartramian Sandpiper. Abundant migrant. August-September and April-May. Trygnites subruficollos. Buff-breasted Sandpiper. 125. Trygnites subruficollos. Buff-breasted Sandpiper. Rather rare migrant in the interior (Gainesville [Ragsdale]). Abun- dant around Galveston (Singley), abundant on the lower Rio Grande in Cameron county (Sennett). BAYLOR UNIVERSITY BULLETIN Actitis macularia. Spotted Sandpiper. Abundant migrant. Rare summer resident in several sections of the State. The migrants usually go south through Waco about the last of October, returning in March. 127. Numenius longirostris. Long-billed Curlew. Common migrant, very abundant in former years. Winters in the Rio Grands counties. I have observed them as late as May 27 in Midland and Jeff Davis counties. Formerly bred north of the Cana- dian, in the Panhandle. 128. 129. Numenius hudsonicus. Hudsonian Curlew. Rare migrant. Recorded from Waco (Strecker) and San Antonio (Dresser). Numenius borealis. Eskimo Curlew. 129. Numenius borealis. Eskimo Curlew. Common migrant through extreme eastern section of the State. ORDER Gallinae. Gallinaceous Birds. FAMILY Tetraonidae. Grouse, Patridges, etc. FAMILY Jacanidae. Jacanas. n Jacana. 140. Jacana spinosa. Mexican Jacana. Valley of the Lower Rio Grande (Merrill). ORDER Gallinae. Gallinaceous Birds. FAMILY Aphrizidae. Surf Birds and Turnstones. lla. Ruddy Turnstone. FAMILY Aphrizidae. Surf Birds and Turnstones. lla. Ruddy Turnstone. 138. Arenaria morinella. Ruddy Turnstone. Rather uncommon winter resident on the coast. FAMILY Haematopodidae. Oystercatchers. FAMILY Haematopodidae. Oystercatchers. 139. Haematopus palliatus. American Oystercatcher. Summer resident of the coast. FAMILY Charadriidae, Plovers. 130. 131. Squatarola squatarola. Black-bellied Plover. Rather common migrant along the coast. (Galveston Island [Sing- ley] and Corpus Christi [Chapman] ). Charadrius dominicus. American Golden Plover. 131. Charadrius dominicus. American Golden Plover. Common migrant, especially along the coast. Reported by the ma- jority of the observers. 132. Oxyechus vociferus. Killdeer. Abundant resident throughout the State. Aegia itis semipalmata. Semipalmated Plover. 132. 133. Oxyechus vociferus. Killdeer. Abundant resident throughout the State. Aegia itis semipalmata. Semipalmated Plover. 133. 134. Aegia itis semipalmata. Semipalmated Plover. Common migrant. Winters in the coast region. Aegialitis meloda circumcincta. Belted Piping Plover. 133. 134. Aegia itis semipalmata. Semipalmated Plover. Common migrant. Winters in the coast region. Aegialitis meloda circumcincta. Belted Piping Plover. 134. Aegialitis meloda circumcincta. Belted Piping Plover. Rare migrant throughout the eastern section of State. Recorded from Galveston Island by Singley. 135. Aegialitis nivosa. Snowy Plover. Resident on the Gulf coast from Galveston southward. More abun- dant in winter thanin summer. Eggs in Baylor museum from eastern Refugio county. 136. Octhodromus wilsonius. Wilson’s Plover. Resident along the coast, breeding abundantly on the islands and beaches. , THE BIRDS OF TEXAS 23 137. Podasocys montanus. Mountain Plover. Common migrant in Western Texas, a few remaining through the winter. Tolerably common in summer in the Trans-Pecos country, on the southern plains and in the Panhandle west of the edge of the plains. FAMILY Tetraonidae. Grouse, Patridges, etc. 141. 142. Colinus virginianus. Bobwhite. Resident of the northeastern section of the State. Colinus virginianus texanus. Texan Bobwhite. 142. Colinus virginianus texanus. Texan Bobwhite. Texas, except extreme northeastern and western sections of the State. Resident. 143. Callipepla squamata. Scaled Partridge. Western Texas, south to the Rio Grande, east locally into the central part. of the State. Rare in the Panhandle, on the edge of the plains (Armstrong county), resident. Rarely staggles into the eastern district (Bonham [Cooke] ). 144. Callipepla squamata castanogastris. Chestnut-bellied Scaled Partridge. Lower Rio Grande, north to Bee county, northwest to Eagle Pass. Resident. 145. Lophorty gambelii. Gambel Partridge. El Paso County, in the mountain ranges, resident. Cyrtonyx montezumae mearnsi. Mearn’s Partridge. 146. Cyrtonyx montezumae mearnsi. Mearn’s Partridge. Trans-Pecos region, in the Davis, Chisos and Guadalupe mountains, east to Tom Green and Concho counties (Lloyd) and Mason (Henry). Resident. : BAYLOR UNIVERSITY BULLETIN 24 147. 148. Tympanuchus americanus. Prairie Hen. Formerly abundant on the prairies of northern Texas, but now exceed- ingly rare. Tympanuchus americanus attwateri. Attwater Prairie Hen. 148. 149. Tympanuchus americanus attwateri. Attwater Prairie Hen. Coast region of Eastern Texas, in the interior to Houston, etc. Resi- dent. Tympanuchus pallidicinctus. Lesser Prairie Hen. 149. Tympanuchus pallidicinctus. Lesser Prairie Hen. “Seen in October and November in Concho county, and also in winter on Middle Concho in Tom Green county. Abundant near Colorado City, on the Texas and Pacific Railroad. I believe this record extends the range to the southwest. Westward it was abundant to the foot- hills of the Davis Mountains. Said to have been driven from the Panhandle by the numerous prairie fires.” (Lloyd, The Auk, Vol. IV, p. 187.) This was written more than twenty-five years ago but I have received specimens from the localities mentioned within the last‘three years. i The Lesser Prairie Hen is resident along the foot of the plains in the Panhandle country, south of the Canadian. FAMILY Phasianidae. Pheasants, Turkeys, etc. FAMILY Phasianidae. Pheasants, Turkeys, etc. 150. 151. Meleagris gallopavo silvestris. Wild Turkey. North-eastern section of the State, resident, formerly abundant, now rare. Meleagris gallopavo merriami. Merriam Turkey. 151. 152. Meleagris gallopavo merriami. Merriam Turkey. Occurs and probably breeds in the Guadalupe Mountains, (Bailey). 152. Meleagris gallopavo intermedia. Rio Grande Turkey. Lowlands of Southern Texas (Lower Rio Grande, Live Oak County, San Antonio, etc.) west to Concho and Tom Green counties (Lloyd). Resident. FAMILY CRACIDAE. Curassows, Guans, Etc. FAMILY CRACIDAE. Curassows, Guans, Etc. 153. _Ortalis vetula mccallii. Chachalaca. Valley of the Lower Rio Grande (Cameron and Hidalgo counties). Resident. in recent years, great flocks of pigeons and doves have been reported from Southern Texas. In some journals, it was reported that in the Rio Grande Valley, great ‘droves’ of white-winged doves were doing great damage to the crops. In September, 1911, flocks of wild pigeons were reported from the vicinity of San Angelo at Lavinia. The lat- ter, from the descriptions I received from there, were evidently a straggling flock of Band-tailed Pigeons. This leads me to wonder if most of Lloyd's ‘‘wild pigeons’’ did not belong to this species and not the Passenger Pigeon. In its proper place I speak of the invasion of Texas by the Inca Dove. ORDER COLUMBAE. Pigeons. 154. Columba fasciata. Band-tailed Pigeon. Abundant in the trans-Pecos region, in the Davis, Chisos and Guada- lupe Mountains. Probably resident. THE BIRDS OF TEXAS 25 155. Columba flavirostris. Red-billed Pigeon. Lower Rio Grande (Cameron and Hidalgo counties, etc.), summer resident. 156. Ectopistes migratorius. Passenger Pigeon. Formerly abundant migrant through Eastern Texas, now extinct. Singley’s record for 1886 (Lee county) is the latest I can find. Lloyd mentions the occurrence of myriads of wild pigeons in Neuces canyon in winter. (This was written in 1882). Zenaidura macroura carolinensis. Mourning Dove. 157. 158. Zenaidura macroura carolinensis. Mourning Dove. Abundant resident throughout the State. Leptotila fulviventris brachyptera. White-fronted Do 157. 158. Zenaidura macroura carolinensis. Mourning Dove. Abundant resident throughout the State. Leptotila fulviventris brachyptera. White-fronted Dove. 158. Leptotila fulviventris brachyptera. White-fronted Dove. Valley of the Lower Rio Grande, resident from Melopelia leucoptera. White-winged Dove. 158. 159. Leptotila fulviventris brachyptera. White-fronted Dove. Valley of the Lower Rio Grande, resident from February to November. Melopelia leucoptera. White-winged Dove. 158. 159. Leptotila fulviventris brachyptera. White-fronted Dove. Valley of the Lower Rio Grande, resident from February to November. Melopelia leucoptera. White-winged Dove. 159. 160. Melopelia leucoptera. White-winged Dove. Southern section of the State. Very abundant summer resident of the Lower Rio Grande counties, northwest to Laredo. Rare summer visitor at San Antonio. Breed at Cotulla, Carrizo Springs, etc. Columbigallina passerina terrestris. Ground Dove. 160. Columbigallina passerina terrestris. Ground Dove. Coast region of Eastern Texas, south almost to Corpus Christi. Columbigallina passerina pallescens. Mexican Ground Dove. 160. Columbigallina passerina terrestris. Ground Dove. Coast region of Eastern Texas, south almost to Corpus Christi. Columbigallina passerina pallescens. Mexican Ground Dove. 161. Columbigallina passerina pallescens. Mexican Ground Dove. Lower Rio Grande, north, rarely, to San Antonio and northwest to Eagle Pass. Summer resident. 162. Scardafella inca. Inca Dove. Southern portion of the State. Some years ago Attwater recorded a single specimen from San Antonio, but in late years this dove is a reg- ular visitor there. In the winter of 1904 large numbers of these birds made their appearance at Waco, remaining until the following April. In November 1905 they again appeared but in smaller numbers, this time remaining until the latter part of May and, as Iam informed by reliable parties, nesting in North Waco. They are said to breed near San Antonio at the present time. FAMILY CATHARTIDAE. American Vultures. 163. Cathartes aura septentrionalis. Turkey Vulture. Abundant resident. Less common than Catharista urubu near the coast. 164. Catharista urubu. Black Vulture. Abundant resident, especially on the Lower Rio Grande and in the coast region of Eastern Texas. Rare west of the middle section. ORDER COLUMBAE. Pigeons. Schutze (The Condor, VI, 172) records the Inca Dove as now common in Travis and Comal counties and in the vicinity of Bryan, breeding. 162. Scardafella inca. Inca Dove. Southern portion of the State. Some years ago Attwater recorded a single specimen from San Antonio, but in late years this dove is a reg- ular visitor there. In the winter of 1904 large numbers of these birds made their appearance at Waco, remaining until the following April. In November 1905 they again appeared but in smaller numbers, this time remaining until the latter part of May and, as Iam informed by reliable parties, nesting in North Waco. They are said to breed near San Antonio at the present time. Schutze (The Condor, VI, 172) records the Inca Dove as now common in Travis and Comal counties and in the vicinity of Bryan, breeding. BAYLOR UNIVERSITY BULLETIN 26 FAMILY FALCONIDAE. Hawkes, Eagles, Falcons, Etc. FAMILY FALCONIDAE. Hawkes, Eagles, Falcons, Etc. 165. Elanoides forficatus. Swallow-tailed Kite. Formerly abundant summer resident, now rather rare, in the eastern and southern sections of the State. Always breeding in proximity to rivers and streams. In March 1891, the writer saw a flock of at least two hundred birds flying over a marsh in the eastern portion of Mc- Lennan county. 166. 167. Elanus leucurus. White-tailed Kite. Summer resident of the southern half of the State, nowhere abundant. Record from the Lower Rio Grande, Houston, San Angelo, Lee county, etc., as a breeding bird. 167. Ictinia mississippiensis. Mississippi Kite. Resident in the extreme southern section. Summer resident in the Panhandle (Amarillo, etc.,) in Lee county (Singley), near Houston (Nehrling) and locally throughout the State. Circus hudsonius. Marsh Hawk. Resident, breeding locally. Very abundant in winter, especially in the western half of the State. Accipiter velox. Sharp-shinned Hawk. Common winter resident. May possibly breed in isolated sections. Accipiter cooperii. Cooper’s Hawk. Accipiter cooperii. Cooper’s Hawk. Resident, breeding locally, principally in the northern and northeast- ern sections. Accipiter a 171. Accipiter cooperii. Cooper’s Hawk. Resident, breeding locally, principally in the northern and northeast- ern sections. Accipiter atricapillus striatulus. 171. 172. Accipiter atricapillus striatulus. Western Goshawk. Record from Western Texas by William Lloyd. Asturina plagiata. Mexican Goshawk. 172. Asturina plagiata. Mexican Goshawk. Occasional visitor to the southern section of the State. THE BIRDS OF TEXAS 27 173. Parabuteo unicinctus harrisi. Harris Hawk. Southern half of the State, resident. Breeding as far north as San Antonio (Attwater), Corpus Christi (Hancock), Comal and Hays counties. Tolerably common in Bee and Refugio counties. 174. Buteo borealis. Red-tailed Hawk. Resident east of the plains. Recorded as an abundant breeding bird on the Lower Rio Grande and in Eastern Texas. Rare in summer in the vicinity of Waco. 175. Buteo borealis kriderii. Krider's Hawk. Rare resident east of the plains. Breeds as far south as Refugio coun- ty (Carroll). Red-tailed hawks from Refugio county in my county in my collection indicate that both borealis and kriderii occur there during the breeding season. I do not think much of kviderii as a variety. 176. 177. Buteo borealis calurus. Western Red-tail. Western half of the State, resident. Bueto borealis harlani. Harlan Hawk. 177. Bueto borealis harlani. Harlan Hawk. Resident in the eastern and Gulf coast sections. Recorded from Brownsville (Merrill), Gainesville (Ragsdale), etc. One record from Waco. 178. FAMILY FALCONIDAE. Hawkes, Eagles, Falcons, Etc. Bueto lineatus alleni. Florida Red-shouldered Hawk. The eastern and central sections of the State, resident. South in winter to the Rio Grande. According to Carroll does not breed as far south as Refugio. Most abundant breeding hawk from Waco to Gid- dings in the river bottoms. Buteo lineatus elegans. Red-bellied Hawk. 179. 180. Buteo lineatus elegans. Red-bellied Hawk. Occasionally straggles into the western section of the State. Buteo abbreviatus. Zone-tailed Hawk. 180. 181. Buteo abbreviatus. Zone-tailed Hawk. Southern Texas, breeding north (rarely) to Comal and Hays counties. Recorded by Lloyd as a fall visitant to the western part of the State. Buteo albicaudatus sennettii. Sennett White-tailed Hawk. 181. Buteo albicaudatus sennettii. Sennett White-tailed Hawk. Rio Grande Valley, resident, not uncommon as far north as Bee and Refugio counties. Swainson’s Hawk. ' 182. Buteo swainsonii. Swainson’s Hawk. ' Western half of the State, resident. Especially abundant on the southern plains and in the Davis Mountain region. East in winter to Waco and southeastward to the Gulf coast. BAYLOR UNIVERSITY BULLETIN 28 183. Buteo platypterus. Broad-winged Hawk. Winter resident from the northern border south to the Rio Grande. 184. Urubitinga anthracina. Mexican Black Hawk. 184. Urubitinga anthracina. Mexican Black Hawk. Occasional along the Valley of the Lower Rio Grande. 185. Archibuteo ferrugineus. Ferruginous Rough-leg. Western half of the State, east to Bexar and Comal counties, etc. Breeds abundantly in the Panhandle (Potter and Armstrong counties). 186. Aguila chrysaetos. Golden Eagle. Western half of the State, resident in the mountains of the trans- Pecos region and in the canyons of the Panhandle. Straggles east to Waco, Refugio, etc. Hancock’s Corpus Christi record is based on the Bald Eagle which breeds commonly in that section. 187. Thrasaetos harpyia. Harpy Eagle. 187. Thrasaetos harpyia. Harpy Eagle. Delta of the Rio Grande, one specimen, only record from North Amer- ica (Oswald, Am. Nat., 1875, p. 151.) 188. Haliaeetus leucocephalus. Bald Eagle. Southern and Eastern Texas, resident, locally distributed. Breeds in Neuces, San Patricio, Bee, Refugio, Goliad, and other counties in the southeastern section, this region seeming to be the center of their dis- tribution in Texas. Recorded as common resident in Western Texas by Lloyd, although the present writer has never observed it in that section. 189. Falco mexicanus. Prairie Falcon. Western Texas, resident along the edge of the plains in the Panhandle, on the southern plains and in the trans-Pecos region. FAMILY FALCONIDAE. Hawkes, Eagles, Falcons, Etc. I am a little doubtful of Nehrling’s Houston breeding record. 190. Falco peregrinus anatum. Duck Hawk. 190. Falco peregrinus anatum. Duck Hawk. Winter visitor, not common. May possibly be resident west of the edge of the plains. 191. Falco columbarius. Pigeon Hawk. Winter resident, not common. 192. Falco richardsonii. Richardson Merlin. 192. Falco richardsonii. Richardson Merlin. Rare winter resident. Recorded from Refugio county by Carroll. 193. Falco fusco-caerulescens. Aplomado Falcon. 192. Falco richardsonii. Richardson Merlin. Rare winter resident. Recorded from Refugio county by Carroll. 193. Falco fusco-caerulescens. Aplomado Falcon. 193. Falco fusco-caerulescens. Aplomado Falcon. Lower Rio Grande Valley (Cameron and Hidalgo counties) westward to El Paso. In the west, breeds north on the plains as far as Midland and Ector counties. Summer resident. 193. Falco fusco-caerulescens. Aplomado Falcon. Lower Rio Grande Valley (Cameron and Hidalgo counties) westward to El Paso. In the west, breeds north on the plains as far as Midland and Ector counties. Summer resident. THE BIRDS OF TEXAS 29 194. 195. Falco sparverius. American Sparrow Hawk. Eastern half of the State, breeding locally as far south as the east- central section. Winters throughout its range. Abundant. Falco sparverius phalaena. Desert Sparrow Hawk. 195. Falco sparverius phalaena. Desert Sparrow Hawk. Western Texas, probably breeding in favorable localities. Polyborus cheriway. Audubon Caracara. 196. Polyborus cheriway. Audubon Caracara. Southern section of the State. Breeds, rarely, as far north as Waco. Resident. 197. Pandion haliaetus carolinensis. American Osprey. Winter resident. Said to breed in certain localities on the coast. FAMILY Strigidae. Barn Owls. 198. Aluco pratincola. American Barn Owl. Common resident, distributed throughout the State. FAMILY Bubonidae. Horned Owls, etc. 199. Asio wilsonianus. American Long-eared Owl. Resident, breeding commonly in wooded regions. Asio flammeus. Short-eared Owl. 200. Asio flammeus. Short-eared Owl. Winter resident, common. Strix varia alleni. Florida Barred Ow 200. Asio flammeus. Short-eared Owl. Winter resident, common. 201. Strix varia alleni. Florida Barred Owl. q Coast region of Eastern Texas, west to Waco. Typical specimens from Orange county in the writer's collection. Some of those from Waco “are not very typical. Strix varia helveolum. Texan Barred Owl. 202. Strix varia helveolum. Texan Barred Owl. Type from Corpus Christi. Lower Rio Grande Valley north to south central section of the State. Resident. Strix occidentale. Spotted Owl. 203. 204. Strix occidentale. Spotted Owl. Occurs and probably breeds in the Guadalupe Mountains. (Bailey.) Otus asio. Screech Owl. Northeastern Texas, resident. 203. Strix occidentale. Spotted Owl. Occurs and probably breeds in the Guadalupe Mountains. (Bailey.) 203. 204. Strix occidentale. Spotted Owl. Occurs and probably breeds in the Guadalupe Mountains. (Bailey.) Otus asio. Screech Owl. Northeastern Texas, resident. 204. Otus asio. Screech Owl. Northeastern Texas, resident. 205. Otus asio mecallii. Texan Screech Owl. Central, Southern and Western Texas, resident. Specimens from Waco are fairly typical. Screech owl. 206. Otus flammeolus. Flammulated Screech owl. Occurs and probably breeds in the Guadalupe Mountains (Bailey). BAYLOR UNIVERSITY BULLETIN 30 207. Bubo virginianus. Great Horned Owl. Eastern half of the State, resident. Bubo virginianus pallescens. Western Horned Owl. 207. Bubo virginianus. Great Horned Owl. Eastern half of the State, resident. 208. Bubo virginianus pallescens. Western Horned Owl. Type from San Antonio. Western and Southern Texas, resident. 209. Nyctea nyctea. Snowy Owl. This northern species rarely straggles into Texas during severe winters. Recorded from San Antonio by Dresser. 210. ail. Speotyto cunicularia hypogaea. Burrowing Owl. Texas with the exception. of the Eastern and Northern wooded districts. Especially abundant on the plains. Common in the mountain districts. Not uncommon in the vicinity of Waco, abundant near San Antonio, only in winter at Refugio. ail. 212. Glaucidium phalaenoides. Ferruginous Pygmy Owl. Lower Rio Grande, resident. Not common. Micropallas whitneyi. Elf Owl. 212. Micropallas whitneyi. Elf Owl. Lower Rio Grande, westward along the border. Resident; range not well known. FAMILY Psittacidae. Parrots and Paroquets. 213. Conuropsis carolinensis. Carolina Paroquet. Formerly abundant in wooded districts of Eastern Texas. Now extinct. FAMILY Cuculidae. Cuckoos, Anis, etc. 214. 215. Crotophaga sulcirostris. Groove-billed Ani. Lower Rio Grande Valley, summer resident. Recorded as a fall visitor to Tom Green and Concho counties by Lloyd (Oct. 1885-1886). 215. Geococcyx californianus. Roadrunner. Distributed over the greater portion of the State except the eastern wooded district. Rather common as far east as Waco. Especially abundant in Western and Southern Texas and in some portions of the Panhandle. 216. Coccyzus americanus. Yellow-billed Cuckoo. Summer resident of the eastern half of the State. THE BIRDS OF TEXAS 3 217. Coccyzus americanus occidentalis. California Cuckoo. Western Texas, summer resident, not very common. 218. Coccyzus erythrophthalmus. Black-billed Cuckoo. Rare migrant. Occurs as far west as San Angelo. (Lloyd) FAMILY Trogonidae. Trogons. 219. Trogon ambiguus. Coppery-tailed Trogon. Occasional on the Lower Rio Grande (Ringgold Barracks and Las Cuevas [Merrill] ). FAMILY Alcedinidae. Kingfishers. 220. 221. Ceryle alcyon. Belted Kingfisher. Permanent resident, more abundant in summer. Found along water- courses throughout the State. Ceryle americana septentrionalis. Texan Kingfisher. 221. Ceryle americana septentrionalis. Texan Kingfisher. Tolerably common as far north as Bexar, Comal and Hays counties. Recorded from Corpus Christi by Hancock but not recorded from other localities in the same vicinity. Ceryle torquata. Ringed Kingfisher. 222. Ceryle torquata. Ringed Kingfisher. Casual on the Lower Rio Grande (one specimen in the Philadelphia Academy of Sciences shot near Laredo by Mr. Geo. B. Benners.) ORDER Pici. Woodpeckers, Wrynecks, etc. FAMILY Picidae. Woodpeckers. 223. Campephilus principalis. Ivory-billed Woodpecker. Formerly an inhabitant of the thickly wooded portions of the Eastern and Southern sections of the State, (Harris, Montgomery, Jasper counties, etc.) now almost extinct. outhern Hairy Woodpecker. 224, Dryobates villosus auduboni. Southern Hairy Woodpecker. Eastern Texas, abundant winter visitor in the vicinity of Waco. It is not mentioned in Carroll’s list of Refugio county birds nor Attwater’s list of the birds of San Antonio. Recorded as breeding in Lee county by Singley. It is an abundant bird in summer in Smith and Hender- son counties. copus. Cabanis Woodpecker. 225. Dryobates villosus hyloscopus. Cabanis Woodpecker. Occurs and probably breeds in the Guadalupe Mountains (Bailey, Biological Survey of Texas.) BAYLOR UNIVERSITY BULLETIN 32 226. Dryobates pubescens. Southern Downy Woodpecker. Eastern Texas, common in winter in the vicinity of Waco. One speci- men recorded from San Antonio and one from the Middle Concho River, these localities indicating in all probability its Southern and Western migration ranges in the State. Recorded as a breeding bird in Singley’s Lee county list but I am a little doubtful of its status as such. 227. Dryobates borealis. Red-cockaded Woodpecker. Pine forests and swamp regions of Eastern Texas (Houston, Hemp- stead, Nacogdoches, etc). Resident. 228. Dryobates scalaris bairdi. Texan Woodpecker. Southern and Western Texas, north on the mesquite plains of middle Texas to the northern boundary and along the valley of the Canadian River across the Panhandle. East, locally, as far as Waco, where it is an uncommon breeding bird. 229. Sphyrapicus varius. Yellow-bellied Sapsucker. A common migrant through the eastern half of the State. A few winter in the vicinity of Waco. 230. Sphyrapicus varius nuchalis, Red-naped Sapsucker. Recorded as a rare winter resident in the vicinity of Fort Davis by Lloyd. 231. Sphyrapicus ruber thyroideus. Williamson Sapsucker. Irregular winter visitant to the western counties. Collected in Tom \| Green, Concho and Uvalde counties by Lloyd. 232. Phloeotomus pileatus. Pileated Woodpecker. Eastern Texas, resident. A few are still found in the thick Tehua- cana bottoms, nine miles from Waco, but the species seems destined to follow the way of the Ivory-bill. 233. Melanerpes erythrocephalus. Red-headed Woodpecker. Northern and Eastern Texas, more abundant in summer than in winter. Recorded as a rare winter resident of Refugio county by Carroll. FAMILY Picidae. Woodpeckers. Said to be exceedingly rare in the vicinity of San Antonio by Beckham but Attwater does not record it from that locality at all. Most abundant woodpecker at Waco. In the Paloduro section of the Panhandle, it is not uncommon during the summer months, following up the river valleys almost to the New Mexican line. THE BIRDS OF TEXAS 33 234. 235. Melanerpes formicivorus. Ant-eating Woodpecker. Trans-Pecos Texas, common in the Davis, Chisos, and Gaudalupe Mountains. Resident. Asyndesmus torquatus. Lewis Woodpecker. 235. 236 Asyndesmus torquatus. Lewis Woodpecker. Recorded from San Angelo by Lloyd. Bailey records a single specimen from the Davis Mountains. Centurus carolinus. Red-bellied Woodpecker. 236 Centurus carolinus. Red-bellied Woodpecker. Eastern section of the State, south to Victoria and Calhoun counties; resident; more abundant in winter than summer. Two stragglers were noted at Corpus Christi by Beckham. Abundant resident in the San Angelo country but not appearing to go west of the Concho, ac- cording to Lloyd. 237. 238. Centurus aurifrons. Golden-fronted Woodpecker. Southern Texas, resident from San Antonio southward. According to Lloyd it ranges west to the Castle Mountains. Rather abundant in the coast counties from Refugio south to the mouth of the Rio Grande. Colaptes auratus. Southern Flicker. 238. Colaptes auratus. Southern Flicker. Common resident of the eastern section of the State. Winter resident at Waco and San Antonio, but only a few records from the southern coast region (Corpus Christi [Chapman], Refugio one specimen, [Carroll]). In some manuscript notes of J. A. Singley, now in the writer’s possession, mention is made of the breeding of this species near Giddings, Lee county, in recent years. Colaptes cafer collaris, Red-shafted Flicker. 239, Colaptes cafer collaris, Red-shafted Flicker. Irregular migrant, some years straggling almost to the eastern border of the State. Common at Waco about every three or four years. Common at San Antonio and San Angelo. A few Waco specimens are typical, but the majority are hybrids of this and C. auratus. Probably breeding in the Davis and Guadalupe Mountains. ORDER Macrochires. Goatsuckers, Swifts, ete. FAMILY Caprimulgidae. Goatsuckers. 240. Antrostomus carolinensis. Chuck-will’s-widow. Eastern section of the State, summer resident as far south as Neuces county. West to Waco and San Antonio, breeding in both localities. Arrives from the south about the middle of March. Carroll thinks that a few winter in Refugio county. BAYLOR UNIVERSITY BULLETIN 34 241. Antrostomus vociferus. Whip-poor-will. Migrant through the eastern half of the State, west to Waco and San Antonio, reported rare from both localities. 242. Antrostomus vociferus macromystax. Stephen’s Whip-poor-will. Common in the Chisos Mountains and noted also in the Gaudalupe range. (Oberholser, The Auk, XX, p. 300.) 243. Phalaenoptilus nuttallii. Poor-will. The Texan range of this subspecies is very imperfectly known. Attwater records it as a common summer resident in the vicin- nity of San Antonio. Cooke records it from Mason and San Angelo. Butcher observed it at Laredo from September to Feb- ruary. 244. Phalaenoptilus nuttallii nitidus. Frosted Poor-will. Western Texas, east during the migrations to San Antonio. The types were from Neuces River bottoms. Nyctidromus albicollis merrillii, Paraque. 245. Nyctidromus albicollis merrillii, Paraque. Lower Rio Grande (Cameron and Hidalgo counties) and coast prairie north to Refugio county where it is rare. Summer resident. 246. Chordeiles virginianus. Nighthawk. Eastern section of the State during the migrations. Summer resident of the extreme northeastern section. Recorded as a migrant at San Antonio. 247. Chordeiles virginianus chapmani. Florida Nighthawk. Coast region from Jefferson to Corpus Christi, summer resident. 248. Chordeiles virginianus henryi. Western Nighthawk. Abundant migrant and common summer resident as far east as Waco and San Antonio. Carroll’s records of this species from Refugio county are not altogether reliable as birds from that locality, collected by the writer, are clearly referable to chapmani. 249, Chordeiles scutipennis texensis. Texan Nighthawk. Southern and Western Texas. North in the coast prairie re- gion to Bee and Refugio counties. Common on the Lower Rio Grande and west of the Pecos. Rachford’s record of the breed- ing of this species at Jefferson, Texas, (Davie, “Nests and Eggs of North American Birds,” fifth edition, p. 287) I do not consider reliable. THE BIRDS OF TEXAS 35 FAMILY Micropodidae. Swifts. 250. ‘ Chaetura pelagica. Chimney Swift. Migrant in Eastern Texas, in the spring arriving in March. On the journey south they pass through about the 20th of September. Rather common at Waco. Reported as rare at San Antonio by Attwater. 251. Aeronautes melanoleucus. White-throated Swift. Of regular occurence in the Davis, Chisos and Guadalupe Monutains. (Oberholser, The Auk, XIX, p. 300.) FAMILY Trochilidae. Hummingbirds. 252. Cyanolaemus clemenciae. Blue-throated Hummingbird. Common in the higher parts of the Chisos Mountains, Brewster county (Oberholser, The Auk, XIX, p. 300.) 253. Archilocus colubris. Ruby-throated Hummingbird. Eastern half of the State, summer resident. Waco (Strecker). Breeds commonly as far west as Tom Green county (Cooke, “Bird Migration in Mississippi Valley,” from Lloyd’s notes.) San Antonio, breeding, (Dresser and Attwater). Summer resident as far south as Beeville and Refugio. No breeding record for the Brownsville country. Ar- rives in March, in some years does not migrate south before the mid- .dle of November. 294. Archilocus alexandri. Black-chinned Hummingbird. Western section of the State, east during the migrations to Waco (Strecker) and Refugio (Carroll). Breeds in the vicinity of San An- tonio (Attwater). A common summer resident of the trans-Pecos re- gion and the southern plains (Midland). 200. Selasphorus platycercus. Broad-tailed Hummingbird. Common summer resident of the Davis, Chisos and Guadalupe Moun- tains. (Bailey, Biological Survey of Texas.) 256. Selasphorus rufus. Rufous Hummingbird. Occurs and probably breeds in the Guadalupe Mountain Transition. (Bailey, Biological Survey of Texas. ) BAYLOR UNIVERSITY BULLETIN 36 257. Stellula calliope. Calliope Hummingbird. Heloise’s Hummingbird (Atthis heloisa) was formerly included in the A. O. U. Check-List on the strength of the report of the capture of a supposed female specimen at El Paso in 1851 (Baird, Brewer and Ridg- way, Land Birds, Vol. II, p. 465, etc.). Adtthis heloisa has since been dropped from the list of North American Birds as the specimen in question proves to be an immature example of Stellula calliope. (See Ridgway, “The Hummingbirds.”) So far as I know, this is the only Texas record for this species. 258. Calothorax lucifer. Lucifer Hummingbird. Summer resident of the Chisos Mountains (Oberholser, The Auk, XIX, p. 300.) 259. 260. Amazilis tzacatl. Reiffer Hummingbird. Casual on the Lower Rio Grande. Specimen recorded from the vicini- ty of Fort Brown by Dr. J. C. Merrill. Amazilis cerniventris chalconota. Buff-bellied Hummingbird. 260. Amazilis cerniventris chalconota. Buff-bellied Hummingbird. Lower Rio Grande Valley. Summer resident in the vicinity of Browns- ville. I can find no records other than those from Cameron county. FAMILY Tyrannidae. Flycatchers. 261. Muscivora forficata. Scissor-tailed Flycatcher. Texas, distributed over the greater portion of the State. Summer resident. In the east-central section, where it is abundant, it arrives from the south from the middle to the 25th of March. Usually about the 15th, small flocks of from four to a dozen are first observed, the bulk not arriving for several days after the vanguard appears. During the breeding season each pair selects some particular grove or large solitary tree and holds it against allcomers. At the close of the nesting season the birds again form into small flocks, usually family groups. They migrate southward in flocks of from a dozen to several hundred birds. In 1898, I witnessed a flight of not less than five hundred at one time.They were congregated in a waste field lying between two strips of woodland and after holding a noisy conclave lasting probably half an hour, flew toward the south in a scattered body. THE BIRDS OF TEXAS 37 262. 263. Tyrannus tyrannus. Kingbird. Eastern section of the State, common summer resident. No breeding records from Corpus Christi south to the Rio Grande. Reported as a migrant at San Antonio, breeds at Houston (Nehrling) and Refugio (Carroll). Rather common resident in the neighborhood of Waco. 263. Tyrannus melancholicus couchii. Couch’s Kingbird. Rare summer resident on the Lower Rio Grande. Reported as breed- ing at Lomita ranch (Sennett). 264. Rare summer resident on the Lower Rio Grande. Reported as breed- ing at Lomita ranch (Sennett). Tyrannus verticalis, Arkansas Kingbird. 264. Tyrannus verticalis, Arkansas Kingbird. Western section of the State, east during the migrations to San Angelo and San Antonio. Abundant summer resident of the Panhandle and the southern plains, and of the mountains west and south of the Pecos. 265. Tyrannus vociferans. Cassin’s Kingbird. Trans-Pecos region, summer resident. This species was an abundant migrant at Paisano and in Du Bois canyon, in the Davis Mountains, during the last three days of April, 1905. 266. Pitangus sulphuratus derbianus. Derby Flycatcher. Lower Rio Grande (Cameron and Hidalgo counties), rare summer resident. 267. Myiarchus crinitus. Crested Flycatcher. Eastern half of the State, breeding south to Refugio and west to San Angelo. Very abundant in the extreme eastern section and as far west as Waco. 268. Myiarchus mexicanus. Mexican Crested Flycatcher. Lower Rio Grande (Cameron and Hidalgo counties), common summer resident. 269. Myiarchus cinerascens. Ash-throated Flycatcher. FAMILY Tyrannidae. Flycatchers. Western half of the State, summer resident. Reported as breeding at San Antonio, Mason, San Angelo and Bonham. I am inclined to doubt the Bonham breeding record. In 1905, we found this species very abundant in the Davis mountains in Brewster county. Hun- dreds were passing through during the latter part of April, and many remained to breed. This flycatcher is a rare fall visitor at Waco. BAYLOR UNIVERSITY BULLETIN 38 270. 271. Sayornis phoebe. Phoebe. Eastern half of the State, breeding west to the San Angelo country. Summer resident of the extreme eastern and northeastern sections but rather locally distributed. Reported as migrant and winter resident at Laredo, Houston, Boerne, Corpus Christi, San Antonio, Refugio and Waco. Attwater states that it breeds in caves in the hilly country northwest of San Antonio. Saynrnis nigricans. Black Phoebe. 271. 272. Saynrnis nigricans. Black Phoebe. Western section of the State, recorded as breeding at Spring Creek, Tom Green County, and at San Angelo (Lloyd). I have observed this species in May in the southern plains region (Midland county ), but records of its occurance in Texas are few and far between. Sayornis saya. Say’s Phoebe. 272. 273. Sayornis saya. Say’s Phoebe. Winter resident, ranging east to the Colorado (Lloyd). Recorded as migrant and winter resident in the following localities: Fort Brown (Merrill), Lomita (Sennett), Boerne (Brown), San Angelo (Lloyd), Clay County (Ragsdale), Corpus Christi and Beeville (Beckham), San Antonio (Dresser and Attwater). A rather common summer resident of the Panhandle region west of the foot of the plains. Nuttallornis borealis. Olive-sided Flycatcher. 273. 274, Nuttallornis borealis. Olive-sided Flycatcher. Migrant, not very abundant. Recorded from Fort Brown, Lomita, Gainesville, San Antonio, Concho and Tom Green counties. Guada- lupe and Chisos Mountains, probably breeding, (Bailey). Myiochanes virens. Wood Pewee. 274, 275. Myiochanes virens. Wood Pewee. Eastern half of the State, west to San Angelo, summer resident. Breeding at San Antonio, Houston, Waco, San Angelo, on the Lower Rio Grande, etc. Migrating birds recorded from Bonham, Refugio, Gainsville and Mason. Probably breeds throughout its Texas range. 275. 276. Myiochanes richardsonii. Western Wood Pewee. Migrant, occurring as far east as San Angelo, San Antonio, etc. Probably abundant in the Trans-Pecos region during the migrations. Empidonax flaviventris, Yellow-bellied Flycatcher. 276. 277. Empidonax flaviventris, Yellow-bellied Flycatcher. Migrant through Eastern Texas. Recorded from Gainesville (Rags- dale), San Antonio (Dresser), Laredo (Butcher) and Lomita (Sennett). Empidonax difficilis. Western Flycatcher. 277. FAMILY Tyrannidae. Flycatchers. Empidonax difficilis. Western Flycatcher. Summer resident in Chisos and Guadalupe Mountains (Bailey and Oberholser). THE BIRDS OF TEXAS 39 278. Empidonax virescens. Green-crested Flycatcher. Eastern section of the State, summer resident, breeding south to Houston and San Antonio. Recorded from Fort Clark as late as May 15 (Ridgway). Rather common in Anderson and Smith counties in summer. 279. Empidonax traillii. Traill’s Flycatcher. The range of this western species extends as far east as Tom Green and Concho counties, where Lloyd recorded it as a tolerably common breeder. The majority of the observers seem to have overlooked this Flycatcher. 280. Empidonax minimus. Least Flycatcher. Eastern section, west to San Angelo. Lloyd records it as possibly breeding at San Angelo, Cooke at Bonham. Recorded from Kendall and Bexar counties in April and May. Winters south of the Mexican border. 281. 282. Empidonax hammondii. Hammond's Flycatcher. Western Texas, east during the migrations to Tom Green and Concho counties. Rather abundant in Brewster county in April. Empidonax wrightii. Wright’s Flycatcher. 282. Empidonax wrightii. Wright’s Flycatcher. The records for this species are exceedingly few. Ridgway records specimens in the U. S. Nat. Museum collection from El Paso county. Tom Green county, in autumn (Lloyd). Rare migrant in the eastern Davis Mountains in April. Pyrocephalus rubineus mexicanus. Vermilion Fiycatcher. 283. Pyrocephalus rubineus mexicanus. Vermilion Fiycatcher. Southern portion of the State, north to Bexar and Comal counties, where it is a rather common summer resident. Resident on the lower Rio Grande, according to Merrill. In the coast region occurring almost as far north as Corpus Christi. 284. Camptostoma imberbe. Beardless Flycatcher. Casual on the Lower Rio Grande (Lomita, Hidalgo, Rio Crono Grancano [Ridgway] ). FAMILY Alaudidae. Larks. 285. Otocoris alpestris leucolaema. Desert Horned Lark. Plains and trans-Pecos regions of Texas, southeast in winter to San Angelo and Del Rio: breeding from the northern boundary of the Pan- handle south to Fort Davis, etc. East to Henrietta. BAYLOR UNIVERSITY BULLETIN 40 286. Otocoris alpestris praticola. Prairie Horned Lark. Eastern and Central Texas in winter, south as far as San Antonio. Rather irregular in its appearance at Waco, very rare some winters. 287. Otocoris alpestris giraudi. Texas Horned Lark. Coast prairie region from Galveston south to Brownsville. Resident. Otocoris alpestris occidentalis. Montezuma Horned Lark. 288. Otocoris alpestris occidentalis. Montezuma Horned Lark. Winter resident in the western section of the State. Oberholser (“A review of the Horned Larks of the Genus Ofocoris,” Proc. U. S. Nat. Museum, XXIV, 1902, p. 857.) records examples from Marfa, Laredo, Sierra Blanca, Henrietta and Comanche. FAMILY Corvidae. Crows. Jays, etc. 289. Pica pica hudsonica. American Magpie. Occasionally straggles into Western Texas, in the mountains. (McCall, Proc. Acad. Nat. Sci., Phila., 1851, 217, “Western Texas, lat..59 degrees, 15 min., Nov.”) Ridgway (“Birds of North and Middle America”) i in- cludes Texas in the range of this species. Cyanocitta cristata. Blue Jay. 290. 291. Cyanocitta cristata. Blue Jay. Eastern section of the State. Very abundant in East-central Texas (Bosque, Falls, McLennan, Navarro counties). In Southwestern Texas recorded from Leon Springs, San Antonio, Corpus Christi. Ac- cording to Lloyd its western limit in the State “seems to be near the mouth of the main Concho, where it is tolerably common” (The Auk, IV, 290.) Eastland (Hasbrouck). Specimens from the coast regions and San Antonio were formerly referred to the variety florincola. While not typical cristata, Ridgway in “The Birds of North and Mid- dle America” now refers to these under the head of that subspecies, restricting florincola to the peninsula of Florida. Cyanocitta stelleri diademata. Long-crested Jay. 291. 292. Cyanocitta stelleri diademata. Long-crested Jay. Of tolerably common occurance in the Guadalupe and Davis Moun- tains (Oberholser, The Auk, XIX, p. 300.) Aphelocoma woodhousei. Woodhouse Jay. 292. Aphelocoma woodhousei. Woodhouse Jay. Trans-Pecos country, east to the Davis Mountains (Ridgway). Lloyd's record of the occurence of this species in Tom Green and Concho counties, where it “is resident wherever there is shin-oak, at the heads of nearly all the creeks,” refers to the species now known as A. texana Ridgway. THE BIRDS OF TEXAS 41 293. Aphelocoma cyanotis. Blue-eared Jay. Southern portion of the trans-Pecos region (Paisano, Brewster coun- ty,) across the Edwards Plateau to Kerr and Sonora counties. (Ridgway.) 294. Aphelocoma texana. Texan Jay. Southwestern Texas, from Concho and Kerr counties west to the Davis Mountains (Alpine, Fort Davis, Paisano, etc. [Ridgway.]) In the two weeks the writer and his assistants spent at Piasano in 1905, this was the only species of Jay secured by us, although A. cyanotis is also supposed to occur there. We found A. texana nesting in scrub-oaks in secluded canyons. 295. 296. Aphelocoma sieberii couchii. Couch Jay. Chisos Mountains, Brewster county. (Bailey.) Xanthoura luxuosa glaucescens. Rio Grande Green Jay. 296. Xanthoura luxuosa glaucescens. Rio Grande Green Jay. Lower Rio Grande north as far as Laredo. Recorded from Browns- ville, Hidalgo, Lomita, ete. 297. Corvus corax sinuatus. American Raven. FAMILY Corvidae. Crows. Jays, etc. Western Texas, on the plains, east in winter to San Antonio (Dresser). “At the mouth of Devils’s River and at the head of the Rio Grande, in Texas, immense numbers pass the winter and dis- perse again in the spring. In Western Texas its nest has been taken by Mr. Lloyd.” (Cooke, “Bird Migration in the Mississippi Valley.”) White-necked Raven. 298. Corvus cryptoleucus. White-necked Raven. Abundant resident in Western Texas, especially on the southern plains and west of the Pecos. In 1904, Mr. J. M. Carroll and the writer found hundreds of nests in Midland, Pecos, Ward and Jeff Davis counties, The birds were rather more abundant on the plains than they were in the mountains. In 1905, we observed large numbers in Brewster county. Not common on the northern plains (Panhandle) as far as my observations go. “Resident as far east as Tom Green and Concho counties where it is abundant at all times.............. The: bulk retire in fall in large flocks down the Pecos and Devil’s Rivers, where they winter by thousands.” (Lloyd.) Singley mentions a colony on the military reservation at Rio Grande City. BAYLOR UNIVERSITY BULLETIN 42 299. Corvus brachyrhynchos. American Crow. Eastern and coast prairie districts (Galveston, Navarro, and Mc- Lennan counties, etc.) Crows from Middle Texas are interme- diate between this form and the subsperies hesperis, Ridgway referring to the majority of Texas specimens under that name. Resident. 300. Corvus brachyrhynchos hesperis. Western Crow. The crows of Western and Southwestern Texas are referred to this form by Ridgway. In the synonymy of hesperis, he includes the fol- lowing records; San Antonio (Dresser), McClellan Creek (McCauley), Concho county, breeding (Lloyd), Leon Springs, March, (Beckham), Eastland County (Hasbrouck), Boerne (Brown). 301. Cyanocephalus cyanocephalus. Pinon Jay: Blue Crow. Recorded as tolerably common in the Guadalupe Mountains by Bailey (See The Auk, 1902, XIX, 300, and North American Fauna, No. 25, Biological Survey of Texas.) FAMILY ICTERIDAE. Blackbirds, Orioles, Etc. 302. Dolichonyx oryzivorus. Bobolink. Migrant through Eastern and Middle Texas. Either rare in most localities or else has been overlooked by the majority of the ob- servers. Not uncommon in the vicinity of San Angelo in Octo- ber (Lloyd). Rather rare migrant at Waco (October 2-5, April 30, May 1-3.) 303. Molothrus ater. Cowbird. Eastern and northern sections of the State, breeding south to Houston (2) (Nehrling). Resident. Abundant winter resident from San An- tonio southward. Ridgway refers to this species as breeding in the vicinity of San Antonio but Attwater refers to it only as a winter bird. 304. Molothrus ater obscurus. Dwarf Cowbird. Summer resident of the southern section of the State, west into the trans-Pecos counties. North as far as Houston and San Antonio. Ridgway records it from Lampasas county. thrus robustus. Red-eyed Cowbird. Southern section of the State, breeding north to San Antonio. Sum- mer resident. 305. Callothrus robustus. Red-eyed Cowbird. Southern section of the State, breeding north to San Antonio. Sum- mer resident. THE BIRDS OF TEXAS 43 306. Xanthocephalus xanthocephalus. Yellow-headed Blackbird. Abundant migrant, through the greater portion of the State. Winter resident in the Lower Rio Grande valley and certain portions of trans- Pecos Texas. May breed in some localities, but I have no authentic record of its nesting in the State. 307. Agelaius phoeniceus. Red-Winged Blackbird. 307. Agelaius phoeniceus. Red-Winged Blackbird. Abundant migrant and winter resident. Summer resident in Central Texas (South to San Antonio [?]) breeding north to the northern boundary. 308. Agelaius phoeniceus floridanus. Florida Red-wing. 308. Agelaius phoeniceus floridanus. Florida Red-wing. Coast region south to Galveston, summer resident. I hardly think it probable that these birds winter in their summer quarters but doubt- less go further south into the Lower Rio Grande Valley. 309. Agelaius phoeniceus fortis. Northern Red-wing. 309. Agelaius phoeniceus fortis. Northern Red-wing. Ridgway records this subspecies from El] Paso in February. (Prob- ably based on skins in the National Museum.) 310. Agelaius phoeniceus neutralis. San Diego Red-wing. 310. Agelaius phoeniceus neutralis. San Diego Red-wing. According to Ridgway, this is the breeding Redwing of Western Texas, from Langtry, Val Verde county, west in the trans-Pecos country to El Paso. Southeastward in winter to Brownsville. us richmondi. Vera Cruz Red-wing. 311. Agelaius phoeniceus richmondi. Vera Cruz Red-wing. Coast region and Lower Rio Grande, from Velasco to Brownsville, resi- dent (Ridgway). 312. Sturnella magna. Meadowlark. 312. Sturnella magna. Meadowlark. FAMILY ICTERIDAE. Blackbirds, Orioles, Etc. Migrant and winter resident in Eastern Texas, apparently not very common. Probably resident in the counties bordering on the State of Louisiana. The literature in regard to the Texas meadowlarks is in hopeless confusion. At Waco, Sturnella magna is a rare winter visitor. Many of the Western Texas records of this species are based on the Western Lark. magna hoopesi. Texas Meadowlark. 313. Sturnella magna hoopesi. Texas Meadowlark. Rio Grande Valley from Brownsville west to El Paso, resident. North in the coast region to Refugio and Calhoun counties, perhaps further. Sturnella neglecta. Western Meadowlark. 314. Sturnella neglecta. Western Meadowlark. Abundant winter resident throughout the State. Summer resident from the wooded and coast prairie regions of Eastern and Southeastern Texas, westward. Rather uncommon at Waco in summer but abundant in the central prairie districts and on the plains. BAYLOR UNIVERSITY BULLETIN 44 315. Icterus melanocephalus audubonii. Audubon’s Oriole. Lower Rio Grande, casually north to San Antonio, resident. 316. Icterus parisorum. Scott’s Oriole. Trans-Pecos region (El Paso: Pecos River [Ridgway]). Sum- mer resident. Orioles supposed to be of this species were ob- served by our party in the Chisos Mountains in the early part of May, 1905. 317. Icierus cucullatus sennettii. Sennett’s Hooded Oriole. Lower Rio Grande (Cameron and Hidalgo counties) northwest to Laredo. Summer resident. 318. Icterus spurius. Orchard Oriole. Summer resident of the eastern half of the State, breeding from the northern boundary south to the Rio Grande, west to San Angelo, Laredo, etc. Mr. William Winston and the writer found 58 nests of this species at Waco on the 21 day of May, 1897, nearly all of them in mesquite trees. On another occasion I found a female. incubating her four eggs in an old mockingbird nest in a Bois d’ Arc hedge. Her own domicile had probably been destroyed by boys soon after its completion and she was compelled to make shift. 319. Icterus galbula. Baltimore Oriole. Eastern Texas, migrant, not common. Breeds in the extreme north- _ eastern section of the State. At Waco an exceedingly rare migrant, only five specimens being noted in twenty years. Cooke records it as a migrant at Gainesville and Bonham. ' 320. Icterus bullockii. Bullock’s Oriole. Abundant summer resident of the western and southwestern sections of the State. East to Refugio and San Antonio, breeding in both localities. Rare spring visitor at Waco, not known to breed in the vicinity. FAMILY ICTERIDAE. Blackbirds, Orioles, Etc. On the southern plains this is one of the most abundant summer birds. Near Midland, we found six nests in onesmall grove of fifteen trees. Nests were found in mesquite and cat-claw bushes only a few feet from the ground and in some cases within a dozen feet of an occupied nest of Swainson’s Hawk. 321. Euphagus carolinus. Rusty Blackbird. Winters on the eastern coast and in the interior districts of extreme Eastern Texas. South to Harris county (Nehrling). gus carolinus. Rusty Blackbird. Winters on the eastern coast and in the interior districts of extreme Eastern Texas. South to Harris county (Nehrling). THE BIRDS OF TEXAS 45 322. Euphagus cyanocephalus. Brewer’s Blackbird. Middle, Western, and Southern Texas. Abundant winter resident on the Lower Rio Grande, in the coast region north to Refugio and Bee Counties, middle southern Texas in Bexar, Kerr and Kendall counties, etc. Rather rare winter visitor at Waco. Breeds on the southern plains and in the trans-Pecos districts where it is a perma- nent resident. 323. Quiscalus quiscula aeneus. Bronzed Grackle. Abundant resident of Eastern, Northern and Central Texas, breeding as far south as Bexar and Harris counties. West in winter to Tom Green and Concho counties and south into the coast prairie region. Rather rare resident of the Panhandle (Armstrong and Potter counties, etc.) 324. Megaquiscalus major. Boat-tailed Grackle. Coast region south to Houston and Galveston, (where it is said to breed abundantly). Intergrading with VM. m. macrourus (Ridgway). 325. Megaquiscalus major macrourus. Great-tailed Grackle. Coast region from Corpus Christi southward, the Lower Rio Grande and the southern district north to San Antonio, resident. Mr. Attwater informed Mr. J. J. Carroll that he was positive that he had observed this species at Waco, but both Carroll and I were sceptical. In the spring of 1904, Prade reported seeing a couple of specimens and in 1906 I had the pleasure of discovering a small flock in the Brazos bottom. They were associated with Brewer's Blackbirds and Cow- birds. I presume that all of these were straggiers. FAMILY FRINGILLIDAE. The Sparrows and Finches. 326. Carpodacus purpureus. Purple Finch. Irregular winter visitor (Gainesville and Waco). ch. acus cassinii. Cassin’s Purple Finch. Straggles into Northeastern Texas during the winter months. Gaines- ville (Ragsdale). inch. 327. Carpodacus cassinii. Cassin’s Purple Finch. Straggles into Northeastern Texas during the winter months. Gaines- ville (Ragsdale). 328. Carpodacus mexicanus frontalis. House Finch. Western Texas—trans-Pecos region and southern plains, east to Fort Clark. Resident. 328. Carpodacus mexicanus frontalis. House Finch. Western Texas—trans-Pecos region and southern plains, east to Fort Clark. Resident. BAYLOR UNIVERSITY BULLETIN BAYLOR UNIVERSITY BULLETIN 46 329. Loxia curvirostra minor. American Cross-bill. In the winter of 1889-90, the late Chas. D. Oldright obtained spec- imens of this species at Waco (Recorded in the Ornithologist and Ooligist for March, 1890). Prade also reports Cross-bills from Waco. 330. Loxia curvirostra stricklandi. Mexican Cross-bill. Bailey records this sub-species from the Chisos Mountains, probably breeding. 331. Astragalinus tristis. American Goldfinch. This Goldfinch straggles into Northeastern and extreme Eastern Texas in winter (Gainesville, Tyler, Palestine, etc.) 332. Astragalinus tristis pallidus. Western Goldfinch. Western half of the State in winter, resident through the season East to Laredo, San Antonio and the vicinity of Corsicana. 333. Astragalinus psaltria. Arkansas Goldfinch. Western Texas, east to the Angelo country. Recorded as a summer resident of the trans-Pecos region, the southern plains and the San Angelo district. Winters principally in the Rio Grande Valley. 334. Astragalinus psaltria mexicanus. Mexican Goldfinch. Southern and Central Texas (Kinney, Mason, Eastland, Bexar and Travis counties). Summer resident. 335. Astragalinus psaltria arizonae. Arizona Goldfinch. This sub-species straggles east and southeastward during the migra- tions to the Rio Grande Valley (Lomita [Sennett] and San Antonio [Attwater] ). Spinus pinus. Pine Siskin. 336. Spinus pinus. Pine Siskin. Irregular winter visitor to the extreme eastern and northern Gulf coast sections of the State. 337. Calcarius lapponicus. Lapland Longspur. Irregular winter visitor, straggling south into the central portion of the State (Gainesville [Ragsdale] Navarro County, Nov.-Mar. [Ogilby] ). 338. Calcarius pictus. Smith’s Longspur. Northern Texas in winter (Bonham, Gainesville). 339. Calcarius ornatus. Chestnut-collared Longspur. South in winter to Central and Southern Texas (Waco, Gainesville, Boerne, San Antonio). THE BIRDS OF TEXAS 47 340. 341. Rhynchophanes mccownii. McCown’s Longspur. South in winter through middle Texas to the Rio Grande (Laredo, Gal- veston, Gainesville, Waco, etc.) Pooecetes gramineus. Vesper Sparrow. 341. Pooecetes gramineus. Vesper Sparrow. This sub-species migrates through the extreme eastern section to the Gulf coast counties where numbers spend the winter. 342. Pooecetes gramineus confinis. Western Vesper Sparrow. This species winters throughout the State with the exception of the extreme eastern and northern Gulf coast sections. Said to breed near San Antonio and locally through Western Texas. 343. Passerculus sandvichensis savanna. Savannah Sparrow. Winter resident in the extreme eastern section and on the Gulf coast south to Corpus Christi. Passerculus sandvichensis alaudinus. Western Savannah Sparrow. 334. Passerculus sandvichensis alaudinus. Western Savannah Sparrow. BAYLOR UNIVERSITY BULLETIN Common winter resident, occurring throughout the State with the ‘possible exception of the extreme eastern counties. Both this and the eastern form occur at Waco. The present sub-species is recorded from various localities on the coast from Corpus Christi southward, the Rio Grande counties, etc. Recorded as breeding near Houston (Nehrling) and in Western Texas (Lloyd). 345. Centronyx bairdii. Baird’s Sparrow. Migrant through the central and western sections of the State, winter- ing in the trans-Pecos country. East to Gainesville. Recorded by McCauley as breeding in Northern Texas (Canyon City). 346. Coturniculus savannarum bimaculatus. Western Grasshopper Sparrow. Resident. Recorded as summer resident at Boerne, Gainsville, San Angelo, Waco, San Antonio, in Navarro county and various localities in Western Texas. Winters principally in the Rio Grande country. 347. Ammodramus henslowii. Henslow’s Sparrow. Rare winter visitor (Cook County [Ragsdale] Navarro County [Ogil- by]). mus leconteii. LeConte’s Sparrow. 348. Ammodramus leconteii. LeConte’s Sparrow. Common winter resident south to San Antonio and Corpus Christi. Also recorded from Cooke, Navarro, Falls and McLennan counties. 349. Ammodramus caudacutus nelsoni. Nelson's Sparrow. Winter resident along the Gulf coast, south to Neuces Bay (Chapman). BAYLOR UNIVERSITY BULLETIN 48 350. Ammodramus maratimus sennettii. Texas Seaside Sparrow. Coast of Texas, probably resident. Recorded in summer from Galves- ton, Rockport, Tally’s Island, Corpus Christi, Neuces Bay, Refugio county, ete. 351. Ammodramus maratimus fisherii. Fisher’s Seaside Sparrow. Corpus Christi, one specimen (Chapman. As A. m. peninsulae Chap- man, See Ridgway). Probably winters along the northern Gulf coast. 352. Chondestes grammacus. Lark Sparrow. 352. Chondestes grammacus. Lark Sparrow. The eastern Lark Sparrow is recorded as a migrant at Bonham and San Antonio. Winter birds from Waco are intermediate between this and strigatus. 353. Chondestes grammacus strigatus. Western Lark Sparrow. Abundant summer resident. Recorded as breeding from the Lower Rio Grande north to Gainesville, and west through the Panhandle and the more level districts of trans-Pecos Texas. We collected specimens in the Davis Mountains during the breeding season. Winters prin- cipally in the southern section of the State. 354. Zonotrichia querula. Harris's Sparrow. Abundant winter resident of the middle districts, south to Bexar and Kendall counties. 355. Zonotrichia leucophrys. White-crowned Sparrow. Abundant winter resident, from the northern boundary south to the Rio Grande country. 356. Zonotrichia leucophrys gambellii. Gambel’s Sparrow. Winter resident of the western half of the State, southeast ward during the migrations to the Rio Grande Valley. BAYLOR UNIVERSITY BULLETIN “Across the great Plains to Eastern Texas” according to Ridgway, but I have never taken a speci- men in Central Texas. 357. Zenotrichia albicollis. White-throated Sparrow. Common winter resident from Northern Texas to the Rio Grande country. 358. Spizella monticola cchracea. Western Tree Sparrow. Winter resident. Recorded from Gainesville and Tom Green and Concho counties. Winter resident. Recorded from Gainesville and Tom Green and Concho counties. 359. Spizella socialis. Chipping Sparrow. Coast region of Eastern Texas in winter. South to the Lower Rio Grande. THE BIRDS OF TEXAS 49 360. 361. Spizella socialis arizonae. Western Chipping Sparrow. Migrant through the western half of the State. Winters in the Rio Grande Valley. During the migrations east to Gainesville,Boerne, Waco, etc. Recorded as a summer resident in Tom Green, Concho and Bexar counties. Spizella pallida. Clay-colored Sparrow. 361. 362, Spizella pallida. Clay-colored Sparrow. Migrates southeastward to the Rio Grande Valley, where it is a com- mon winter resident. Recorded as breeding in the Panhandle region, “Red River Canyon, edge of the Staked Plains, Paloduro and Red River Valley” (McCauley, Bull. U.S. Geol. and Geog. Surv. Terr., III, 1877, 664.) Spizella breweri. Brewer's Sparrow. 362, 363. Spizella breweri. Brewer's Sparrow. Migrant through Middle and Western Texas. East to Gainesville, Boerne. Reported from El Paso and Tom Green and Concho counties in winter. Spizella pusilla, Field Sparrow. 363. Spizella pusilla, Field Sparrow. Middle and Eastern Texas, summer resident from San Antonio north- ward. South In winter to the Gulf coast (Neuces, San Patricio, and Refugio counties.) Spizella pusilla arenacea. Western Field Sparrow. Spizella pusilla arenacea. Western Field Sparrow. Western Texas, east during the migrations to Boerne, Laredo and the Angelo country. Winters in the Rio Grande region. Spizella wortheni. Mexican Field Sparrow. Spizella wortheni. Mexican Field Sparrow. Western Texas (Sharpe. Cat. Birds, Brit. Mus., XII, 1888, 666.) Junco hyemalis. Slate-colored Junco. Junco hyemalis. Slate-colored Junco. , Winter resident of Eastern and Southern Texas, south to Bexar and Harris counties, west to Tom Green and Concho counties. Junce oreganus shufeldti. Shufeldt’s Junco. 368. Junce oreganus shufeldti. Shufeldt’s Junco. Recorded from Boerne, San Antonio, Gainesville and San Angelo in winter. Junco caniceps. Gray-headed Junco. 368. Junco caniceps. Gray-headed Junco. Winters in El Paso county. May breed in the Guadalupe Mountains ? (Florence Merriam Bailey, Handbook of the Birds of the Western United States.) Junco montanus. Montana Junco. 369. Junco montanus. Montana Junco. BAYLOR UNIVERSITY BULLETIN According to Ridgway, this species occurs east to Middle Texas in winter. The only authenic specimens that \| have seen are two col- lected near Waco by Prade and now in my collection. BAYLOR UNIVERSITY BULLETIN 50. 370. Junco dorsalis. Red-backed Junco. Fort Davis, winter resident (Lloyd). Occurs and probably breeds in the Guadalupe Mountains (Bailey). 371. Amphispiza bilineata. Black-throated Sparrow. Middle and Eastern Texas, except along the Gulf coast. Resident. (Recorded from the Lower Rio Grande, San Antonio, Mason, Eastland county, etc.). 372. Amphispiza bilineata deserticola. Desert Sparrow. Trans-Pecos region, the southern plains and the Panhandle west of the edge of the Staked Plains, summer resident. Winters in the southern part of its breeding range. Amphispiza belli nevadensis. Sage Sparrow. 373. Amphispiza belli nevadensis. Sage Sparrow. Recorded from Fort Davis by Lloyd. Winter resident. 374. Aimophila aestivalis bachmanii. Bachman’s Sparrow. Open woodlands of Eastern and Middle Texas, very locally distributed. Probably resident. Recorded from Cooke, Navarro, Tom Green and Concho counties. Aimophila botterii. Botteri’s Sparrow. Lower Rio Grande Valley (Fort Brown). Summer resident. Aimophila cassinii. Cassin’s Sparrow. Texas, except the extreme eastern section, summer resident. The writer has personally observed this species in McLennan, Falls, Rob- ertson, Somervell, Armstrong, Potter, Midland, Burnet, Travis, Llano, Bexar, Bee, Refugio and Matagorda counties, and in the country west of the Pecos River. 377. Aimophila ruficeps scottii. Scott’s Sparrow. (Resident) in El Paso County (Ridgway). The same author states that specimens from Presidio and Mitchell counties are intermediate between this and eremoeca. 378. Aimophila ruficeps eremoeca. Rock Sparrow. Limestone hill districts of Middle Texas, from Kinney and Maverick counties on the Rio Grands, northeastward to Cooke county and west- ward at least to Tom Green county, south in winter to Mexico. (Ridgway.) The writer has observed this species in Burnet and Travis counties but never in the limestone districts of Coryell and Bos- que counties. THE BIRDS OF TEXAS 51 379. Melospiza cinerea melodia. Song Sparrow. Eastern half of the State, south to the Rio Grande, winter resident. West to Waco and San Antonio. 380. Melospiza cinerea montana. Mountain Song Sparrow. The western section of the State in winter (Fort Clark, Fort Davis, etc.) Recorded from Navarro county by Ogilby but his specimens were probably not typical. BAYLOR UNIVERSITY BULLETIN Song sparrows from Waco are duller colored and have smaller bills than eastern examples but I do not believe that we get typical examples of montana this far east. 381. Melospiza lincolnii. Lincoln’s Sparrow. Abundant winter resident, recorded principally from the eastern half of the State. (Recorded from San Antonio, Brownsville, Waco, Gainesville, Dallas, etc). 382. 383. Melospiza georgiana. Swamp Sparrow. Winter resident, apparently not common or else it has been overlooked by nearly all of the collectors. Recorded from San Antonio, San An- gelo and (?) Fort Davis. 383. Passerella iliaca. Fox-colored Sparrow. Common winter resident. Recorded from as far south as Bexar and Kendall counties. Very common at Waco and in the more eastern counties. Arremonops rufivirgatus. Texas Sparrow. 384. Arremonops rufivirgatus. Texas Sparrow. Southern Texas, summer resident (Webb, Kinney, Starr, Zapata, Cameron, Hidalgo, Neuces, San Patricio counties). 385. Pipilo erythrophthalmus. Towee: Chewink. Eastern and Central Texas, winter resident. Recorded from as far south as San Antonio and west to San Angelo. 386. Pipilo maculatus arcticus. Arctic Towhee. Winters through the middle district south to Bexar and Bandera coun- ties and in the coast region (Corpus Christi). East to Waco. 387. Pipilo maculatus megalonyx. Spurred Towhee. Western Texas, east in winter to Kendall county. A common sum- mer resident of the trans-Pecos country. 388. Pipilo fuscus mesoleucus. Canyon Towhee. Western Texas—trans-Pecos region east to Tom Green county. Res- ident. 52 BAYLOR UNIVERSITY BULLETIN 389. 390. Oreospiza chlorura. Green-tailed Towhee. Western half of the State eastward during the migrations to the Low- er Rio Grande, San Antonio, etc. Breeds in the trans-Pecos region. Recorded from the Middle Concho and the east side of the Pecos, probably breeding, by Lloyd. Cardinalis cardinalis. Cardinal. 390. 391. Cardinalis cardinalis. Cardinal. North-eastern Texas, resident. 390. 391. Cardinalis cardinalis. Cardinal. North-eastern Texas, resident. 391. Cardinalis cardinalis canicaudus. Gray-tailed Cardinal. ‘Texas, except the western and northeastern parts, resident. Speci- mens from Waco come as near being “Gray-tailed Cardinals” as specimens from Rockport, near the type locality, in the writer's collection. 392. 393. Pyrrhuloxia sinuata. Pyrrhuloxia. Trans-Pecos region, resident. Recorded from El Paso by Ridgway. The writer has specimens from the arid strip between Alpine and Ele- phant Mesa, Brewster county. 393. 394. Pyrrhuloxia sinuata texana. Texan Pyrrhuloxia. BAYLOR UNIVERSITY BULLETIN Sonthern Texas, north in the coast region to Refugio and Bee coun- ties, in the interior to Bexar and Kendall counties, west to the Angelo country and Pecos river. 394. 395. Zamelodia ludoviciana. Rose-breasted Grosbeak. Rare migrant. Recorded from San Antonio by Dresser and Attwater. The writer has a male example in full plumage that was shot near Waco by Earl T. Prade. 395. 396. Zamelodia melanocephala. Black-headed Grosbeak. Western Texas, east to Colorado City, San Saba, Mason, Tom Green and Concho counties. Guiraca coerulea. Blue Grosbeak. 396. 397. Guiraca coerulea. Blue Grosbeak. Extreme eastern and northeastern Texas, summer resident. Guriaca coerulea lazula. Western Blue Grosbeak 397. 398. Guriaca coerulea lazula. Western Blue Grosbeak. Texas, except the extreme eastern and northeastern parts, summer resident. (Gainesville, Waco, Austin, Burnet, Navaro county, San Antonio, Fort Brown, Lomita, Hidalgo, Refugio, Laredo, Davis Moun- tains, etc.) Cyanospiza cyanea. Indigo Bunting. 398. Cyanospiza cyanea. Indigo Bunting. Common migrant through the eastern half of the State, west to Tom Green county. Breeds in the northeastern section. THE BIRDS OF TEXAS 53 399. 400. Cyanospiza amoena. Lazuli Bunting. Western Texas, migrant. Summer resident in the trans-Pecos coun- try. Cyanospiza versicolor. Varied Bunting. 400. Cyanospiza versicolor. Varied Bunting. Lower Rio Grande (Cameron and Hidalgo counties), summer resident. 401. 402. Cyanopiza ciris. Painted Bunting. Eastern, Central and Southern Texas, summer resident. Recorded from the Lower Rio Grande, the Red River Vailey in Northern Texas, the eastern and central district, etc. Migrating westward over the southern plains and through the trans-Pecos country. Sporophila morrelleti. Morrelet’s Seed-eater. 402. Sporophila morrelleti. Morrelet’s Seed-eater. Lower Rio Grande (Cameron and Hidalgo counties), summer resi- dent. 403. Spiza americana. Dickcissel. Prairie districts of Eastern Texas from the northern boundary to the Rio Grande. (Recorded as breeding in Cooke, Mc Lennan, a Bexar, Bee, Harris, Hidalgo counties.) 404. Calamospiza melancorys. Lark Bunting. This species migrates through the middle and western districts and coast region from Corpus Christi southward. East to Bonham and Waco. Winters principally in the southern counties. Ihave exam- ples in full breeding plumage collected in the Davis Mountains and at Midland in April and May. The Lark Bunting is probably a breeding bird on the central plains and in the Panhandle. Recorded as a rare summer resident at Mason. 405. Passer domesticus. House Sparrow. This introduced species is now diffused over the entire State. BAYLOR UNIVERSITY BULLETIN I have observed specimens at several small stations in the northern Pan- handle, at Alpine, in Brewster county, and at Boracho, E] Paso county. FAMILY Tanagridae. Tanagers. 406. Piranga ludoviciana. Louisiana Tanager. Trans-Pecos region, summer resident. Lloyd records a single speci- men shot on the south Concho. Breeds in the Transition Zone of both the Davis and Guadalupe Mountains (Bailey). BAYLOR UNIVERSITY BULLETIN 54 407. Piranga erythromelas. Scarlet Tanager. Eastern half of the State, rather rare migrant. West to Eagle Pass (Butcher). Moderately common migrant at Houston (Nehrling). Rare at Waco (only observed twice in twenty years) and at San An- tonio. Dresser’s El Paso breeding record is ridiculous. 408. Piranga hepatica. Hepatic Tanager. Trans-Pecos region, summer resident, breeding in the Davis, Chisos and Guadalupe Mountains (Bailey). 409. 410. Pirango rubra. Summer Tanager. Eastern section of the State, summer resident, breeding from the northern boundary south to the Lower Rio Grande, west to San An- gelo, Laredo, etc. Authentic breeding records from Fort Brown, Lomita, San Antonio, Refugio (Strecker), Gainesville, Mason, Houston, Burnet (Strecker), etc. Piranga rubra cooperi. Cooper’s Tanager. 410. Piranga rubra cooperi. Cooper’s Tanager. Trans-Pecos region, summer resident. We collected an interesting series of these Tanagers in several localities in Brewster county, and I observed numerous specimens in El Paso and Jeff Davis counties in 1904. FAMILY Laniidae. Shrikes. 421. Lanius ludovicianus excubitorides. White-rumped Shrike. Abundant winter resident, occurring throughout the State. Breeds locally in Eastern and Southeastern Texas. Common summer resi- dent of the southern plains and trans-Pecos regions (Midland, Ector, Ward, Reeves, Jeff Davis counties). 422. Lanius ludovicianus migrans. Migrant Shrike. El Paso, February: Fort Clark, January (Ridway). FAMILY Ptilogonatidae. The Silky Flycatchers. 420. Phainopepla nitens. Phainopepla. Trans-Pecos region, summer resident, in winter east to Eagle Pass etc. My specimens are from the Davis Mountains (Paisano, May.). FAMILY Ampelidae. Waxwings. 419. Ampelis cedrorum. Cedar Waxwing. Abundant from October to May, distributed throughcut the State. Resident in some of the mountain ranges of the trans-Pecos region. FAMILY Ptilogonatidae. The Silky Flycatchers. FAMILY Hirundinidae. Swallows. 411. 412. Progne subis. Purple Martin. Summer resident, distributed over the entire State. Petrochelidon lunifrons. Cliff Swallow. 412. 413. Petrochelidon lunifrons. Cliff Swallow. The entire State, with the exception of the Upper Rio Grande Valley from the mouth of the Pecos westward, summer resident. Especially abundant in the canyons and breaks on the plains. Petrochelidon lunifrons tachina. Lesser Cliff Swallow. 413. 414, Petrochelidon lunifrons tachina. Lesser Cliff Swallow. Upper Rio Grande Valley, from the mouth of the Pecos westward, summer resident. Recorded from the Pecos River in Presidio county, Brewster county (Paisano), and Val Verde county (Langtry). (Ridg- way and Oberholser). Hirundo erythrogastra. Barn Swallow. 414, Hirundo erythrogastra. Barn Swallow. Abundant migrant. Breeds locally (San Antonio, Boerne, Valentine [Strecker], and Tyler [Strecker]). 415. Iridoprocne bicolor. Tree Swallow. Common migrant. Winter resident in the coast region. Recorded as a common summer resident at Corpus Christi by Hancock. rocne bicolor. Tree Swallow. Common migrant. Winter resident in the coast region. Recorded as a common summer resident at Corpus Christi by Hancock. THE BIRDS OF TEXAS 55 416, Tachycineta thalassina lepida. Violet-green Swallow. Western Texas, occurring as a fall migrant as far east as Concho county (Lloyd). Probably breeds in the mountains in the trans- Pecos country. 417. 418. Riparia riparia. Bank Swallow. Abundant migrant. Common summer resident, breeding in favorable localities throughout the eastern, central and southern sections. In Concho county recorded only as a migrant. Stelgidopteryx serripenis. Rough-winged Swallow. 418. Stelgidopteryx serripenis. Rough-winged Swallow. Abundant migrant. Summer resident, breeding locally. FAMILY Vireonidae. Vireos. 423. Vireosylva olivacea. Red-eyed Vireo. Eastern Texas, summer resident in the wooded regions. West during the the migrations to Tom Green and Concho counties. een Vireo. . 424. Vireosylva flavoviridis. Yellow-green Vireo. . Accidental in Southern Texas (Fort Brown, one specimen, August 23, 1877, Dr. J. C. Merrill). BAYLOR UNIVERSITY BULLETIN 56 425. Vireosylva gilva Warbling Vireo. Eastern section of the State, summer resident, breeding south to San Antonio (Dresser). 426. Vireosylva gilva swainsonii. ‘Western Warbling Vireo. Western Texas, east to Webb and Tom Green counties. Common in the mountains of trans-Pecos Texas. 427. Lanivireo solitarius. Solitary Vireo. Winter resident of Eastern and Southern Texas, south to the Lower Rio Grande. Not common. 428. Lanivireo solitarius plambeus. Plumbeous Vireo. Tolerably common in the mountains of the trans-Pecos region. Sum- mer resident. 429. Lanivireo flavifrons. Yellow-throated Vireo. Eastern and Southeastern Texas, summer resident as far south as Houston and San Antonio. 430. Vireo atricapillus. Black-capped Vireo. Middle Texas, summer resident. Recorded from Cooke, Comanche, Comal, Bexar, Medina, Erath, Bandera, Kendall, Concho, Tom Green, Travis, and McLennan counties. El Paso and San Pedro River (Baird). 431. Vireo neveboracensis. White-eyed Vireo. Common resident of the eastern half of the State, west in fall to the San Angelo country. Breeds south to Refugio, Bee, and Live Oak counties, in this district intergrading with the next sub-species. 432. Vireo noveboracensis micrus. Small White-eyed Vireo. Rio Grande Valley (Cameron and Hidalgo counties to Kinney county) (Ridgway). Resident. 433. Vireo huttoni stephensi Stephen’s Vireo. Recorded from Fort Davis by Lloyd. The specimens were identified by Ridgway. Probably winter birds. 434. Vireo bellii. Bell’s Vireo. Prairie districts of Eastern and Central Texas, west to the Angelo country, south to the Rio Grande. Summer resident. 435. Vireo bellii medius. Texas Vireo. Southwestern Texas, in Brewster, Presidio and Kinney counties. Type from Boquillas, Brewster county. (Oberholser and Ridgway). THE BIRDS OF TEXAS 57 436. 437. Vireo bellii arizonae. Arizona Vireo. El Paso and Fort Hancock (Ridgway). Vireo vicinior. Gray Vireo. 437. Vireo vicinior. Gray Vireo. Western Texas (A. O. U. Check-List). FAMILY MNIOTILTIDAE. Wood Warblers. 438. Mniotilta varia. Black-and-White Warbler. Eastern Texas, abundant migrant; summer resident in the extreme northeastern and eastern sections. A few probably spend the winter in the Lower Rio Grande country. 439. Protonotaria citrea. Prothonotary Warbler. Summer resident of Eastern Texas, breeding west to Navarro and Mc- Lennan and south to Harris and Fort Bend counties. 440. Helinaia swainsonii. Swainson’s Warbler. Rice, Navarro county, J. Douglas Ogilby (Ridgway, Bull. Nutt. Ornith, Club, VI, 1881, pp. 54-55) Port Bolivar, April 17, 1904 (Cooke, “Dis- tribution and Migration of North American Warblers.”) Prob- ably stragglers. ing Warbler. 441. Helmitheros vermivorus. Worm-eating Warbler. Rather rare migrant in the eastern section. Said to breed in the neighborhood of Houston (Nehrling) but I do not believe it. 442. Helminthophila pinus. Blue-winged Warbler. Eastern Texas, migrant, apparently not very common. (Recorded from Hidalgo, Corpus Christi, San Antonio, Waco.) rbler. 443. Helminthophila chrysoptera. Golden-winged Warbler. Rather common migrant through the eastern part of the State. ricapilla. Nashville Warbler. 444. Helminthophila rubricapilla. Nashville Warbler. Eastern half of Texas, abundant migrant, recorded in nearly every published list or report. West as far as San Angelo. er. 445. Helminthophila rubricapilla gutturalis. Calaveras Warbler. Common migrant in the western half of the State. East to San An- tonio, Concho county, etc. bler. 446. Helminthophila celata. Orange-crowned Warbler. Migrant through the eastern section, west to San Antonio. A few winter on the Lower Rio Grande. BAYLOR UNIVERSITY BULLETIN 58. 447. Helminthophila celata lutescens. Lutescent Warbler. Recorded from the Guadalupe Mountains, probably breeding, by Bailey. I have examples collected in the Davis Mountains in the early part of May. 448. Helminthophila peregrina. Tennessee Warbler. _ Migrant through Eastern Texas, west to the Angelo country. A few winter on the Lower Rio Grande. 449. Compsothlypis americana ramalinae. Western Parula Warbler. Common summer resident of the eastern and southeastern sections. Reported as breeding at Houston, Galveston, Leon Springs, San An- tonio, in Lee and McLennan counties, etc. 450. Compsothlypis pitiayumi nigrilora. Sennett’s Warbler. Summer resident of the Lower Rio Grande counties (Cameron, Starr, Hidalgo.) Not uncommon. 451. Dendroica aestiva. Yellow Warbler. Summer resident in the eastern section of the State. Migrant south of San Antonio. 452. Dendroica aestiva sonorana. Sonoran Yellow Warbler. Western Texas (Frontera, Laredo, Fort Hancock), summer resident. 453. Dendroica coerulescens. Black-throated Blue Warbler. Rather rare migrant through the eastern section (San Antonio, Waco, etc.) 454. Dendroica coronata. Myrtle Warbler. Abundant migrant. FAMILY MNIOTILTIDAE. Wood Warblers. Winter resident from the northern boundary to the Rio Grande country. I have one specimen from Paisano in the Davis Mountains. 455. Dendroica audubonii. Audubon’s Warbler. Western Texas, common migrant. Recorded from as far east as San Angelo. Probably breeds in the Guadalupe Mountains (Bailey). 456. Dendroica magnolia. Magnolia Warbler. 456. Dendroica magnolia. Magnolia Warbler. Rather common migrant through the eastern section (San Antonio, Waco, Fort Brown, etc.) 457. Dendroica caerulea. Cerulean Warbler. Rare migrant through the eastern section of the State, west to the Angelo country (Lloyd). ' THE BIRDS OF TEXAS 59 458. Dendroica pensilvanica. Chestnut-sided Warbler. Rare migrant. The only records I can find are from the Lower Rio Grande (Hidalgo [Sennett], Fort Brown [Merrill]). Probably over- looked by some of the observers, but I am positive that it does not occur in the vicinity of Waco. 459. Dendroica castanea. Bay-breasted Warbler. Rather uncommon migrant in the eastern section (Houston, San An- tonio, Waco, Fort Brown). 460. 461. Dendroica fusca. Blackburnian Warbler. This species migrates through the eastern half of the State, but is by no means common (Boerne, Gainesville, Waco, Fort Brown). Dendroica dominica albilora. Sycamore Warbler. 461. Dendroica dominica albilora. Sycamore Warbler. Eastern and Southeastern Texas, summer resident, not uncommon. Recorded as breeding at San Antonio. near Houston, in Lee county, ete. \| Dendroica graciae. Grace’s Warbler. 462. 463. Dendroica graciae. Grace’s Warbler. Recorded from the Guadalupe Mountains, E] Paso county, probably breeding (Bailey). Dendroica chrysoparia. Golden-cheeked Warbler. 463. Dendroica chrysoparia. Golden-cheeked Warbler. Central and Southern Texas, summer resident. Recorded from Bosque, McLennan (one specimen), Comal, Kerr, Kendall, Tom Green, Concho, etc., counties. Dendroica striata. Black-poll Warbler. 464. Dendroica striata. Black-poll Warbler. Rare migrant in the eastern section (Fort Brown, Waco, etc.) 465. Dendroica virens. Black-throated Green Warbler. The eastern and central portions of Texas, west to the Angelo country, common migrant. 466. Dendroica townsendi. Townsend’s Warbler. Not uncommon migrant through the western section. Reported as a rare summer resident of Tom Green county by Lloyd. 467. Dendroica vigorsii. Pine Warbler. Eastern Texas, summer resident of the pine-woods district. West dur- ing the migrations to Lee county. 468. Dendroica discolor. Prairie Warbler. Cooke (“Distribution and Migration of North American Warblers”) reports having examined a single example of this Warbler from Texas, but fails to state specific locality. BAYLOR UNIVERSITY BULLETIN 60 469. Seiurus aurocapillus. Ovenbird. Migrant through the eastern half of the State, rather common. FAMILY MNIOTILTIDAE. Wood Warblers. (Waco, San Antonio, Giddings [Singley MSS.] Concho county, etc.) 470. Seiurus noveboracensis. Water Thrush. 470. Seiurus noveboracensis. Water Thrush. Common migrant through the eastern section, west to San Antonio, Waco, etc. Winter resident on the Lower Rio Grande. In mild winters remaining in Central Texas through the season. 471. Seiurus noveboracensis notabilis. Grinnell’s Water Thrush. Migrant through Eastern and Southeastern Texas. (Hidalgo, San An- tonio, etc.) 472. Seiurus motacilla. Louisiana Water Thrush. Migrant through Eastern and Southeastern Texas. (Hidalgo, San An- tonio, etc.) 472. Seiurus motacilla. Louisiana Water Thrush. 472. Seiurus motacilla. Louisiana Water Thrush. Eastern Texas, common migrant, rare summer resident. 473. Oporornis formosa. Kentucky Warbler. 473. Oporornis formosa. Kentucky Warbler. Summer resident, breeding locally as far south as Houston, west to Waco. 474. Oporornis philadelphia. Mourning Warbler. 474. Oporornis philadelphia. Mourning Warbler. Common migrant through the eastern section. 474. Oporornis philadelphia. Mourning Warbler. Common migrant through the eastern section. 475. Oporornis tolmei. Macgillivray’s Warbler. Abundant migrant in the western section, summer resident in the trans-Pecos mountains. Straggling eastward during the migrations to Gainesville, San Antonio. 476. Geothlypis trichas occidentalis. Western Yellow-throat. Migrant through the western section, east to Waco, Gainesville, Gid- dings. 477. Geothlypis tikes brachidactyla. Northern Yellow-throat. 477. Geothlypis tikes brachidactyla. Northern Yellow-throat. Abundant migrant through the eastern and central sections. Sum- mer resident, according to Cooke. Abundant migrant through the eastern and central sections. Sum- mer resident, according to Cooke. 478. Chamaethlypis poliocephala. Ralph’s Ground Chat. A Mexican species found in summer in the vicinity of Brownsville, Cameron county. 479, Icteria virens. Yellow-breasted Chat. 479, Icteria virens. Yellow-breasted Chat. ‘Eastern Texas, summer resident, breeding from the northern boundary south to the Lower Rio Grande. West to Austin and Waco. At the latter place it is an abundant migrant but is very rare during the breeding season. 480. Icteria virens longicauda. Long-tailed Chat. 480. Icteria virens longicauda. Long-tailed Chat. Western Texas, summer resident. (Breeding in Tom Green and Con- cho counties, etc.) East during the migrations through the Lower Rio Grande. THE BIRDS OF TEXAS 61 Wilsonia citrina. Hooded Warbler. Eastern section of the State, common migrant, rare summer resident. Wilsonia pusilla. Wilson’s Warbler. Eastern section of the State, common migrant, rare summer resident. Wilsonia pusilla. Wilson’s Warbler. Common migrant through the eastern section. Wilsonia pusilla. Wilson’s Warbler. Common migrant through the eastern section. Wilsonia pusilla pileolata. Pileolated Warbler. 484. Wilsonia pusilla pileolata. Pileolated Warbler. FAMILY Cinclidae. Dippers. 488. Cinclus mexicanus unicolor. American Dipper. Recorded from western Texas by McCall (Proc. Acad. Nat. Sci. Phila., V. 1851, p. 216). \| Western Texas is also included in the range of this species by Ridgway (Birds of North and Middle America). FAMILY Motacillidae. Wagtails. Pipits, etc. 485. Anthus rubescens. American Pipit. Abundant migrant and common winter resident, distributed over the entire state. Anthus spragueii. Sprague’s Pipit: Missouri Skylark. 487. Anthus spragueii. Sprague’s Pipit: Missouri Skylark. Migrant and winter resident, not as abundant as the last species. Recorded from Cameron, Kendall, Tom Green, Concho, Harris, Bexar, Neuces, and McLennan counties. FAMILY MNIOTILTIDAE. Wood Warblers. Guadalupe Mountains, probably breeding (Bailey). We observed a small flock of these warblers in the Davis Mountains in the early part of May and obtained two males. Wilsonia canadensis. Canadian Warbler. 484. 485. Wilsonia canadensis. Canadian Warbler. Eastern half of the State, migrant, not uncommon. (Houston, San Antonio, Hidalgo, San Angelo, Fort Brown.) I have never observed a specimen of this warbler in East-central Texas. Setophaga ruticilla. American Redstart. 485. Setophaga ruticilla. American Redstart. Common migrant through the eastern half of the State. FAMILY Motacillidae. Wagtails. Pipits, etc. FAMILY Mimidae. Mocking Thrushes. 489. Oroscoptes montanus. Sage Thrasher. Western Texas, in summer to Tom Green and Concho counties: in winter east to the eastern and coast sections. (Corpus Christi, Re- fugio, Waco, San Antonio, etc.) 490. Mimus polyglottos. Mockingbird. Eastern and coast sections, south to Galveston, resident. BAYLOR UNIVERSITY BULLETIN 62 491. Mimus polyglottos leucopterus. Western Mockingbird. This subspecies is a resident of the entire State with the exception of the extreme eastern section. Waco specimens are intermediate between this and typical polyg/lottos. 492. Dumetella carolinensis. Catbird. Eastern Texas, south along the coast to the RioGrande. Vest to Waco and San Antonio. Resident in some of the eastern counties, breeding as far south as Houston (Nehrling). At Waco and San Antonio, mi- grant and irregular winter resident, not common. 493. Toxostoma rufum. Brown Thrasher. Common migrant and winter resident in the extreme eastern section of the State. South to Bexar and Harris counties. Rare winter resi- dent in the vicinity of Waco. 494. 495. Toxostoma longirostre sennettii. Sennett Thrasher. Rio Grande Valley and southern Gulf coast of Texas. (Neuces, Cam- eron, Hidalgo, Starr, Webb, Zapata counties, occasionally along the coast to Galveston [Ridgway.]) 495. Toxostoma curvirostre. Curve-billed Thrasher. Rio Grande Valley of Texas—Cameron, Hidalgo, Starr, Webb, Maver, ick, south Gulf coast (Neuces, San Patricio and Refugio counties)- westward along the Rio Grande through the trans-Pecos region to El Paso. 496. Toxostoma crissale. Crissal Thrasher. Deserts of Western Texas (Fort Hancock, Ysleta, Franklin Mountains El] Paso and Brewster counties, etc [Ridgway] ). FAMILY Troglodytidae. Wrens. 497. Heleodytes brunneicapillus couesi. Texas Cactus Wren. Rio Grande Valley, north to San Antonio, west across the southern plains and trans-Pecos regions. Resident. Salpinctes obsoletus. Rock Wren. 498. Salpinctes obsoletus. Rock Wren. Western Texas, east to the central districts (Bexar, Travis, Kendall counties). A common bird in the trans-Pecos region and in the can- yons on the plains. Summer resident, wintering in the Rio Grande Valley and southward. 499. Catherpes mexicanus albifrons. Giraud’s Canyon Wren. Recorded from Langtry, Val Verde county, by 499. Catherpes mexicanus albifrons. Giraud’s Canyon Wren. Recorded from Langtry, Val Verde county, by Oberholser. THE BIRDS OF TEXAS 63 500. Catherpes mexicanus polioptilus. Intermediate Canyon Wren. Central and Central Southern Texas, west thryough the trans-Pecos country and north through the Panhandle. In Central Texas recorded from Bosque, Travis and Burnet counties, and from Kerr, Kendall and Bexar in the more southern portion of the same region. Resident. 501. Thryothorus ludovicianus. Carolina Wren. Eastern and Middle Texas (west to Val Verde and Tom Green coun- ties). Repiaced on the Lower Rio Grande by the Lomita Wren. Res- ident. 502. Thryothorus ludovicianus lomitensis. Lomita Wren. Lower Rio Grande Valley (Lomita ranch, Hidalgo, Brownsville, Gran- cano [Ridgway] ). 503. Thryomanes bewickii. Bewick’s Wren. Eastern and Central Texas (Waller, Brazos, Jefferson, Orange, Brazo- ria, ete., counties) in winter. 504. Thryomanes bewickii cryptus. Texan Wren. Texas, except extreme western portion. Resident. Very abundant in the southern and central sections of the State. Thryomanes bewickii eremophilus. Desert Wren. 505. Thryomanes bewickii eremophilus. Desert Wren. Extreme western portion of Texas, east to central parts in winter. (Oberholser). 506. Troglodytes aedon. House Wren. Eastern Texas in coast region, fall and winter (Neuces Bay, Dec.; Alice, Oct. 5,; Santa Rosa, Cameron county, Sept. 27 [Ridgway ]). parkmanii. Western House Wren. 507. Troglodytes aedon parkmanii. Western House Wren. Abundant migrant. Winter resident in the southern section of the State. May possibly breed in Northern Texas. Wren. 508. Nannus hiemalis. Winter Wren. Rare winter visitor, occurring as far south as San Antonio. Marsh Wren. 508. Cistothorus stellaris. Short-billed Marsh Wren. Rare migrant and winter resident, principally in the coast region. Recorded from Lee county (Singley), San Antonio (Attwater), etc. Prairie Marsh Wren. 510. Telmatodytes palustris iliacus. Prairie Marsh Wren. Fort Brown and Corpus Christi (Ridgway). Winter birds. plesius. Western Marsh Wren. 511. Telmatodytes palustris plesius. Western Marsh Wren. Western and Central Texas, migrant and winter resident. FAMILY Sittidae. Nuthatches. 514. Sitta carolinensis. White-breasted Nuthatch. Eastern Texas, west to Gainesville, resident. According to Beckham, occurs south to San Antonio and Leon Springs. In the twenty years the writer has lived at Waco, he has never observed a single specimen of this species. 515. Sitta carolinensis nelsoni. Rocky Mountain Nuthatch. Trans-Pecos region in the Davis, Chisos and Guadalupe Mountains (Bailey). East to Concho and Tom Green counties in winter (Lloyd. As Sitta carolinensis aculeata). Sitta canadensis. Red-breasted Nuthatch. 516. Sitta canadensis. Red-breasted Nuthatch. Irregular winter resident in Eastern and Southeastern Texas, south to San Antonio. During severe winters this species is not uncommon in the vicinity of Waco. 517. Sitta pusilla. Brown-headed Nuthatch. Eastern Texas (Tyler, Jasper, Jefferson, Harris, Montgomery, Rockland counties, etc.), resident. 518. Sitta pygmaea. Pygmy Nuthatch. Common in the Guadalupe Mountains. Resident. (Oberholser, The Auk, XIX, 1902, p. 301). FAMILY Certhiidae. Creepers. 513. Certhia familiaris americana. Brown Creeper. Winter resident in Eastern Texas, south to Bexar, Medina and Kendall counties. FAMILY Troglodytidae. Wrens. (San An- tonio [Attwater], Waco, etc.) 64. BAYLOR UNIVERSITY BULLETIN 512. Telmatodytes palustris thryophilus. Louisiana Marsh Wren. Sabine, Texas, collection U.S. Nat. Museum (Oberholser, Proc. Biol. Soc. Wash., XVI, 1903, p. 149). FAMILY Paridae. Titmice. 519. Baelophus bicolor. Tufted Titmouse. Eastern section of Texas, south and west to Matagorda, Victoria and Bexar counties. Resident. Rather common at Waco. 520. Baelophus atricristatus. Black-crested Titmouse. Rio Grande Valley, from Brownsville west to El Paso. Very abundant in the trans-Pecos country, resident. 520. Rio Grande Valley, from Brownsville west to El Paso. Very abundant in the trans-Pecos country, resident. THE BIRDS OF TEXAS 65 521. 522. Baelophus atricristatus sennettii. Sennett’s Titmouse. Central Texas, east to the valley of the Brazos (Young county to Harris county), south to Neuces and Bee counties, west to Tom Green and Concho counties, north to Young county. (Ridg- way.) Baelophus inornatus griseus. Gray Titmouse. 522. 523. Baelophus inornatus griseus. Gray Titmouse. Fairly common in the Guadalupe Mountains (Oberholser). Penthestes carolinensis agilis. Texan Chickadee. 523. 524. Penthestes carolinensis agilis. Texan Chickadee. Eastern and Central Texas (south to Bee and Refugio counties, west to Bexar, Kendall and eastern Comal counties). Parus gambeli. Mountain Chickadee. 524. 525. Parus gambeli. Mountain Chickadee. Guadalupe and Davis Mountains (Oberholser, The Auk, XIX, 1902, 301.) Psaltriparus melanotis lloydi. Lloyd's Bush-Tit. 525. 526. Psaltriparus melanotis lloydi. Lloyd's Bush-Tit. Guadalupe, Davis and Chisos Mountains (Bailey.) Psaltriparus plumbeus. Lead-colored Bush-Tit. 526. 527. Psaltriparus plumbeus. Lead-colored Bush-Tit. Mountains of the trans-Pecos region. We collected examples of both species of Bush-Tits in the Davis Mountains in April and May. Auriparus flaviceps. Verdin. 527. Auriparus flaviceps. Verdin. Southern Texas, north to Bexar and Refugio counties, west to El Paso. Summer resident at Refugio and San Antonio but probably winters on the Lower Rio Grande. FAMILY Sylviidae. Warblers, Kinglets, Gnatcatchers. 528. Regulus satrepa. Golden-crowned Kinglet. Winter visitor, rather irregular in appearance, occurring south to Houston and San Antonio. Severe winters a few remain at Waco throughout the season. 529. Regulus calendula. Ruby-crowned Kinglet. Abundant migrant and winter resident, from the northern boundary to the Rio Grande. 530. Polioptila caerulea. Blue-gray Gnatcatcher. Eastern half of the State, summer resident, breeding from the northern boundary south to the Rio Grande. 531. Polioptila caerulea obscura. Western Gnatcatcher. Trans-Pecos region, summer resident. 531. Polioptila caerulea obscura. Western Gnatcatcher. Trans-Pecos region, summer resident. BAYLOR UNIVERSITY BULLETIN 66 532. Pelioptila plumbea. Plumbeous Gnatcatcher. Abundant summer resident of the high mountains of the trans-Pecos region, east during the migrations to Rio Grande City (Oberholser: Ridgway). FAMILY Turdidae. Thrushes, Bluebirds, etc. Myadestes townsendii. Townsend's Solitaire. Western Texas, resident, rare in summer. Reported as breeding at Saragossa by Lloyd. Prade shot one example in the Davis Mountains in the middle of May. Hylocichla mustelina. Wood Thrush. 534. Hylocichla mustelina. Wood Thrush. Eastern section of the State, summer resident. Reported as a mi- grant from most localities south of the northeastern corner. Rare migrant at Waco. 535. Hylocichla fuscescens. Wilson’s Thrush. Migrant, not very common, through the eastern half of the State. Hylocichla fuscescens salicicola. Willow Thrush. 537. Hylocichla fuscescens salicicola. Willow Thrush. Rare migrant. The only authentic record I could find was the speci- men from Cooke county (Gainesville) mentioned in Cook’s “Bird Mi- gration in the Mississippi Valley.” Hylocichla aliciae. Gray-cheeked Thrush. 537. Hylocichla aliciae. Gray-cheeked Thrush. Migrant through the eastern half of the State. Rare at Gaines- ville (Ragsdale), common at San Antonio (Attwater), rare at Waco. Hylocichla ustulatus swainsonii. Olive-backed Thrush. Migrant, not common. Reported from Gainesville (Ragsdale), Tom Green county (Lloyd) and Waco. Hylocichla guttata. Kadiak Dwarf Thrush. Migrant througn the western half of the State. Ridgway mentions this thrush from the following localities: El Paso, Tom Green and Concho counties, San Antonio, Leon Springs, Langtry, Mouth of the Pecos. Hylocichla guttata auduboni. Rocky Mountain Hermit Thrush. Hylocichla guttata auduboni. Rocky Mountain Hermit Thrush. Migrates through Western Texas as far east as San Antonio. Bailey records it from the Guadalupe Mountains, probably breed- ing. THE BIRDS OF TEXAS 67 541. 542. Hylocichla guttata pallasii. Hermit Thrush. Migrant through the eastern half of Texas. Recorded from San An- tonio, January to March; Corpus Christi, March; Leon Springs, March; Gainesville, March; Fort Clark, December 27; Kendall county, March. (Ridgway and Cooke). Observed by the writer in McLennan and Falls counties in mild winters from the last of November to the middle of March. Planesticus migratorius. American Robin. 542. 543. Planesticus migratorius. American Robin. Abundant migrant and winter resident of the eastern half of the State, west as far as Tom Green and Concho counties. Planesticus migratorius propinguus. Western Robin. 543. Planesticus migratorius propinguus. Western Robin. Abundant migrant and winter resident of Western Texas, occurring as far east as Kendall county. Guadalupe Mountains, probably breed- ing (Bailey). I have specimens of this sub-species that were shot at Paisano as late as May 5, and am positive that others were seen later in the month in the same vicinity. 544. 545. FAMILY Turdidae. Thrushes, Bluebirds, etc. Sialia sialis. Bluebird. Eastern half of the State, west to Val Verde county and the edge of the plains, resident, breeding locally from the northern border to the Rio Grande. Sialia mexicana bairdi. Chestnut-backed Bluebird. 545. Sialia mexicana bairdi. Chestnut-backed Bluebird. Western Texas, east during the migrations to Kendall, Tom Green and Concho couuties. Breeds in the mountains of the trans-Pecos region (Bailey). 546. Sialia arctica. Mountain Bluebird. Migrant and winter resident in trans-Pecos section. During the migra- tions more or less irregularly over nearly the whole State (south to Corpus Christi and Bonham.) BAYLOR UNIVERSITY BULLETIN Hypothetical List 1. (382.1) Rhynchopsitta pachyrhyncha Swainson. Thick-billed Parrot. May straggle across the Rio Grande River from Mexico. Has been col- lected in Arizona. May straggle across the Rio Grande River from Mexico. Has been col- lected in Arizona. 2. (420c) Chordeiles virginianus sennettii Coues. Sennett’s Nighthawk. Great Plains, south to Texas (A. O. U. Check-List). No specific local- ities for this species. 3. (—) Otocoris alpestris chrysolaama Wagler. Mexican Horned Lark. 3. (—) Otocoris alpestris chrysolaama Wagler. Mexican Horned Lark. =Alauda minor Giraud. “Texas.” Inhabits the southern portion of the Mexican Plateau. Only three of Giraud’s “Sixteen Species of Tex- as Birds” have ever been collected within the limits of Texan territory. These are Icterus audubonii Giraud, Catherpes mexicanus albifrons (Giraud) (=Certhia albifrons Giraud) and Astragalinus psaltria mex- icanus Swainson (—Fringilla texensis Giraud ). 4. ([450]) Myiozetetes texensis Giraud. Giraud’s Flycatcher. 4. ([450]) Myiozetetes texensis Giraud. Giraud’s Flycatcher. =Muscicapa texensis Giraud. “Texas.” Southern Mexico. A. O. U. Check-List on authority of Giraud’s “Texas” specimen. 5. ([455]) Myiarchus lawrenceii Giraud. Lawrence Flycatcher. 5. ([455]) Myiarchus lawrenceii Giraud. Lawrence Flycatcher. =Muscicapa lawrenceii Giraud. “Texas.” Included in the North American list on the above authority. An inhabitant of Eastern and Southern Mexico. 6. ({470]) Empidonax fulvifrons Giraud. Fulvous Flycatcher. =Muscicapa fulvifrons Giraud. “Texas.” Probably mountains of Northeastern Mexico (Ridgway). Northeastern Mexico (Ridgway). 7. (490) Corvus ossifragus Wilson. Fish Crow. This species is resident on the coast of Louisana. 7. (490) Corvus ossifragus Wilson. Fish Crow. This species is resident on the coast of Louisana. 8. (541) Ammodramus princeps Maynard. Ipswich Sparrow. Recorded from Dallas by Sennett (The Auk, III, 1886, p. 135). Prob- ably an error, arising, as suggested by Cooke (“Bird Migration in the Mississippi Valley, 1888, p. 188) from a transposition of labels. THE BIRDS OF TEXAS 69 9. (565) Spizella atrigularis Cabanis. Black-chinned Sparrow. North to the Upper Rio Grande in Texas. (Cooke, “Bird Migration in the Mississippi Valley”). I can find no definite locality records for this species, if it really occurs in Western Texas. 10. ({606]) Euphonia elegantissima Bonaparte. Blue-headed Euphonia. =Pipra galericulata Giraud. “Texas.” Inhabits Southern Mexico. 11. (650) Dendroica tigrina Gmelin. Cape May Warbler. 10. ({606]) Euphonia elegantissima Bonaparte. Blue-headed Euphonia. =Pipra galericulata Giraud. “Texas.” Inhabits Southern Mexico. 11. (650) Dendroica tigrina Gmelin. Cape May Warbler. 11. (650) Dendroica tigrina Gmelin. Cape May Warbler. I can find no Texas records for this species. 12. Hypothetical List (651) Peucedramus olivacea Giraud. Olive Warbler. 11. (650) Dendroica tigrina Gmelin. Cape May Warbler. I can find no Texas records for this species. 12. (651) Peucedramus olivacea Giraud. Olive Warbler. 12. (651) Peucedramus olivacea Giraud. Olive Warbler. =Sylvia olivacea Giraud. “Texas.” Highlands of Guatamala and Mexico, north to mountains of Central Arizona. 13. (672) Dendrioca palmarum Gmelin. Palm Warbler. 13. (672) Dendrioca palmarum Gmelin. Palm Warbler. This warbler winters in Louisiana and (probab 14, (665) Dendroica nigrescens Townsend. Black-throated 13. (672) Dendrioca palmarum Gmelin. Palm Warbler. This warbler winters in Louisiana and (probably) Eastern Texas. 14, (665) Dendroica nigrescens Townsend. Black-throated Gray Warbler. 13. (672) Dendrioca palmarum Gmelin. Palm Warbler. This warbler winters in Louisiana and (probably) Eastern Texas. 14, (665) Dendroica nigrescens Townsend. Black-throated Gray Warbler. 14, (665) Dendroica nigrescens Townsend. Black-throated Gray Warbler. =Sylvia halseii Giraud. “Texas.” Western mountain States. 15. (688) Setophaga picta Swainson. Painted Redstart. 15. (688) Setophaga picta Swainson. Painted Redstart. =Muscicapa leucosmus ‘Giraud. “Texas.” ‘Higher mountains ‘of . Northern Mexico and Southern and ‘Central Arizona. 16. ([689]) Setophaga miniata Swainson. Red-bellied ‘Redstart. =Muscicapa derhamii Giraud. “Texas.” Mexico. ellina rubrifrons Giraud. Red-faced Warbler. 17. (690) Cardellina rubrifrons Giraud. Red-faced Warbler. =Muscicapa rubrifrons Giraud. “Texas.” Higher mountains of Northern Mexico, Southern Arizona, etc. ber Swainson. Red Warbler. 18. ({691]) Ergaticus ruber Swainson. Red Warbler. =Parus leucotis Giraud. “Texas.” An inhabitant of the highlands of Mexico. Basileuterus culicivorus brasherii Giraud. Brasher’s Warbler. 19. ({692]) Basileuterus culicivorus brasherii Giraud. Brasher’s Warbler. —Muscicapa brasierii Giraud. “Texas.” Northeastern Mexico. bellii Giraud. Bell’s Warbler. 19. ({692]) Basileuterus culicivorus brasherii Giraud. Brasher’s Warbler. —Muscicapa brasierii Giraud. “Texas.” Northeastern Mexico Giraud. Bell’s Warbler. uscicapa brasierii Giraud. “Texas.” Northeastern Mexico. rus bellii Giraud. Bell’s Warbler. 20. ({693]) Basileuterus bellii Giraud. Bell’s Warbler. —Muscicapa bellii Giraud. “Texas.” Southeastern Mexico. 20. ({693]) Basileuterus bellii Giraud. Bell’s Warbler. —Muscicapa bellii Giraud. “Texas.” Southeastern Mexico. Ba —Muscicapa bellii Giraud. “Texas.” Southeastern Mexico. OMISSIONS Page 24. (Bottom of page.y After ORDER Columbae add FAMILY Columbidae. Page 37. (Under Tyrannus tyrannus.) Last sentence should read: “Rather com- mop summer resident at Waco. OMISSIONS Page 24. (Bottom of page.y After ORDER Columbae add FAMILY Columbidae. Page 37. (Under Tyrannus tyrannus.) Last sentence should read: “Rather com- mop summer resident at Waco. OMISSIONS Page 24. (Bottom of page.y After ORDER Columbae add FAMILY Columbidae. Page 37. (Under Tyrannus tyrannus.) Last sentence should read: “Rather com- mop summer resident at Waco.
https://openalex.org/W1988267138	https://europepmc.org/articles/pmc3358313?pdf=render	English	null	Wheat TaRab7 GTPase Is Part of the Signaling Pathway in Responses to Stripe Rust and Abiotic Stimuli	PloS one	2,012	cc-by	8,328	Abstract CARS-3-1-11) and the 111 Project from Ministry of Education of China (No. B07049). The funders had no role in study design ysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Kangzs@nwsuaf.edu.cn * E-mail: Kangzs@nwsuaf.edu.cn . These authors contributed equally to this work. . These authors contributed equally to this work. converts Rab to its GTP-bound, active conformation, allowing it to interact with its downstream effectors. Rabs regulate cell proliferation, cytoskeleton organization, intracellular membrane trafficking and vesicle motility along the actin/microtubule cytoskeletons, vesicle tethering, transport, and fusion [6,7]. Many downstream effectors of Rab7 in mammals have been extensively characterized and shown to interact with their partners to exert biological functions. Rab7 and its downstream effectors are important factors in the pathogenesis of microorganisms. Rab7 is a key regulator in the process of phagosome maturation [8–11]. Rab7 and the Rab interacting lysosomal protein (RILP) are essential factors in regulating the maturation of the phagosome into a lysophagosome [12,13]. In addition, the homotypic fusion and vacuole protein sorting (HOPs) may play dual roles as upstream GEF and downstream tethering effector of Rab7 to facilitate endosomal membrane fusion [14]. Furong Liu1., Jun Guo1., Pengfei Bai1, Yinhui Duan2, Xiaodong Wang1, Yulin Cheng1, Hao Feng1, Lili Huang1, Zhensheng Kang1* Furong Liu1., Jun Guo1., Pengfei Bai1, Yinhui Duan2, Xiaodong Wang1, Yulin Cheng1, Hao Feng1, Lili Huang1, Zhensheng Kang1* 1 State Key Laboratory of Crop Stress Biology for Arid Areas and College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China 2 State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Science, Northwest A&F University, Yangling, Shaanxi, People’s Republic of China PLoS ONE \| www.plosone.org Abstract Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst) the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI) of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like) from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance. Citation: Liu F, Guo J, Bai P, Duan Y, Wang X, et al. (2012) Wheat TaRab7 GTPase Is Part of the Signaling Pathway in Responses to Stripe Rust and Abiotic Stimuli. PLoS ONE 7(5): e37146. doi:10.1371/journal.pone.0037146 Editor: Zhengguang Zhang, Nanjing Agricultural University, China Editor: Zhengguang Zhang, Nanjing Agricultural University, China Received February 6, 2012; Accepted April 17, 2012; Published May 22, 2012 Copyright: 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted production in any medium, provided the original author and source are credited. Funding: This work was financially supported by National Natural Science Foundation of China (No. 30930064 and 31171795), the Agriculture Research System (No. CARS-3-1-11) and the 111 Project from Ministry of Education of China (No. B07049). The funders h data collection and analysis, decision to publish, or preparation of the manuscript. financially supported by National Natural Science Foundation of China (No. 30930064 and 31171795), the earmarked fund for China em (No. May 2012 \| Volume 7 \| Issue 5 \| e37146 Cloning and phylogenetic analyses of TaRab7 Cloning and phylogenetic analyses of TaRab7 A wheat small GTP binding protein gene, TaRab7, was cloned by the reverse-transcription technique. Sequence analysis in- dicated that TaRab7 includes a complete 618 bp open-reading frame (ORF) which encodes a putative protein composed of 206 amino acids. The predicted protein of TaRab7 had a theoretical molecular weight of 23.13 kD with an isoeletric point (pI) of 5.13. The analysis by InterProScan indicated that TaRab7 contains domains for guanine nucleotide binding that are conserved among members of the Rab subfamily. In addition, it is highly homologous at the protein level to Rab7 in Oryza sativa, Hordeum vulgare, Arabidopsis thaliana and other organisms (Fig. 1). In all eight Rab subfamilies in Arabidopsis, one protein sequence was selected from each subfamily, four Rab proteins from both rice and Brachypodium distachyon, and two other Rab7 proteins from soybean and barley were downloaded from the GenBank to construct a neighbor-joining phylogenetic tree (Fig. 2). The results suggested a high level of conservation among Rab7 proteins from different plant species (Arabidopsis RabG3b protein is related to mammalian Rab7 [19]), especially between the monocotyledonous wheat and Brachypodium distachyon. The BSMV:TaRab7-inoculated plants also displayed mild cholorotic mosaic symptoms by 9 dpi but expressed no significant defects in further leaf growth (Fig. 6). qRT-PCR was applied to examine the relative expression levels of TaRab7 in the fourth leaves of infected plants. In comparison with BSMV:c-infected wheat leaves, the expression level of TaRab7 in leaves infected with BSMV: TaRab7 was reduced at an average of 70% at each time point, respectively, after inoculation with the incompatible Pst race CYR23 (Fig. 7). Then the fourth leaf of wheat plants mock inoculated with buffer or infected with BSMV:c or BSMV:TaRab7 were inoculated with urediospores of Pst races CYR23 (incompatible) and CYR31 (compatible). Conspicuous HR was elicited by CYR23 on leaves previously infected with BSMV:c and BSMV:TaRab7 (Fig. 8). However, limited fungal sporulation was observed around the necrotic spots only on leaves infected with BSMV:TaRab7 by 10 dpi (Fig. 8). In contrast, wheat Transcriptional responses of TaRab7 to infection by Pst Quantitative RT-PCR analysis showed that the gene was strongly up-regulated in the incompatible interaction (Fig. 3). The expression of TaRab7 was up-regulated as early as 6 hours post- inoculation (hpi) until 24 hpi and was more than 4-fold higher than the control group before 24 hpi. The expression level peaked at 12 hpi. Functional Characterization of Triticum TaRab7 environmental stresses [17]. In Arabidopsis, Kwon et al. [18] analyzed a SA-responsive protein involved in plant-pathogen interactions RabG3b, which shows strong similarity to the Rab7 in mammals and yeast which contain conserved motifs for GTP binding and hydrolysis. Compared to wild-type and transgenic plants overexpressing dominant negative RabG3b, transgenic plants overexpressing wild-type and constitutively active RabG3b displayed expanded programmed cell death (PCD) – hypersensi- tive response (HR) upon infection and accelerated leaf senescence. Their results suggest that RabG3b may regulate PCD associated with pathogen response and senescence. PCD-HR is one of numerous complicated defense responses that allow plants to survive invasion by various infectious pathogens. incompatible interaction at 12 hpi and 24 hpi and was even down- regulated to some extent after 12 hpi. The results indicated that the higher expression of TaRab7 was associated with defense to the stripe rust fungus. BSMV-mediated silencing of TaRab7 To further dissect the function of TaRab7 during the interaction between the stripe rust fungus and wheat, a preliminary functional characterization of the TaRab7 gene was silenced with the barley stripe mosaic virus-induced gene silencing (VIGS) system. It is an effective reverse genetics tool in barley and wheat [20,21]. We tested the silencing of the wheat phytoene desaturase gene (PDS) in cv. Suwon 11 by inoculating the second leaf of the wheat seedlings with BSMV: TaPDS. At 9 days post-inoculation (dpi), the mild chlorotic mosaic symptoms became visible on the third leaf, and at 15 dpi systematic photo-bleaching was observed. Wheat seedlings inoculated with sterile buffer developed normal leaves under the same conditions, indicating that silencing of TaPDS occurred specifically in BSMV:TaPDS infected plants. Two-leaf stage wheat seedlings were infected with recombinant BSMV: TaRab7 virus, which contains a 154-bp fragment amplified with primers TaRab7- VIGS-F and TaRab7-VIGS-R (Table 1), which are located in regions that are not conserved in the small GTP binding protein family. Subcellular localization of TaRab7-GFP fusion protein Subcellular localization of TaRab7-GFP fusion protein To examine the subcellular localization of TaRab7, we constructed the pCaMV35S:TaRab7-GFP fusion vector and control plasmid pCaMV35S: GFP, and introduced them into onion epidermal cells by particle bombardment. The TaRab7 protein was predominantly localized in the cytoplasm, perinuclear area and nucleus, whereas the protein in the control was uniformly distributed throughout the cell (Fig. 5). Expression of TaRab7 is responsive to various environmental stresses The expression pattern of TaRab7 was induced by environmen- tal stresses (Fig. 4). In the presence of PEG-6000, TaRab7 was up- regulated by the drought stress. The transcription of TaRab7 in wheat leaves increased dramatically by 2 hours post-treatment (hpt) and peaked at 12 hpt with 13-fold higher than that of the control. After high salinity treatment, the expression level of TaRab7 was overall 5-fold higher than the control group at 2 hpt and peaked at 12 hpt. When the leaves were exposed in low temperature (4uC), the expression level of TaRab7 was also up- regulated as early as 2 hpt. Together, these results suggest a general role of TaRab7 in stress responses. Wheat stripe rust fungus, Pucinia striiformis f. sp. tritici (Pst), is one of the most devastating pathogens of wheat, causing significant yield loss in wheat production worldwide. One of the most effective methods to control stripe rust is the use of resistant wheat cultivars. Comprehending molecular mechanisms of interactions between wheat and the stripe rust pathogen is important to the rational use of resistance genes in the improvement of cultivars. Although the structure and function of Rab7 in trafficking has been studied, little is known about its function in the interaction between wheat and the stripe rust fungus. In this study, we isolated and characterized a wheat Rab7 homologue, designated TaRab7. The expression profile of TaRab7 was determined in wheat seedlings inoculated with Pst and plants subjected to environmen- tal stimuli. Subcellular localization of TaRab7 was also de- termined to reveal its function in the interaction, and expression of TaRab7 in an incompatible wheat-Pst interaction was decreased by virus induced gene silencing (VIGS), further indicating its important role in wheat defense against the rust pathogen. PLoS ONE \| www.plosone.org Introduction Small GTP-binding proteins are monomeric G proteins with molecular masses of 20–40 kDa. Small G proteins in eukaryotes from yeast to human constitute a superfamily with at least five families (Ras, Rho, Rab, Sar1/Arf and Ran) including more than 100 members [1]. Although plants have only four of these families of small G proteins, they have a unique subfamily of Rho GTPases instead of the Ras family. Rac1, a member of the Rho family, has been shown to play an essential role in the defense of rice against pathogens [2,3]. The functions of other superfamilies of small G proteins in plant toward pathogens have not been determined. The Rab proteins belong to the small guanosine triphosphatases (GTPases) superfamily. Rabs are thought to act as molecular switches, which play an essential role in both endocytic and exocytic traffic in eukaryotic cells, being active in their GTP- bound state and inactive in their GDP-bound state [4]. Because Rabs do not have high intrinsic guanine nucleotide exchange or hydrolytic activities, they are regulated by other proteins, such as guanine nucleotide exchange factors (GEFs) and GTPase-activat- ing proteins (GAPs). In their GDP-bound state, Rabs are typically soluble and bound to guanine nucleotide dissociation inhibitor (GDI). At the acceptor membrane, the Rab-GDI complex is thought to interact with GDI displacement factor, which removes GDI and allows Rab membrane insertion [5]. Next, a GEF Approximately 70 members have been identified in mammals, and Rab7 is one of the Rab proteins that have been investigated extensively. Rab7 is regarded as a key regulator in endo-lysosomal trafficking based on the extensive investigations in the past decades [15]. Rab7 mediates the regulated internalization and degradation of nutrient transporters and triggers nutrient starvation that facilitates cell death [16]. A tonoplast-localized rice Rab7 homologue is up-regulated in response to cold, salt, and drought stress, suggesting that it plays a role in plant adaptation to various PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 1 Functional Characterization of Triticum TaRab7 Cloning and phylogenetic analyses of TaRab7 It can be inferred that TaRab7 plays an important role in regulating defense responses during the interaction of wheat and Pst. leaves inoculated with compatible race CYR31 expressed normal disease development and produced numerous regular uredia (Fig. 8), indicating that the silencing of TaRab7 has no obvious effect on the compatible interaction. Race-specific resistance to the stripe rust fungus was not blocked or eliminated by BSMV:TaRab7 infection or by silencing of TaRab7. Nevertheless, knocking down TaRab7 expression allowed limited fungal growth and uredium development and therefore, reduced resistance indicated that resistance of wheat to stripe rust race CYR23 is weakened after knocking down. It can be inferred that TaRab7 plays an important role in regulating defense responses during the interaction of wheat and Pst. Cloning and phylogenetic analyses of TaRab7 On the other hand, the relative expression of TaRab7 in the compatible interaction was much lower than that in the PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 May 2012 \| Volume 7 \| Issue 5 \| e37146 2 Functional Characterization of Triticum TaRab7 Figure 1. Alignment of the amino acid sequence of TaRab7 and selected Rab7 proteins. Alignment of the amino acid sequence aestivum Rab7 (TaRab7) with other Rab7 proteins from Oryza sativa (OsRab7), Hordeum vulgare (HvRab7), Arabidopsis (RabG3b), Homo sapie and Saccharomyces cerevisiae (ScYpt7). Sequences were aligned using DNAMAN. Identical residues in all organisms are shaded. Red indicate sequence motifs involved in nucleotide binding and hydrolysis that are conserved in Rab GTPases. doi:10.1371/journal.pone.0037146.g001 Figure 1. Alignment of the amino acid sequence of TaRab7 and selected Rab7 proteins. Alignment of the amino acid sequence of Triticum aestivum Rab7 (TaRab7) with other Rab7 proteins from Oryza sativa (OsRab7), Hordeum vulgare (HvRab7), Arabidopsis (RabG3b), Homo sapiens (HsRab7) and Saccharomyces cerevisiae (ScYpt7). Sequences were aligned using DNAMAN. Identical residues in all organisms are shaded. Red underlines indicate sequence motifs involved in nucleotide binding and hydrolysis that are conserved in Rab GTPases. doi:10.1371/journal.pone.0037146.g001 observed at 24 hpi on leaves inoculated with avirulent race CYR23. At 48 hpi and 120 hpi, the average necrotic area per infection site did not show significant difference after TaRab7 was silenced. There were significant differences in fungal development and hyphal growth between BSMV:c-inoculated plants and TaRab7 knocked-down plants at 24 hpi and 48 hpi (Table 2) when plants inoculated with CYR23. However, at 120 hpi, hyphae were too long and intertwined to be measured. These results indicated that the susceptibility of cv. Suwon11 to CYR23 was enhanced when TaRab7 was silenced. Therefore, the gene TaRab7 might be involved in defense responses against Pst. leaves inoculated with compatible race CYR31 expressed normal disease development and produced numerous regular uredia (Fig. 8), indicating that the silencing of TaRab7 has no obvious effect on the compatible interaction. Race-specific resistance to the stripe rust fungus was not blocked or eliminated by BSMV:TaRab7 infection or by silencing of TaRab7. Nevertheless, knocking down TaRab7 expression allowed limited fungal growth and uredium development and therefore, reduced resistance indicated that resistance of wheat to stripe rust race CYR23 is weakened after knocking down. Discussion Histological observations of fungal growth and host responses in plants with decreased expression of TaRab7 Leaf segments inoculated with race CYR23 were examined microscopically in order to determine histological changes associated with enhanced susceptibility of TaRab7 knocked-down plants to P. striiformis f. sp. tritici (Fig. 9). Necrotic cells were not Histological observations of fungal growth and host responses in plants with decreased expression of TaRab7 In this study, we cloned and characterized an 1149-bp full- length sequence of a wheat small GTP binding protein gene TaRab7. The amino acid sequence alignment demonstrated that TaRab7 has the conserved guanine nucleotide binding motifs and domains of small G proteins from animals and plants, which are Leaf segments inoculated with race CYR23 were examined microscopically in order to determine histological changes associated with enhanced susceptibility of TaRab7 knocked-down plants to P. striiformis f. sp. tritici (Fig. 9). Necrotic cells were not PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 May 2012 \| Volume 7 \| Issue 5 \| e37146 3 Functional Characterization of Triticum TaRab7 Figure 2. A representative phylogenetic tree of TaRab7 and selected Rab7 proteins. A representative phylogenetic tree of TaRab7, Rab family member proteins in Arabidopsis, Oryza Sativa and Brachypodium distachyon, and Rab7 proteins from soybean (GmRab7) and barley (HvRab7). GeneBank accession numbers are provided after the gene names. doi:10.1371/journal.pone.0037146.g002 Figure 2. A representative phylogenetic tree of TaRab7 and selected Rab7 proteins. A representative phylogenetic tree of TaRab7, Rab family member proteins in Arabidopsis, Oryza Sativa and Brachypodium distachyon, and Rab7 proteins from soybean (GmRab7) and barley (HvRab7). GeneBank accession numbers are provided after the gene names. doi:10.1371/journal.pone.0037146.g002 pathogens [17,18]. The high homology of TaRab7 to the rice and Arabidopsis genes let us to make a hypothesis that the wheat gene has the similar role in response to environmental stresses and important for interaction with its regulators and effectors. The phylogenetic analysis showed that TaRab7 has the highest similarity to small GTP binding protein OsRab7 in rice and to RabG3b in the Rab subfamily proteins in Arabidopsis. Based on previous research, the OsRab7 plays an important role in response to environmental stresses and AtRabG3b is vital for plant defense to Figure 4. Assays for the transcript levels of TaRab7 during different treatment stages. Quantitative reverse-transcription poly- merase chain reaction analyses of TaRab7 expression in wheat exposed to environmental stresses. The data were normalized using the expression level of wheat translation elongation factor 1 alpha-subunit. Mean and standard deviation were calculated with data from three independent biological replicates. High salinity, 200 mM NaCl; drought, 20% PEG6000; low temperature, 4uC. doi:10.1371/journal.pone.0037146.g004 p Figure 3. Assays for the transcript levels of TaRab7 during different infection stages. Histological observations of fungal growth and host responses in plants with decreased expression of TaRab7 Expression levels of TaRab7 in wheat leaves inoculated with CYR23 (incompatible reaction) and CYR31 (compatible reaction) sampled at 0, 6, 12, 24, 48, 72, and 120 hpi. Relative expression was calculated by the comparative threshold (DDCT) method. Mean and standard deviation were calculated with data from three independent biological replicates. doi:10.1371/journal.pone.0037146.g003 Figure 4. Assays for the transcript levels of TaRab7 during different treatment stages. Quantitative reverse-transcription poly- merase chain reaction analyses of TaRab7 expression in wheat exposed to environmental stresses. The data were normalized using the expression level of wheat translation elongation factor 1 alpha-subunit. Mean and standard deviation were calculated with data from three independent biological replicates. High salinity, 200 mM NaCl; drought, 20% PEG6000; low temperature, 4uC. doi:10.1371/journal.pone.0037146.g004 Figure 3. Assays for the transcript levels of TaRab7 during different infection stages. Expression levels of TaRab7 in wheat leaves inoculated with CYR23 (incompatible reaction) and CYR31 (compatible reaction) sampled at 0, 6, 12, 24, 48, 72, and 120 hpi. Relative expression was calculated by the comparative threshold (DDCT) method. Mean and standard deviation were calculated with data from three independent biological replicates. doi:10.1371/journal.pone.0037146.g003 PL S ONE \| l Figure 4. Assays for the transcript levels of TaRab7 during different treatment stages. Quantitative reverse-transcription poly- merase chain reaction analyses of TaRab7 expression in wheat exposed to environmental stresses. The data were normalized using the expression level of wheat translation elongation factor 1 alpha-subunit. Mean and standard deviation were calculated with data from three independent biological replicates. High salinity, 200 mM NaCl; drought, 20% PEG6000; low temperature, 4uC. doi:10.1371/journal.pone.0037146.g004 Figure 3. Assays for the transcript levels of TaRab7 during different infection stages. Expression levels of TaRab7 in wheat leaves inoculated with CYR23 (incompatible reaction) and CYR31 (compatible reaction) sampled at 0, 6, 12, 24, 48, 72, and 120 hpi. Relative expression was calculated by the comparative threshold (DDCT) method. Mean and standard deviation were calculated with data from three independent biological replicates. doi:10.1371/journal.pone.0037146.g003 PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 4 Functional Characterization of Triticum TaRab7 Figure 5. Subcellular localization of TaRab7 protein. Onion epidermal cells were transformed with plasmids expressing the fusion protein and green fluorescent protein (GFP) by bombardment. All images were observed with a confocal microscope. (A). Onion epidermal cells expressing the GFP alone driven by the 35 S promoter. (B). Onion epidermal cells expression the TaRab7-GFP fusion protein. Histological observations of fungal growth and host responses in plants with decreased expression of TaRab7 doi:10.1371/journal.pone.0037146.g005 Figure 5. Subcellular localization of TaRab7 protein. Onion epidermal cells were transformed with plasmids expressing the fusion protein and green fluorescent protein (GFP) by bombardment. All images were observed with a confocal microscope. (A). Onion epidermal cells expressing the GFP alone driven by the 35 S promoter. (B). Onion epidermal cells expression the TaRab7-GFP fusion protein. doi:10.1371/journal.pone.0037146.g005 pathogen infection. The serious experiments conducted in this study proved the hypothesis. the control while in the compatible group the gene was down- regulated compared with 6 hpi. It is supposed that there is a required minimum of expression level to initiate or contribute to the expression of wheat response to Pst while it did not reach in the compatible reaction. In this sense, when TaRab7 was silenced, the expression level of TaRab7 was not high enough to inhibit the Plant defense responses against microbial infection are often triggered during the initial stages of plant-pathogen interactions [22], and the early recognition and penetration between wheat and Pst occurred from 6 to 12 hpi [23]. qRT-PCR was utilized to analyze the expression pattern of TaRab7 in wheat seedlings inoculated with Pst race CYR23 (incompatible) and CYR31 (compatible). The results showed that, in the incompatible interaction, TaRab7 was up-regulated at 6 hpi and the expression remained at a high level, however, in the compatible interaction, TaRab7 was also about 4-fold higher than that in the control group at 6 hpi but it began to down-regulate since then. During the interaction between the host and pathogen, once the defense mechanism was triggered, both in the compatible and incompat- ible interactions TaRab7 was up-regulated, however, at 12 hpi in the incompatible interaction the gene was about 9-fold higher over Functional Characterization of Triticum TaRab7 Leaves inoculated with urediospores of avirulent race CYR23 or virulent race CYR31. Leaves inoculated with BSMV: c or BSMV:TaRab7 and urediospores of avirulent race CYR23 or virulent race CYR31. Mock: wheat leaves treated with FES buffer. Typical leaves were photographed at 15 dpi. doi:10.1371/journal.pone.0037146.g008 become less resistant to a pathogen, the fungal hypha grows longer and produces more branches; once they have contacted more cells, they will induce more cell death. spread of the pathogen in the incompatible interaction. This observation suggests that TaRab7 might play a positive role in wheat defense to Pst after the initial penetration and recognition between plant and stripe rust pathogen in the incompatible interaction. Protein-protein interaction cascades are crucial for signaling pathways. Like other Rab proteins, the Rab7 is activated by a set of upstream regulators that converge on its GEF and GAP. Rab7 exerts its biological functions through interaction with diverse downstream effectors [15]. Upon upstream signal stimulation, the TaRab7 changes into active form and leads to the conformational change of the downstream effector-binding region so that this region interacts with the downstream effector(s). In this way, TaRab7 may drive vesicle transport and membrane fission and fusion to convey various materials to the site of defense. Several plant defense response genes are reported to express substantially under abiotic stresses and their transcript accumula- tion can be activated by different environmental stresses and pathogens. These findings suggest that different stress signaling pathways share certain components and that plant responses to biotic and abiotic stresses overlap. For instance, the CPN1 gene in Arabidopsis was proposed as a link between resistance to pathogens and acclimation to low-humidity and low-temperature conditions [24]. The expression of TaRab7 is highly induced by environmen- tal stresses such as high salt, low temperature and dehydration. Thus, those results demonstrate that TaRab7 might play an important role in regulating some downstream genes during the plant’s tolerance to different environmental stimuli. In this way, TaRab7 could participate in the basal resistance of wheat to these stresses. y Rabs appear to be concentrated to specific subcellular compartments in eukaryotic cells [7]. They are targeted to microdomains in specific organelles via interactions with their effectors. Reports have suggested that Rab7 plays a role in vesicular transport from early endosomes to late endosomes in the cytoplasm [25,26]. Functional Characterization of Triticum TaRab7 Functional Characterization of Triticum TaRab7 Functional Characterization of Triticum TaRab7 Figure 7. Relative transcript levels of TaRab7 assayed by qRT- PCR after TaRab7 gene silencing by the BSMV-VIGS method. RNA samples were isolated from the fourth leaves of the wheat seedlings infected with BSMV:TaRab7 at 0, 24, 48 h and 120 post- inoculation (hpi) after inoculation with CYR23. CK: wheat leaves infected with BSMV:c were sampled immediately (0 hpi) after inoculation with CYR23. Error bars represent the variations among three independent replicates. doi:10.1371/journal.pone.0037146.g007 Figure 7. Relative transcript levels of TaRab7 assayed by qRT- PCR after TaRab7 gene silencing by the BSMV-VIGS method. RNA samples were isolated from the fourth leaves of the wheat seedlings infected with BSMV:TaRab7 at 0, 24, 48 h and 120 post- inoculation (hpi) after inoculation with CYR23. CK: wheat leaves infected with BSMV:c were sampled immediately (0 hpi) after inoculation with CYR23. Error bars represent the variations among three independent replicates. doi:10.1371/journal.pone.0037146.g007 Figure 8. Leaves inoculated with urediospores of avirulent race CYR23 or virulent race CYR31. Leaves inoculated with BSMV: c or BSMV:TaRab7 and urediospores of avirulent race CYR23 or virulent race CYR31. Mock: wheat leaves treated with FES buffer. Typical leaves were photographed at 15 dpi. doi:10.1371/journal.pone.0037146.g008 Figure 7. Relative transcript levels of TaRab7 assayed by qRT- PCR after TaRab7 gene silencing by the BSMV-VIGS method. RNA samples were isolated from the fourth leaves of the wheat seedlings infected with BSMV:TaRab7 at 0, 24, 48 h and 120 post- inoculation (hpi) after inoculation with CYR23. CK: wheat leaves infected with BSMV:c were sampled immediately (0 hpi) after inoculation with CYR23. Error bars represent the variations among three independent replicates. doi:10.1371/journal.pone.0037146.g007 Figure 8. Leaves inoculated with urediospores of avirulent race CYR23 or virulent race CYR31. Leaves inoculated with BSMV: c or BSMV:TaRab7 and urediospores of avirulent race CYR23 or virulent race CYR31. Mock: wheat leaves treated with FES buffer. Typical leaves were photographed at 15 dpi. doi:10.1371/journal.pone.0037146.g008 Figure 7. Relative transcript levels of TaRab7 assayed by qRT- PCR after TaRab7 gene silencing by the BSMV-VIGS method. RNA samples were isolated from the fourth leaves of the wheat seedlings infected with BSMV:TaRab7 at 0, 24, 48 h and 120 post- inoculation (hpi) after inoculation with CYR23. CK: wheat leaves infected with BSMV:c were sampled immediately (0 hpi) after inoculation with CYR23. Error bars represent the variations among three independent replicates. doi:10.1371/journal.pone.0037146.g007 Figure 8. Figure 6. Mild chlorotic mosaic symptoms were observed on the leaves at 9 dpi. Mild chlorotic mosaic symptoms were observed on the leaves inoculated with BSMV: c or BSMV:TaRab7 at 9 dpi. Photobleaching was evident on leaves infected with BSMV: TaPDS at 15 dpi but not on mock-inoculated leaves. doi:10.1371/journal.pone.0037146.g006 Table 1. PCR primers used for TaRab7 cloning, expression vector construction and qRT-PCR amplification. Primers Sequences (59R39) TaRab7-ORF-F ATGTCGACCTCGCGCAGG TaRab7-ORF-R CTAGCATGCACACCCGCCT TaRab7-SL-F AACTGCAGATGTCGACCTCGCGCAGG TaRab7-SL-R CGGGATCCGCATGCACACCCGCCTCTCTG TaRab7-VIGS-F ATATTAATTAAGCGATTAGCTGTCTTTGTCCTT TaRab7-VIGS-R TATGCGGCCGCTTGCCGACTCAAGAAACAGG TEF1-F TGGTGTCATCAAGCCTGGTATGGT TEF1-R ACTCATGGTGCATCTCAACGGACT TaRab7-qRT-F ATCCTCGCACGAACCCCTT TaRab7-qRT-R CCTTGGTGACGAAATCCGCA Sequences underlined are restriction sites for cloning. doi:10.1371/journal.pone.0037146.t001 Table 1. PCR primers used for TaRab7 cloning, expression vector construction and qRT-PCR amplification. Primers Sequences (59R39) TaRab7-ORF-F ATGTCGACCTCGCGCAGG TaRab7-ORF-R CTAGCATGCACACCCGCCT TaRab7-SL-F AACTGCAGATGTCGACCTCGCGCAGG TaRab7-SL-R CGGGATCCGCATGCACACCCGCCTCTCTG TaRab7-VIGS-F ATATTAATTAAGCGATTAGCTGTCTTTGTCCTT TaRab7-VIGS-R TATGCGGCCGCTTGCCGACTCAAGAAACAGG TEF1-F TGGTGTCATCAAGCCTGGTATGGT TEF1-R ACTCATGGTGCATCTCAACGGACT TaRab7-qRT-F ATCCTCGCACGAACCCCTT TaRab7-qRT-R CCTTGGTGACGAAATCCGCA Sequences underlined are restriction sites for cloning. doi:10.1371/journal.pone.0037146.t001 Table 1. PCR primers used for TaRab7 cloning, expression vector construction and qRT-PCR amplification. Table 1. PCR primers used for TaRab7 cloning, expression vector construction and qRT-PCR amplification. Figure 6. Mild chlorotic mosaic symptoms were observed on the leaves at 9 dpi. Mild chlorotic mosaic symptoms were observed on the leaves inoculated with BSMV: c or BSMV:TaRab7 at 9 dpi. Photobleaching was evident on leaves infected with BSMV: TaPDS at 15 dpi but not on mock-inoculated leaves. doi:10.1371/journal.pone.0037146.g006 May 2012 \| Volume 7 \| Issue 5 \| e37146 PLoS ONE \| www.plosone.org 5 PLoS ONE \| www.plosone.org Functional Characterization of Triticum TaRab7 The results of the subcellular localization of TaRab7 based on the transient assays showed that TaRab7 is located in both the cytoplasm and at the periphery of the nucleus. The gene transport materials in the cytoplasm and the location in perinuclear area suggest that the gene is associated with lysosomes or plays a role in the regulation of the perinuclear lysosome compartment. In Hela cells, Rab7 was associated with lysosomes, which aggregated and fused in the perinuclear region. In the absence of a functional Rab7 protein, the lysosomes become dispersed. In this sense, Rab7 is a key regulatory protein for proper aggregation and fusion of late endocytic structures in the perinuclear region and consequently for the biogenesis and maintenance of the lysosomal compartment [27]. Lysosomes of most cells function principally in intracellular digestion and contain several enzymes, mainly acid hydrolases, and the function of lysosomes is not restricted to protein degradation; they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types [28]. Nascent phagosomes must undergo a series of fusion and fission When TaRab7 was knocked down by BSMV-VIGS, the hyphal length and hyphal branching increased, indicating that the susceptibility to Pst was enhanced after inoculation with avirulent Pst race CYR23. On TaRab7 knocked-down plants, few small uredia were observed on seedling leaves. In this sense, TaRab7 is important for wheat resistance to the stripe rust fungus. Hypersensitive cell death was further examined for discontinuity of cytoplasmic strands and from auto fluorescence of dead cells. There were no necrotic cells by 24 hpi on leaves inoculated with avirulent race CYR23 (Fig. 9), but by 48 hpi and 120 hpi, we observed the dead cells with auto fluorescence. As reported in Arabidopsis by Kwon et al. [18], RabG3b may regulate PCD associated with pathogen response and senescence [18]. However, the average necrotic area per infection site was not reduced significantly after TaRab7 was silenced. The necrotic cell is viewed as a significant site for plant to defense. However, as plant cells PLoS ONE \| www.plosone.o May 2012 \| Volume 7 \| Issue 5 \| e37146 May 2012 \| Volume 7 \| Issue 5 \| e37146 6 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org Functional Characterization of Triticum TaRab7 Figure 9. Histological observation of leaves treated with recombinant BSMV viruses and infected with avirulent race CYR23. Functional Characterization of Triticum TaRab7 Typical leaves were examined at 24, 48 and 120 hpi. Treatments a, b, c: BSMV:c infected leaves inoculated with CYR23 examined at 24, 48 and 120 h, respectively. Treatment were d, e, f, BSMV: TaRab7 infected leaves inoculated with CYR23 examined at 24, 48, 120 h. SV, substomatal vesicle; IH, infection hypha; HMC, haustorial mother cell; NC, necrotic cell; SH, second hypha; HC, haustorium cell. Bars = 20 mm. doi:10.1371/journal.pone.0037146.g009 Figure 9. Histological observation of leaves treated with recombinant BSMV viruses and infected with avirulent race CYR23. Typical leaves were examined at 24, 48 and 120 hpi. Treatments a, b, c: BSMV:c infected leaves inoculated with CYR23 examined at 24, 48 and 120 h, respectively. Treatment were d, e, f, BSMV: TaRab7 infected leaves inoculated with CYR23 examined at 24, 48, 120 h. SV, substomatal vesicle; IH, infection hypha; HMC, haustorial mother cell; NC, necrotic cell; SH, second hypha; HC, haustorium cell. Bars = 20 mm. doi:10.1371/journal.pone.0037146.g009 one function of TaRab7 in wheat against the pathogen. When some pathogens invade the plant, they secrete some materials into plant cells that facilitate infection. Accordingly, TaRab7 may regulate lysosome and phagosomes to render these toxic materials dysfunctional. The explanation for localization of TaRab7 in the nucleus requires further examination. reactions to acquire the microbicidal properties required for the innate immune response. Here reports demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP, which in turn bridges phagosomes with dyneindynactin, a micro- tubule-associated motor complex [10]. RILP has been studied because it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP [29]. And this may explain Following the demonstration of the pivotal role of TaRab7 in wheat defense response, dissecting of its signaling pathway, such as identifying its upstream regulators, downstream effectors, and interacting or interconnected partners becomes the next major task. Table 2. RNA extraction, cDNA synthesis and qRT-PCR RNA extraction, cDNA synthesis and qRT-PCR RNA extraction, cDNA synthesis and qRT-PCR Total RNA was isolated with the Trizol reagent (Invitrogen. Carlsbad, CA, U.S.A) and digested with DNase I (TaKaRa, Dalian, China) as described [36]. Then formamide denaturing gel electrophoresis was used to check the integrity of total RNA, and the concentration was determined with a NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific, USA). Re- verse transcriptase (TaKaRa, Dalian, China) was used to synthesize the first strand cDNA. After 1:10 dilution, 2 ml of the synthesized cDNA was used for qRT-PCR. Quantification of gene expression was performed with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.). PCR conditions were 1 cycle at 95uC for 1 min, 40 cycles at 95uC for 10 s, 60uC for 20 s, 72uC for 40 s, and then followed by 1 cycle at 95uC for 15 s, 60uC for 1 min, 95uC for 15 s and finally 60uC for 15 s. A 107-bp fragment of wheat translation elongation factor 1 alpha- subunit (TEF1) mRNA (GenBank accession No. M90077.1) was amplified as an internal reference for the qRT-PCR analysis. Dissociation curves were generated for each reaction to ensure specific amplification, and threshold values (CT) generated from the 7500 Software Toll (Applied Biosystems) were used to quantify relative gene expression by the comparative 2-DDCT method [37]. BSMV-mediated TaRab7 gene silencing A 216-bp cDNA fragment of TaRab7 was cloned in its 39 untranslated region and then replaced the TaPDS coding sequence in BSMV:TaPDS to construct the plasmid designed for gene silencing as Holzberg [20] (Primer: TaRab7-VIGS, Table 1). After verifying the correctness of the constructed plasmid, capped in vitro transcripts were produced from linearized plasmids containing the tripartite BSMV genome [42] by the mMessage mMachine T7 in vitro transcription kit (Ambion, Austin, TX, U.S.A). Two-leaf-stage wheat seedlings were infected with BSMV as described [21]. After 24 hours in darkness, they were placed in a growth chamber at 2562uC. The experiment was repeated three times. For each BSMV viruses construct 24 seedlings were inoculated and the control group seedlings were inoculated with 16 FES buffer [43]. The BSMV transcripts were inoculated on the second leaf of two-leaf wheat seedlings by gently rubbing the leaves with gloved fingers [20,21,44]. Then the fourth leaf was infected with urediniospores of CYR 23 at 9 dpi. Infection types of stripe rust were examined at 15 dpi. Functional Characterization of Triticum TaRab7 Functional Characterization of Triticum TaRab7 Plant materials and inoculation Two Pst pathotypes (CYR23 and CYR31) and wheat (Triticum aestivum) cultivar Suwon 11 were used in the study. Suwon 11 that possesses stripe rust resistance gene YrSu [30,31], is highly resistant to CYR23 (incompatible reaction) and susceptible to CYR31 (compatible reaction) [32]. Wheat seedlings were grown and maintained as described [33]. For biological stress treatments, wheat plants were inoculated with sterile distilled water as mock- inoculation controls, and a paintbrush was used to inoculate the freshly collected urediniospores onto the surface of the primary leaves of 2-week-old wheat seedlings. Wheat leaves were excised at 0, 6, 12, 24, 48, 72 and 120 hours post inoculation (hpi) [34]. For high salinity and drought-stress treatments, the roots of the seedlings were soaked in 200 mM NaCl or 20% PEG6000. For low temperature stress, the seedlings were transferred to an incubator under long-day conditions (16 h light/8 h dark cycle) at 4uC. Wheat seedlings treated with various stress elicitors along with control plants were sampled at 0, 2, 6, 12, and 24 hour post treatment (hpt). All samples were quickly frozen in liquid nitrogen and stored at 280uC until they were used for RNA isolation. Each treatment was performed with three independent biological replicates [35]. Materials and Methods analyses. MEGA4 [41] was used for phylogenetic analysis using the Neighbor-Joining (NJ) method. InterProScan and ProtParam were used to analyze the amino acid sequence of TaRab7. The TaRab7 gene sequence was deposited in GenBank (GenBank accession number JQ364960). Histological observations of fungal growth and host response p The leaves inoculated with BSMV were sampled at 0, 24, 48, 120 hpi with Pst. The samples were stained and the leaf segments were observed with an Olympus BX-51 microscope (Olympus Corp., Tokyo) for infection sites and lengths and branches of infection hyphae [23]. A minimum of 50 infection sites were examined on each of five randomly selected leaf segments for every treatment. The area of necrotic death was observed by epifluorescence microscopy through the auto fluorescence of the attacked mesophyll cells (excitation filter, 485 nm; dichromic mirror, 510 nm; and barrier filter, 520 nm). Only infected sites in which appressoria successfully penetrated were examined for the formation of substomatal vesicles, infection hyphae, haustorium mother cells and hyphal branches. Standard deviations and Tukey’s test for statistical analysis were performed with the SPSS software. PLoS ONE \| www.plosone.org Functional Characterization of Triticum TaRab7 Histological observations during the incompatible interaction between wheat and the stripe rust fungus when the transcription of TaRab7 was repressed.a Hyphal length (mm)b Hyphal branches Percentage of the occurrence of haustorial mother cells Treatmentc 24 hpi 48 hpi 24 hpi 48 hpi 24 hpi 48 hpi BSMV: c 23.9 24.0 1.6 1.9 81% 85% BSMV: TaRab7 31.4* 33.2* 2.0* 2.2* 90% 92% aAbbreviations: hpi.: hours post inoculation. bDistance from the base of substomatal vesicles to hyphal tips. Values with * are significantly different at P = 0.05 according to the Tukey’s test. cBSMV: c and BSMV: TaRab7, leaves inoculated with BSMV: c or BSMV: TaRab7 followed by infection with CYR23. doi:10.1371/journal.pone.0037146.t002 aAbbreviations: hpi.: hours post inoculation. bDistance from the base of substomatal vesicles to hyphal tips. Values with * are significantly different at P = 0.05 according to the Tukey’s test. cBSMV: c and BSMV: TaRab7, leaves inoculated with BSMV: c or BSMV: TaRab7 followed by infection with CYR23. doi:10.1371/journal.pone.0037146.t002 May 2012 \| Volume 7 \| Issue 5 \| e37146 PLoS ONE \| www.plosone.org 7 Subcellular localization of TaRab7 The cDNA fragments containing the TaRab7 open reading frame (ORF) were amplified by PCR with specific primers containing PstI and BamHI sites (TaRab7-SL, Table 1), and then ligated to the 59 end of green fluorescent protein (GFP) coding region. The fused gene was subcloned into the pCaMV35S: GFP vector and placed under the control of the CaMV 35S promoter. Onion inner peels were incubated inside-out on MS medium plates for 4–6 h before bombardment. The constructs of pCaMV35S:TaRab7-GFP and GFP alone (pCaMV35S:GFP) were transformed into onion epidermal cells by the particle bombard- ment at a helium pressure of 7.6 MPa (1100 psi) using the PDS- 100/He system (Bio-Rad, Hercules, USA). Transformed materials were incubated in darkness at 24uC for 18–24 h in a growth chamber. GFP signals were detected with a Zeiss LSM 510 confocal laser microscope using a 488 nm filter (Zeiss, Oberko- chen, Germany). The experiment was performed twice and with three replicates each time. May 2012 \| Volume 7 \| Issue 5 \| e37146 References Eur J Plant Pathol 120: 241–247. 14. Peralta ER, Martin BC, Edinger AL (2010) Differential effects of TBC1D15 and mammalian Vps39 on Rab7 activation state, lysosomal morphology, and growth factor dependence. 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(2007) Mycobacterium bovis BCG disrupts the interaction of Rab7 with RILP contributing to inhibition of phagosome maturation. J Leukocyte Biol 82: 1437–1445. 36. Wang X, Zheng W, Buchenauer H, Zhao J, Han Q, et al. (2008) The development of a PCR-based method for detecting Puccinia striiformis latent infections in wheat leaves. Functional Characterization of Triticum TaRab7 Functional Characterization of Triticum TaRab7 Author Contributions Conceived and designed the experiments: FRL JG ZSK. Performed the experiments: FRL YHD. Analyzed the data: FRL HF LLH. Contributed reagents/materials/analysis tools: PFB XDW YLC. Wrote the paper: FRL. We thank Dr. Larry Dunkle, U.S. Department of Agriculture-Agricultural Research Service at Purdue University, for critical reading of this manuscript and editorial guidance. We thank Dr. Larry Dunkle, U.S. Department of Agriculture-Agricultural We thank Dr. Larry Dunkle, U.S. Department of Agriculture-Agricultural R h S i P d U i i f i i l di f hi We thank Dr. Larry Dunkle, U.S. Department of Agriculture-Agricultural R h S i P d i i f i i l di f hi Research Service at Purdue University, for critical reading of this manuscript and editorial guidance. Research Service at Purdue University, for critical reading of this manuscript and editorial guidance. Research Service at Purdue University, for critical reading of this manuscript and editorial guidance. Cloning and sequencing analysis Special primers of TaRab7 (TaRab7-ORF, Table 1) were designed according to the sequence from cDNA library during wheat-Pst interactions [38]. The fragment of TaRab7 gene was cloned from the first cDNA synthesized with RNA isolated from leaves of wheat Suwon 11 at 24 hpi with CYR23. The PCR products were constructed in the pGEM T-easy vector (Promega, Madison, WI, USA) and transformed into Escherichia coli (JM109) by the CaCl2 procedure [39]. The plasmid was sequenced with an ABI PRISM 3130XL Genetic analyzer (Applied Biosytems, USA). DNA sequences were analyzed with the DNASTAR (http:// www.dnastar.com), BLAST (http://www.ncbi.nlm.nih.gov/blast/ ), and ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html) programs. ClustalW 1.83 [40] and DNAMAN6.0 (Lynnon BioSoft, USA) were used for sequence alignment PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 8 References Scofield SR, Huang L, Brandt AS, Gill BS (2005) Development of a virus- induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138: 2165–2173. 44. Hein I, Barciszewska-Pacak M, Hrubikova K, Williamson S, Dinesen M, et al. (2005) Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley. Plant Physiol 138: 2155–2164. 22. Lamb CJ, Lawton MA, Dron M, Dixon RA (1989) Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56;2): 215–224. PLoS ONE \| www.plosone.org May 2012 \| Volume 7 \| Issue 5 \| e37146 9
W3153018666.txt	https://link.springer.com/content/pdf/10.1007/s10926-021-09973-8.pdf	en		Measurement Properties of the Full and Brief Version of the Work Rehabilitation Questionnaire in Persons with Physical Disabilities	Journal of occupational rehabilitation	2,021	cc-by	6,208	Journal of Occupational Rehabilitation (2021) 31:886–894 https://doi.org/10.1007/s10926-021-09973-8 Measurement Properties of the Full and Brief Version of the Work Rehabilitation Questionnaire in Persons with Physical Disabilities Ellen H. Roels1 · Michiel F. Reneman1 · Marcel W. M. Post1,2 Accepted: 17 March 2021 / Published online: 12 April 2021 © The Author(s) 2021 Abstract Purpose The Work Rehabilitation Questionnaire (WORQ) is a self-report vocational rehabilitation assessment. A comprehensive (WORQ-FULL) and a brief version (WORQ-BRIEF) are available. The purpose of this study was to investigate measurement properties of both versions in persons with physical disabilities. Methods Cross sectional and test–retest design. Adults with physical disabilities in vocational rehabilitation were included. Internal consistency (Cronbach’s alpha), test– retest reliability (intra-class correlation; ICC), agreement between sessions (Bland–Altman Plots), criterion validity (ICC and agreement with Bland–Altman Plots between WORQ-FULL and WORQ-BRIEF) and convergent validity with the Work Ability Index -Single item (WAS) and the EuroQOL 5D-5L were analyzed. Results Out of the 91 individuals who agreed to participate, 74 (81%) returned questionnaire T1 and 49 (54%) participants returned questionnaire T2 within the maximum time interval (= 27 days). At T2, 28 (57%) participants reported no medical changes compared to T1. Median age was 49 (IQR 40–60), 57% were male, 47% had experienced a stroke and 27% a spinal cord injury (n = 49). Internal consistency was good: 0.95/0.95/0.94 for the WORQ-FULL and 0.88/0.89/0.85 for the WORQ-BRIEF (n = 74/n = 48/n = 28, respectively). Test–retest reliabilitywas good: ICC = 0.86/0.85 for the WORQ-FULL and ICC = 0.87/0.86 for the WORQ-BRIEF (n = 49/ n = 28). Bland Altman plots suggested a higher score at T1. As for criterion validity of the WORQ-FULL versus the WORQBRIEF, ICC was good (ICC = 0.84; n = 74), however Bland Altman plots indicated potential bias. Correlations with the WAS/EuroQOL 5D-5L were variable: r = -0.24/r = -0.57 (WORQ-FULL) and r = -0.28/-0.65 (WORQ-BRIEF). Conclusions The WORQ showed good internal consistency and test–retest reliability. Agreement demonstrated large score differences are needed to indicate change beyond random chance at individual level, whereas small changes are sufficient at group level. Criterion validity of the WORQ-FULL versus the WORQ-BRIEF was supported, however, agreement demonstrated moderate to large score differences are needed to indicate change beyond random chance at individual level, whereas small changes are sufficient at group level. This indicates the WORQ-FULL and WORQ-BRIEF are better not used interchangeably. Correlation analyses provided better insight in the validity of the WORQ. Convergent validity was supported for the WORQ-BRIEF with the EuroQoL 5D-5L (r = -0.65). Keywords Questionnaire · Vocational rehabilitation · Employment · Spinal injury · Stroke Introduction * Ellen H. Roels e.h.roels@umcg.nl 1 Department of Rehabilitation Medicine, Center for Rehabilitation, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Center of Excellence for Rehabilitation Medicine, UMC Utrecht Brain Center, University Medical Center Utrecht and De Hoogstraat Rehabilitation, Utrecht, The Netherlands 13 Vol:.(1234567890) Work participation (WP) of persons with a physical disability has many advantages for the individual and society [1–4]. Employment rates of persons with disabilities, however, remain low compared to the general population with reported averages of 35% among people with spinal cord injury (SCI) [5] and 67% among people with stroke [6]. Vocational rehabilitation (VR) can be instrumental to enhance WP outcomes after onset of physical disability. A biopsychosocial approach to VR is warranted to address all aspects of the return to work (RTW) process [7]. Journal of Occupational Rehabilitation (2021) 31:886–894 A VR Core Set including a selection of categories from the International Classification of Functioning, Disability and Health (ICF) [8] that were considered the most relevant to describe functioning of VR clients was developed for use in VR practices [9]. Based on this VR Core Set, the Work Rehabilitation Questionnaire (WORQ) was developed to assess work-related functioning [10]. The WORQ can be used to describe the client’s level of functioning on work related domains at the start of VR; to set VR goals and monitor functioning over time; to facilitate interdisciplinary communication in VR; to stimulate the patient to actively participate in his/her VR process; and to open up a conversation with the rehabilitant simplifying communication [11]. Moreover, the WORQ questionnaire is free and easy to use. The first version of the WORQ was interview administered. The initial psychometric evaluation of the WORQ showed satisfactory test–retest reliability and good internal consistency [10]. To improve its practicality, a self-report version was developed [12]. This version suggested content validity in a sample of people with SCI in the early subacute phase [12]. This version has been translated in many languages [13], including French and Dutch (Flemish version for use in Belgium) [14, 15]. Both translations showed good internal consistency (Cronbach’s alpha 0.97 and 0.95 respectively) and good test–retest reliability (ICC 0.94 and 0.85 respectively) in relatively small samples with varying pathologies. Studies into the construct validity of the WORQ have revealed correlations with several reference measures ranging from 0.28 to 0.81 [10, 14–17]. Based on the full version of WORQ, a brief 13-item version was developed to encounter the challenge of the length of the WORQ [14]. A shorter version may be preferred if it has adequate measurement properties and correlates strong with the comprehensive version. The brief version has shown good internal consistency (Cronbach’s alpha 0.96) and test–retest reliability (ICC 0.91) [17], but agreement between the full and the brief version of the WORQ has not yet been investigated. Because research in different diagnostic groups is needed to assess the validity of the WORQ [17], this study was conducted to test the internal consistency, test–retest reliability, agreement, and convergent validity of the full and brief versions of the WORQ for use in the Netherlands (WORQ-NL) in persons with physical disabilities in a VR setting. Methods Design A cross-sectional and test–retest design was applied. Participants first completed questionnaire T1 (= WORQ-NL part 1 and 2, Work Ability Index -Single item (WAS) and EuroQol-5D-5L) and 1 week after receiving questionnaire 887 T1 by mail, questionnaire T2 (= control questions, WORQNL part 2 and WAS) was sent. Inclusion of 50 persons was aimed at, based on proposed quality criteria for this type of reliability studies [18]. Data collection took place between April 2018 and July 2019. Participants This study included persons, aged 18 to 65 years old, with physical disabilities (spinal cord injury, brain injury, stroke, neuromuscular disease, multiple sclerosis, amputation, or other) who had been referred for vocational rehabilitation (VR) at the Centre for Rehabilitation, University Medical Center Groningen, location Haren, The Netherlands. The participants had to be able to read and write the Dutch language. Procedures The study was introduced by the vocational counselor who gave oral and written information during the intake consultation. Once informed consent was given, the participant received the questionnaire, an instruction leaflet and a return envelope. The participant was requested to fill out the questionnaire independently and return it by postal mail to the researcher. Approximately 1 week after arrival of questionnaire T1, questionnaire T2 with a return envelope was sent to the participant by postal mail, with the request to fill out the questionnaire independently and return it to the researcher within 1 week. No reminder was sent. If questionnaire T2 was filled out 28 or more days after questionnaire T1 was filled out, the participant was excluded because too long time interval between both questionnaires. To trace possible changes in health and work status during the time interval, the following control questions were asked in the second questionnaire: ‘’Did anything regarding your medical condition change during the past 2 weeks when compared to the weeks before? If yes, please specify.’’ and ‘’Did anything regarding your work situation change during the past 2 weeks when compared to the weeks before? If yes, please specify.’’. Instruments WORQ The two versions of the WORQ were used: the 40 items questionnaire (further mentioned as WORQ-FULL) and the brief version questionnaire (further mentioned as WORQBRIEF). The WORQ consists of two parts. Part 1 is identical for the WORQ-FULL and WORQ-BRIEF, and has 17 questions collecting socio-demographic and work-related information. Part 2 of the WORQ-FULL consists of 40 evaluative 13 888 questions (18 body functions and 22 activities and participation items) whereas part 2 of the WORQ-BRIEF consists of 13 evaluative questions (6 body functions and 7 activities and participation items) [14, 17]. The questions in part 2 are answered on a numerical rating scale ranging from 0 (no problem) to 10 (complete problem). The WORQ-NL was developed for use in The Netherlands in collaboration with the developers of the Flemish version [15]. Part 1 was substantially adapted since this part contains questions that are country-specific (e.g. education level grading). For part 2, minimal changes in wording of the Flemish version were negotiated and consensus was reached on a single version for use in Flanders and the Netherlands. However, in the WORQ-NL a response option ‘not applicable’ was added to question 34 (driving) for people without driving license, without a car. The sum score of the WORQFULL was calculated as the sum of the 40 questions of part 2 (higher scores indicate higher work-disability). The sum score of the WORQ-BRIEF was calculated from the WORQFULL by adding the scores of the related 13 questions. Furthermore the mean item score (sum score/ n of items) was computed. WAS The Work Ability Index (WAI) is a well-known instrument used in occupational health care and research to assess work ability of workers during health examinations and workplace surveys developed by the Finnish Institute of Occupational Health [19]. The WAS (Work Ability Index – Single item) is derived from the WAI and consists of one question on the participant’s self-reported current work ability compared to his highest work ability ever (scoring from 0 to 10) [20, 21]. The WAS showed a strong association with the WAI and both have shown a strong predictive value for sick leave and health related quality of life [20]. Euro‑Qol 5D‑5L The Euro-Qol 5D is a simple instrument used to describe health related quality of life. It consists of a self-reported health state description with 5 items (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression) [22]. The Euro-Qol 5D has shown validity and reliability e.g. in the stroke population [22]. The number of answer categories has been increased from 3 levels (EQ-5D-3L) to 5 levels (EQ-5D-5L): 1 = no; 2 = slight; 3 = moderate; 4 = severe problems or 5 = extreme problems [23, 24]; and which has improved its measurement properties [25]. The index valuation score of the EQ-5D-5L was used (a maximal score of 1 stands for perfect health) [26]. 13 Journal of Occupational Rehabilitation (2021) 31:886–894 Data from Medical Files The diagnostic group was the only data from the medical file that was provided to the researcher by the vocational counselor. Statistical Analysis Data were analyzed using SPSS for Windows, version 23.0 (IBM Corp., Armonk, NY). Descriptive analysis were used to describe the study sample. Analyses were performed on the sum-WORQ-FULL—and on the sum-WORQ- BRIEF scores. If a participant provided 2 scores per question, the average of the two scores was used. In case of the EuroQoL, this average was rounded up- or downwards into the nearest integer value. If item 34 was marked as not applicable, this was treated as a missing value. If less than 25% of the item scores were missing, the missing values were replaced by the mean of the valid WORQ item scores. Descriptive, reliability and agreement analyses were repeated in the subgroup of participants not reporting relevant changes in their medical condition. Only few participants mentioned job changes, and these were considered irrelevant for the purpose of this study. Therefore the analyses were not repeated in the subgroup without job changes. Reliability Internal consistency of the WORQ was examined using Cronbach’s alpha. Values of alpha can range from 0 (no internal consistency) to 1 (perfect internal consistency). Internal consistency was considered good when alpha coefficients are between 0.70 and 0.95 [18]. To analyze test–retest reliability, we used the intraclass correlation coefficient (ICC). The ICC was calculated for the WORQ-FULL T1 versus WORQ-FULL T2 and the WORQBRIEF T1 versus WORQ-BRIEF T2. A two-way mixed effects model for absolute agreement was used [27]. An ICC of > 0.70 indicates sufficient reliability and > 0.80 is good [28]. Agreement A paired samples t-test was performed to test for differences in the sum scores for WORQ-FULL T1 versus WORQFULL T2 and for WORQ-BRIEF T1 versus WORQ-BRIEF T2. The values of the paired samples t-test were used for constructing the Bland–Altman plots. Bland–Altman plots were used to analyze agreement between the sum-WORQ scores at T1 and T2. The limits of agreement (LOA) were computed, defined as ± 1.96SD of the mean difference score. To express the figures in terms of effect size (ES), the LOA Journal of Occupational Rehabilitation (2021) 31:886–894 was divided by the SD of the baseline score. Similarly the ES at group level was calculated as 1.96 times the standard error of the difference score (SE = SD/√N) divided by the SD of the baseline score [29]. Cohen’s approach was used to interpret these values: 0.2 is small, 0.5 is moderate and 0.8 is large [28]. Criterion validity To test for criterion validity of the WORQ-FULL versus the WORQ-BRIEF, we used the intraclass correlation coefficient (ICC). This was calculated with the mean item score of both versions (sum score/ n of items) as the sum score of the WORQ-FULL (40 items) is obviously considerably higher than the sum score of the WORQ-BRIEF (13 items). A paired samples t-test was performed to test for differences in means for WORQ-FULL T1 versus WORQ-BRIEF T1. The values of the paired samples t-test were used for constructing Bland–Altman plots. Bland–Altman plots were used to analyze agreement between the WORQ-FULL and BRIEF scores at T1. The LOA and ES was calculated as described above. Validity Convergent validity was tested with correlational analyses between WORQ scores at baseline with the WAS and EuroQoL scores using the Pearson correlation coefficient. Higher scores on the WORQ were expected to correlate with a lower score on the WAS and with a lower score on the EuroQol (negative correlation). A correlation of > 0.6 was expected between the EuroQoL and WORQ and the WAS and WORQ to support convergent validity [30]. Results Data Collection Process Out of the 91 individuals who agreed to participate, 74 (81%) and 60 (66%) returned questionnaire T1 and T2 respectively of whom 49 (54%) participants returned questionnaire T2 within the maximum time interval. Out of the 49 participants that filled in questionnaire T2 within the maximum time interval, 57% (28 out of 49) reported medical changes and 6% (3 out of 49) reported job status changes. The reported job changes were considered minor and not potentially disturbing the reliability results (starting 1–2 h of work per week, having a work visit, starting volunteering). The reported medical changes included: less medical issues; chest infection; knee pain; improvement of muscle strength, sensibility, walking, writing or speaking; physical improvement; less or more pain; more energy; improvement 889 of condition; perspective change; better tolerance for stimuli; more emotional; infection and improvement of functioning. Descriptive Analysis The demographic, employment and VR-related characteristics of the participants are displayed in Table 1. The participants were mainly residing in the community, however also patients undergoing inpatient (n = 2) rehabilitation were included. Median age was 49 (IQR 40–60), 57% were male, 47% had experienced a stroke and 27% spinal cord injury (n = 49). Few missing values were seen, 53 (0.002%) of all answers in WORQ T1 and 12 (0.0006%) of all answers in WORQ T2. Further, 1 participant ticked two boxes in response to one question, once in the WORQ T1 and once in the WORQ T2. Fifteen (20%) participants scored item 34 as not applicable in WORQ T1 and 11 (18%) in WORQ T2. Reliability and Agreement Table 2 shows the mean and the internal consistency values of the WORQ-FULL and the WORQ-BRIEF questionnaires at T1 and T2. Internal consistency was good for both versions and all samples (≥ 0.85). The median score for all measurements was similar to the mean score and is therefore not further mentioned. The mean score of both the WORQ-FULL and the WORQ-BRIEF were lower at T2 when compared with T1. The mean total score (calculated as the mean of the mean item scores) on the WORQ-FULL was lower compared to the corresponding mean total score on the WORQ-BRIEF at both T1 and T2. Table 3 shows the ICC, paired sample t-test and the LOA values. All ICC values of the WORQ-FULL and the WORQ-BRIEF T1 versus T2 were good (≥ 0.85). ICC for the WORQ-FULL versus WORQ-BRIEF was 0.84 (n = 74). The paired sample t-test showed a statistically significant difference for the WORQ-FULL and the WORQ-BRIEF T1 versus T2 (higher score at T1; p ≤ 0.01) and a statistically significant difference for the WORQ-FULL versus the WORQ-BRIEF (higher score for the WORQ-BRIEF; p = 0.00). Bland–Altman plots are seen in Fig. 1. Because all Bland–Altman plots were similar, only the most relevant are presented, the other plots are available upon request. Participants scored higher at baseline compared to retest, and their mean scores on the WORQ-BRIEF were higher compared to the WORQ-FULL. Visual inspection of these plots suggested no proportional bias for the WORQ-FULL and the WORQ-BRIEF at both time points. However the decreasing trend in Fig. 1c comparing the WORQ-FULL with the WORQ-BRIEF indicated that the higher the scores on the questionnaires, the stronger the difference between the scores on both questionnaires (BRIEF having higher 13 890 Table 1 Participants characteristics Journal of Occupational Rehabilitation (2021) 31:886–894 Median (IQR) or n (%) Total group at baseline n = 74 Test–retest n = 49 Test–retest unchanged n = 28 Age (years) Sex (male) Diagnosis Stroke Spinal cord injury Neuromuscular disease Other Civil status Married/partnership Single/divorced/separated/widowed Work status Employed Self-employed Not (formally) employed Current job status Working full time Part time Therapeutic work Missing Time off work (days) Missing/NA Education Primary school Secondary school High school University Missing In medical treatment (% yes) Missing Time between questionnaires (days) 48 (37.8–56.2) 42 (57%) 33 (45%) 17 (23%) 13 (18%) 11 (15%) 49 (66%) 25 (34%) 56 (76%) 6 (8%) 12 (16%) 5 (7%) 3 (4%) 8 (11%) 4 (5%) 108 (71–252) 8(11%) 1 (1%) 41 (55%) 21 (28%) 9 (12%) 2 (3%) 66 (89%) 4 (5%) NA 49 (40–60) 28 (57%) 23 (47%) 13 (27%) 7 (14%) 6 (12%) 35 (71%) 14 (27%) 38 (78%) 5 (10%) 6 (12%) 4 (8%) 1 (2%) 5 (10%) 2 (4%) 112.5 (64–261) 5 (10%) 1 (2%) 25 (51%) 14 (29%) 7 (14%) 2 (4%) 45 (92%) 2 (4%) 14 (12–16) 49 (40–60) 16 (57%) 12 (43%) 8 (29%) 5 (18%) 3 (11%) 22 (79%) 6 (21%) 21 (75%) 2 (7%) 5 (18%) 3 (11%) 0 (0%) 4 (14%) 1 (4%) 140 (83–414) 4 (14%) 1 (4%) 14 (50%) 10 (36%) 3 (11%) 0 (0%) 26 (93%) 1 (4%) 13.5 (12–15) IQR interquartile range, n number, NA not applicable Table 2 Mean and internal consistency analysis All n = 74 Mean (SD) Mean total score (SD) Cronbach’s alpha Range sum score Returned n = 49 Mean (SD) Mean total score (SD) Cronbach’s alpha No changes n = 28 Mean (SD) Mean total score (SD) Cronbach’s alpha WORQ FULL T1 WORQ BRIEF T1 WORQ FULL T2 WORQ BRIEF T2 149.6 (71)* 3.7* (1.8) 0.95* 5–345* 60.2 (26.5)* 4.6* (2) 0.88* 3–120* 152.3 (71.3)* 3.81* (1.8) 0.95* 61.1 (27.4)* 4.7* (2.1) 0.89* 132.4 (66.3) 3.3 (1.7) 0.95 53.1 (25.5) 4.1 (1) 0.88 152.6 (67.8) 3.8 (1.7) 0.94 61.6 (24.6) 4.7 (1.9) 0.85 135.4 (61) 3.4 (1.5) 0.93 54.8 (23.8) 4.2 (1.8) 0.85 SD standard deviation 1 missing mean score). The LOA were similar and large for all agreement comparisons. For all T1 versus T2 measurements at individual level, large score differences (ES ≥ 0.82) are 13 needed to exceed the limits of agreement indicating change beyond random chance, however at group level small score differences (ES ≥ 0.12) are sufficient to show change beyond Journal of Occupational Rehabilitation (2021) 31:886–894 891 Table 3 Test–retest reliability, t-test and agreement values of T1 versus T2 and of WORQ-FULL versus WORQ-BRIEF All n = 74 Returned n = 49* No changes n = 28 SCI n = 13 Stroke n = 23* ICC (95% CI) Range of differences Mean of differences (SD) 95% CI of difference T1-T2 (p value) Limits of agreement Effect size needed to exceed change I Effect size needed to exceed change G ICC (95% CI) Range of differences Mean of differences (SD) 95% CI of difference T1-T2 (p value) Limits of agreement Effect size needed to exceed change I Effect size needed to exceed change G ICC (95% CI) Range of differences Mean of differences (SD) 95% CI of difference T1-T2 (p value) Limits of agreement Effect size needed to exceed change I Effect size needed to exceed change G ICC (95% CI) ICC (95% CI) WORQ FULL T1 vs T2 WORQ BRIEF T1 vs T2 WORQ FULL T1 vs BRIEF T1** – – – – – – 0.84 (0.13–0.95) − 2.83 to 1.0 − 0.9 (0.7) − 1.05 to − 0.72 (0.00) – – – – 0.50 to − 2.28 0.79 – – 0.09 0.86 (0.69–0.93) − 73 to 93 18.1 (32.3) 8.77–27.50 (0.00) 0.87 (0.67–0.94) − 16 to 35 7.4 (11.5) 4.03–10.69 (0.00) − 45.08 to 81.34 0.90 − 15.12 to 29.84 0.82 0.13 0.12 0.85 (0.64–0.93) − 39 to 93 17.2 (32.8) 4.509–29.90 (0.01) 0.86 (0.64–0.94) − 16 to 30 6.8 (11.5) 2.36–11.24 (0.004) − 46.99 to 81.39 0.95 − 15.64 to 29.24 0.91 0.18 0.17 0.77 (− 0.02 to 0.94) 0.88 (0.68–0.95) 0.82 (0.14–0.95) 0.87 (0.68–0.95) CI confidence interval, G group level, I individual level, ICC intraclass correlation coefficient, SD standard deviation 1 missing *Calculated with the mean item score random chance. For the WORQ-FULL versus the WORQBRIEF at individual level moderate to large score differences (ES = 0.79) are needed to exceed the limits of agreement indicating change beyond random chance, however at group level small score differences (ES = 0.09) are sufficient to show change beyond random change. was supported only for the WORQ-BRIEF with the EuroQoL 5D-5L as we determined a correlation of > 0.6 as cut off value. Convergent Validity This study is the first psychometric testing of the Dutch version of the WORQ (for use in the Netherlands) and also the first to compare the full and the brief versions of the WORQ. The internal consistency was good for both versions in our study. This is in line with results on the WORQ-German (α = 0.88) [10], the WORQ-French (α = 0.97) (14] and the WORQ-VL (α = 0.95) [15]. The Correlations of the WORQ-FULL and the WORQ-BRIEF with the EuroQoL 5D-5L were r -0.57 and r = -0.65 respectively (both p < 0.01) and correlation of the WORQ-FULL and the WORQ-BRIEF with the WAS were r = -0.24 and r = -0.28 respectively (both p < 0.05). Convergent validity Discussion 13 892 Fig. 1 a–c Bland–Altman plots of WORQ-FULL T1 vs T2, WORQBRIEF T1 vs T2 and WORQ-FULL vs BRIEF T1 13 Journal of Occupational Rehabilitation (2021) 31:886–894 results of our study furthermore demonstrate good test–retest reliability for both versions. The ICC values in this study were similar to those observed in previous studies with other language versions or patient samples (0.94 for the WORQ-French [14] and 0.85 for the WORQ-VL [15]). The paired sample t-test however showed a statistical significant difference (higher score at T1; p ≤ 0.01) between the scores for both questionnaires (full and short version). In the Bland–Altman plots this lower sum score in the second measurement is seen for both versions. We could not find previous studies analyzing agreement with Bland–Altman plots. In our study we could potentially explain the decrease in sum score as most participants were actively undertaking clinical or outpatient multidisciplinary rehabilitation and in medical treatment (92–93%) and the time interval between both questionnaires was rather long, allowing potential functional recovery during the rehabilitation program. However, the large effect size of the Bland Altman analysis at individual level could also indicate that the differences seen in the sum scores of the first versus the second measurement can be attributed to random chance and not be clinically relevant. In our study, the large effect size could imply that the WORQ might not be sensitive enough to monitor change at individual level in clinical practice. At group level, e.g. in research, the WORQ however confirmed good sensitivity to change in this study. Previous studies suggest that the WORQ may be used to measure reliable change in work-related functioning and found Minimal Detectable Change values ranging from 4.3% to 8.95% for the WORQ-FULL and 8.5% for the WORQ-BRIEF [14, 16, 17], representing the change that can be interpreted as real change. These studies calculated the Standard Error of Measurement using Cronbach’s α or person separation index as reliability coefficient. They therefore represent cross-sectional data which are less reliable than our agreement data representing the difference between both measurements. When analyzing criterion validity and comparing the WORQ-FULL with the WORQ-BRIEF, good correlations were seen (ICC 0.84). The Bland–Altman graph showed that the average item WORQ-score in the WORQ-BRIEF was higher than in the WORQ-FULL. This could be explained by the WORQ-BRIEF having a high proportion of questions on physical complaints and activities resulting in a higher mean score for participants with physical disabilities. Nevertheless, calculating the proportion of dexterity and mobility related items (assumed to be more relevant in persons with physical disabilities) of the WORQ-FULL and -BRIEF (35% respectively 38%), only a slightly higher % is seen in the WORQ-BRIEF [16, 17]. The large limits of agreement and the potential bias seen in the Bland Altman plot emphasize that the full and brief version of the WORQ are better not used interchangeably, Journal of Occupational Rehabilitation (2021) 31:886–894 but are to be used as separate measurement instruments e.g. when the full version is filled out at intake, also the full version should be used for monitoring progress. The strong negative correlation of the WORQ-BRIEF with the EuroQoL 5D-5L is interpreted as support for the convergent validity. The correlation of the WORQ-FULL fell just below the cut off level used. The correlation with the WAS was far below the pre-defined cut off value. The correlations of the WORQ have shown varying results in other studies: weak to strong correlations were reported with self-reported general functioning (r = 0.66), HADS anxiety (r = 0.56 and 0.55), HADS depression (r = 0.57 and 0.49), and self-evaluated general health (r = 0.48) [14, 16], the Becks Depression Inventory II (r = 0.51), EuroQol-VAS current health (r = -0.42 and -0.49), the Medical Outcomes Study Short Form-36 (r = -0.35) [10], the work subscales of the Michigan Hand Outcomes Questionnaires (r = -0.25), the work subscales of Disabilities of Arm, Hand and Shoulder (r = 0.28), the WHODAS (r = 0.81) and the WHOQoL (r = -0.47) [16]. Low correlation with the WAS can be explained, because the WAS measures estimated work ability compared with life time best, whereas the WORQ is a presentation of present functions without a comparison. In hindsight, our predefined expectation of a correlation with the WAS may have been incorrect. The high correlation with the EuroQoL 5D-5L in combination with the higher mean score of the WORQ-BRIEF, however, might indicate that the WORQ-BRIEF is a good selection of important items to measure vocational functioning in persons with physical disabilities and therefore a good instrument for use in rehabilitation settings in this population. A loss of information, however, might be expected when using the more practical WORQ-BRIEF only (e.g. in tetraplegia as items on hand function are not included in the WORQ-BRIEF). A previous study mentioned the WORQ-BRIEF can be used as screening instrument on work-related problems, but also mentioned that it is debatable if it is more suitable in research than WORQ [17]. In our study we had a relatively large time interval (max 27 days) possibly resulting in a population with more medical changes over time. However, analysis on both groups with and without medical changes revealed minor differences. Furthermore, our sample size was relatively small and we had a limited number of participants per diagnostic subgroup not allowing analysis per subgroup with sufficient statistical power. In addition, participants completed the WORQ-FULL only and the results from the WORQBRIEF were derived from this original instrument. Hence, the actual answers on the shorter version (WORQ-BRIEF) might have been slightly different from the answers on the original version since the answers might be influenced by the presence of other questions. 893 Future research into the convergent validity should reveal more clarity on the relation of the WORQ with other (work-related) constructs specifically for persons with physical disabilities in a multidisciplinary setting. Also in future studies, a revision of item 34 (driving) could be considered, because many persons in our study (18–20%) have indicated this item as not applicable and it can be interpreted ambiguously. The relevance of this is confirmed by a study from the authors of the WORQ, published whilst our study was conducted, that also opted for the ‘’not applicable’’ answer option on this question [14]. At the moment to our knowledge the WORQ is not used (yet) for clinical practice or in social security settings in the Netherlands. It is still in an experimental phase, hence the importance of this study. Based on the results of our study we would consider using the WORQ-BRIEF in clinical settings as a screening instrument during VR in combination with more diagnose specific instruments investigating work related functioning. We would prefer the brief version above the full version based on the results of the convergent validity study, the very high internal consistency score of the WORQ-FULL (α ≥ 94) indicating potential redundancy of items and the practicality of the WORQ-BRIEF. In conclusion, we found that the WORQ-NL showed good internal consistency (Cronbach’s alpha ≥ 0.85) and test–retest reliability (ICC ≥ 0.85). Agreement demonstrated large score differences are needed to indicate change beyond random chance for all T1 versus T2 measurements at individual level, however at group level small score differences are sufficient. Criterion validity of the WORQ-FULL versus the WORQ-BRIEF was supported (ICC = 0.84) however agreement demonstrated that moderate to large score differences are needed to exceed the limits of agreement indicating change beyond random chance at individual level, however small score differences are sufficient at group level. This indicates the WORQ-FULL and WORQ-BRIEF are better not to be used interchangeably, but are to be used as separate measurement instruments e.g. when the full version is filled out at intake, also the full version should be used for monitoring progress. Convergent validity was supported only for the WORQ-BRIEF with the EuroQoL 5D-5L (r = -0.65). Acknowledgments Special thanks to G. van Laar, G. Slot, E. Salomon, N. de Jong and E. ten Have, vocational councilors at the Center for Rehabilitation, UMCG, Groningen, The Netherlands for their assistance in recruiting participants and data collection. Authors’ Contribution EHR performed the data collection, analysis and drafted the paper. MFR and MWMP provided feedback on the data analyses and the paper. Data Availability The datasets analysed during the current study are available from the corresponding author on reasonable request. 13 894 Declarations Conflict of interest The authors declare no conflict of interest. Ethical Approval The Medical Ethics Committee of the University Medical Centre Groningen declared that this protocol did not need formal ethical approval under the Dutch law regulating medical research in human beings (Reference Number M18.224565). Informed Consent Informed consent was obtained from all individual participants included in the study. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References 1. Roels EH, Aertgeerts B, Ramaekers D, Peers K. 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https://openalex.org/W4225683966	https://www.researchsquare.com/article/rs-1204081/latest.pdf	English	null	Cyclic experiments and global buckling design of steel-angle-assembled buckling-restrained braces	Bulletin of earthquake engineering	2,022	cc-by	17,164	Cyclic Experiments and Global Buckling Design of Steel-Angles-Assembled Buckling-Restrained Braces Jing-Zhong Tong (  tongjz@zju.edu.cn ) Zhejiang University https://orcid.org/0000-0003-3190-518X En-Yuan Zhang Zhejiang University Yan-Lin Guo Tsinghua University Chao-Qun Yu Zhejiang University Research Article Keywords: Steel-angels-assembled buckling-restrained braces, Hysteretic behavior, Cyclic experiments, Finite element analysis, Restraining ratio, Global buckling design Posted Date: December 30th, 2021 * Corresponding author E-mail address: tongjz@zju.edu.cn DOI: https://doi.org/10.21203/rs.3.rs-1204081/v1 DOI: https://doi.org/10.21203/rs.3.rs-1204081/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Bulletin of Earthquake Engineering on April 4th, 2022. See the published version at https://doi.org/10.1007/s10518-022-01389-w. Jing-Zhong Tong a,b,*, En-Yuan Zhang a, Yan-Lin Guo b, Chao-Qun Yu a nstitute of Advanced Engineering Structures and Materials, Zhejiang University, Hangzhou 310058, China b Department of Civil Engineering, Tsinghua University, Beijing 100084, China a Institute of Advanced Engineering Structures and Materials, Zhejiang University, Hangzhou 310058, China 4 b Department of Civil Engineering, Tsinghua University, Beijing 100084, China 5 Abstract: Compared with traditional BRB, the steel-angles-assembled buckling-restrained 6 brace (SAA-BRB) is an innovative BRB with light-weight, accurate control of the geometrical 7 dimensions, easy installation and convenient disassembly. The SAA-BRB is composed of an 8 external restraining system and a cruciform-sectional inner core. The external restraining 9 system is assembled by four steel angles with the connection of high-strength bolts, and the 10 spacers are installed between the inner core and the restraining system. In this study, the 11 hysteretic behavior of SAA-BRB was investigated by experiments and finite element (FE) 12 simulations. Firstly, three SAA-BRB specimens with different restraining ratios were tested 13 under cyclic loads to investigate the hysteretic performance. It was found that all specimens 14 exhibited stable responses and satisfactory energy-dissipating capabilities during the whole 15 loading process. Then, a refined FE model was established, and its validity in predicting the 16 hysteretic responses of SAA-BRB was verified by the experiments. Moreover, based on the 17 yielding criteria of the outmost fiber for the restraining member section, a design formula for 18 the restraining ratio requirements to avoid global buckling of the SAA-BRB was deduced. 19 Finally, extensive parametric analysis was conducted to verify the accuracy of the design 20 formula by changing the geometric dimensions (the restraining ratio) of models. It was found 21 that the proposed formula for the restraining ratio requirement could lead to a conservative 22 prediction with reasonable accuracy, thus providing valuable references for global buckling 23 design of SAA-BRBs in engineering practice. 24 Keywords: Steel-angels-assembled buckling-restrained braces; Hysteretic behavior; Cyclic 25 experiments; Finite element analysis; Restraining ratio; Global buckling design 26 27 Keywords: Steel-angels-assembled buckling-restrained braces; Hysteretic behavior; Cyclic 25 experiments; Finite element analysis; Restraining ratio; Global buckling design 26 27 1 1 Introduction 29 As a high-performance member with excellent lateral resistance and energy dissipation 30 capacity, buckling-restrained brace (BRB) has been widely used in engineering practice in 31 many countries such as Japan, the US, and China over the years (Kiggins and Uang 2006; 32 Tremblay et al. 2006; Zhao et al. 2017). Generally, the BRB is composed of an inner steel core 33 encased by an external restraining system. The inner core is used to bear the axial load, and the 34 external member is used to limit the lateral displacement of the inner core to prevent it from 35 global buckling (Tong et al. 2020). In the design of BRB, the inner core should reach its full- 36 sectional yield load without undergoing global buckling/local failure mode, ensuring the 37 excellent load-carrying performance and energy-dissipating capacity of BRBs (Black et al. 38 2004; Eryasar and Topkaya 2010; Kiggins and Uang 2006; Tong and Guo 2018). 39 Under regular use or minor earthquakes, BRB can provide sufficient lateral stiffness and 40 horizontal bearing capacity for the frame structure as common braces, and the inner core is in 41 an elastic working state. Under the action of severe earthquakes, BRB can fully develop its 42 plasticity and energy dissipation capacity, reducing damage to the main frame structure 43 (Ariyaratana and Fahnestock 2011; Takeuchi et al. 2010; Zhao et al. 2013). Therefore, the 44 application of BRB not only avoids the global bucking of ordinary braces under severe 45 earthquakes, but achieves excellent energy-dissipating performance to protect the frame 46 structure (Shi et al. 2018). 47 The concept of BRB was first proposed by Yoshino and Karino (1971). In that study, a 48 member with steel plates embedded in reinforced concrete shear walls was proposed. Its 49 working principle is to restrain the buckling of the embedded steel plate through the external 50 concrete wall panel. However, this is not conductive to the material utilization because only 51 the diagonal strip-shaped area of the concrete wall can directly limit the lateral deformation of 52 the embedded steel plate. In this consideration, BRB has gradually evolved into a type in which 53 a slender external restraining system constrains the inner core to further improve the utilization 54 of material. In early development stage of this type of BRBs, a typical restraining member is 55 composed of concrete-filled steel tube or reinforced concrete (Mochizuki et al. Nomenclature Symbol Description Symbol Description A1 Cross-sectional area of a single steel angle A0 Cross-sectional area of the external restraining system Ac Cross-sectional area of the steel core b Width of yield segment of the inner core ba Length of the steel angle flange b1 Width of strengthened segment of the inner core fyr Yield stress of the external restraining system fyc Yield stress of the inner steel core Fy Yield load of the specimen Fmax Maximum load of the specimen g Gap size between the steel core and restraining system har Distance between the edge of restraining system and the bolts circle center H Height of frame connected with the BRB I0 Moment of inertia regarding the external restraining system as a whole I1 Moment of inertia of a single steel angle Ki Axial secant stiffness l Length of SAA-BRB l0 Length of the restraining system ly Length of yield segment of the inner core lu Length of unrestrained segment of the inner core lt Length of transition segment of the inner core ls Length of strengthened segment of the inner core ∆ly Axial deformation of the BRB ∆y Yield displacement of the specimen ∆max Maximum displacement of the specimen +∆i Displacement corresponding to tensile peak point at i-th hysteretic cycle y p A1 Cross-sectional area of a single steel angle A0 Cross-sectional area of the external restraining system Ac Cross-sectional area of the steel core b Width of yield segment of the inner core ba Length of the steel angle flange b1 Width of strengthened segment of the inner core fyr Yield stress of the external restraining system fyc Yield stress of the inner steel core Fy Yield load of the specimen Fmax Maximum load of the specimen g Gap size between the steel core and restraining system har Distance between the edge of restraining system and the bolts circle center H Height of frame connected with the BRB I0 Moment of inertia regarding the external restraining system as a whole I1 Moment of inertia of a single steel angle Ki Axial secant stiffness l Length of SAA-BRB l0 Length of the restraining system ly Length of yield segment of the inner core lu Length of unrestrained segment of the inner core lt Length of transition segment of the inner core ls Length of strengthened segment of the inner core ∆ly Axial deformation of the BRB ∆y Yield displacement of the specimen ∆max Maximum displacement of the specimen +∆i Displacement corresponding to tensile peak point at i-th hysteretic cycle 2 3 -∆i Displacement corresponding to compressive peak point at i-th hysteretic cycl l1 Bolts spacing along the longitudinal direction L Length of frame connected with BRB Mmax Maximum bending moment at mid-span of the restraining system Pcr Euler buckling load of BRB Pcr,0 Euler buckling load of the external restraining system of ordinary BRB Pcr,c Euler buckling load of the inner core Pcr,r Euler buckling load of the external restraining system of SAA-BRB Py Yield load of the inner core Pcmax Load corresponding to compressive peak point of the hysteretic curve Ptmax Load corresponding to tensile peak point of the hysteretic curve +Pi Load corresponding to tensile peak point at i-th hysteretic cycle -Pi Load corresponding to compressive peak point at i-th hysteretic cycle S(ABC+CDA) Area enclosed by a single hysteretic curve and the abscissa S(OBE+ODF) Triangle area enclosed by a dotted line and the abscissa t Thickness of the inner core ta Thickness of steel angle v0 Amplitude of the initial geometrical imperfection of the SAA-BRB vmax Lateral maximum deformation at mid-span of the restraining system W Plastic section modulus of the external restraining system of SAA-BRB ω Reduction factor of global buckling load of external restraining system ζ Restraining ratio [ζ] Restraining ratio requirements λ0 Slenderness ratio regarding the external restraining system as a whole λ1 Slenderness ratio of a segment of single chord member σmax Maximum normal stress of the external restraining system λc Slenderness ratio of the inner core α Angle between the BRB and its connected frame beam -∆i Displacement corresponding to compressive peak point at i-th hysteretic cycl l1 Bolts spacing along the longitudinal direction L Length of frame connected with BRB Mmax Maximum bending moment at mid-span of the restraining system Pcr Euler buckling load of BRB Pcr,0 Euler buckling load of the external restraining system of ordinary BRB Pcr,c Euler buckling load of the inner core Pcr,r Euler buckling load of the external restraining system of SAA-BRB Py Yield load of the inner core Pcmax Load corresponding to compressive peak point of the hysteretic curve Ptmax Load corresponding to tensile peak point of the hysteretic curve +Pi Load corresponding to tensile peak point at i-th hysteretic cycle -Pi Load corresponding to compressive peak point at i-th hysteretic cycle S(ABC+CDA) Area enclosed by a single hysteretic curve and the abscissa S(OBE+ODF) Triangle area enclosed by a dotted line and the abscissa t Thickness of the inner core ta Thickness of steel angle v0 Amplitude of the initial geometrical imperfection of the SAA-BRB vmax Lateral maximum deformation at mid-span of the restraining system W Plastic section modulus of the external restraining system of SAA-BRB ω Reduction factor of global buckling load of external restraining system ζ Restraining ratio [ζ] Restraining ratio requirements λ0 Slenderness ratio regarding the external restraining system as a whole λ1 Slenderness ratio of a segment of single chord member σmax Maximum normal stress of the external restraining system λc Slenderness ratio of the inner core α Angle between the BRB and its connected frame beam Displacement corresponding to compressive peak point at i-th hysteretic cycle Bolts spacing along the longitudinal direction 3 [εc] Maximum axial strain requirement of the BRB φ Ratio of the inner core yield segment length to BRB length β Compression strength adjustment factor ξeq Equivalent viscous damping ratio [εc] Maximum axial strain requirement of the BRB φ Ratio of the inner core yield segment length to BRB length β Compression strength adjustment factor ξeq Equivalent viscous damping ratio 28 4 4 1 Introduction 29 1979; Nagao et 56 5 al. 1992). Since the restraining system is used to limit the lateral deformation of the inner core, 57 a thin layer of unbonded material should be installed at the interface between the inner core 58 and its surrounding concrete to avoid axial force transmission (Fujimoto et al. 1990; Ju et al. 59 2009; Palazzo et al. 2009). However, complex construction procedures caused by wet concrete 60 work, heavy self-weight and inconvenient disassembly result in difficulties when repairing the 61 BRB after an earthquake (Ju et al. 2009; Shi et al. 2018). To solve these problems, researchers 62 proposed BRBs with all-steel composition and conducted experimental and numerical studies 63 (Guo et al. 2016; Guo et al. 2017a, b; Hosseinzadeh and Mohebi 2016; Jia et al. 2017; Judd et 64 al. 2016; Mirtaheri et al. 2017). The all-steel BRB is mainly characterized by a steel core with 65 a circular section or H-section and a restraining member of a circular or square steel tube. Due 66 to its all-steel composition, there is no need for unbonded material at the interface between the 67 inner core and the restraining member as an air gap could be precisely controlled during its 68 fabrication (Guo et al. 2017a; Jia et al. 2017; Mirtaheri et al. 2017). Besides, the self-weight of 69 all-steel BRB is significantly reduced compared with the traditional BRB composed of an inner 70 steel core with the encasement of concrete-filled steel tube. Furthermore, to satisfy the 71 requirements of high-rise buildings or long-span structures with popularity in recent years, 72 assembled BRBs with bolt-connected restraining systems were proposed and investigated by 73 numerous researchers (Chou and Chen 2010; Dehghani and Tremblay 2018; Ding 2014; Guo 74 et al. 2018; Jiang et al. 2017; Metelli et al. 2016; Tong and Guo 2018; Chou et al. 2019). Chou 75 and Chen (2010) developed a sandwiched assembled BRB with an inner core encased by two 76 identical restraining members connected with high-strength bolts, and four BRB specimens 77 were tested to investigate the load-carrying performance. Dehghani and Tremblay (2018) 78 proposed an all-steel assembled BRB composed of a flat plate core and a rectangular restraining 79 member with a hollow steel section. The stable seismic performance and high post-yield 80 stiffness of this BRB were examined by experiments. Jiang et al. 1 Introduction 29 (2017) developed a pinned 81 double-rectangular tube assembled BRB, which exhibited satisfactory energy-dissipating 82 performance under repeated axial-tension loading. Guo et al. (2018) proposed an assembled 83 BRB in which the restraining member consists of four steel channels assembled by bolts. A FE 84 6 6 model that could well predict the hysteretic responses was established and a design procedure 85 was proposed subsequently. For assembled BRBs, the external restraining system generally 86 includes multiple components connected by high-strength bolts. The advantages of assembled 87 BRB include: (1) the fabrication of BRB with the bolt connection is convenient and quick; (2) 88 the BRB can be easily disassembled, reducing the difficulty in transportation and erection; (3) 89 the damaged core can be easily replaced in the post-earthquake structure due to the feature of 90 easy disassembling. It should be noted that the discrete arrangement of the connecting bolts 91 will weaken the rigidity of the restraining member in the design of assembled BRBs. 92 model that could well predict the hysteretic responses was established and a design procedure 85 was proposed subsequently. For assembled BRBs, the external restraining system generally 86 includes multiple components connected by high-strength bolts. The advantages of assembled 87 BRB include: (1) the fabrication of BRB with the bolt connection is convenient and quick; (2) 88 the BRB can be easily disassembled, reducing the difficulty in transportation and erection; (3) 89 the damaged core can be easily replaced in the post-earthquake structure due to the feature of 90 easy disassembling. It should be noted that the discrete arrangement of the connecting bolts 91 will weaken the rigidity of the restraining member in the design of assembled BRBs. 92 In this paper, a new type of assembled BRB called steel-angles-assembled BRB (SAA- 93 BRB) was studied. The SAA-BRB consists of a cruciform-sectional steel core and a restraining 94 system composed of four steel angles. The cyclic loading experiment was carried out on three 95 specimens to investigate the load-carrying performance and hysteretic response of SAA-BRB. 96 Besides, a finite element (FE) model of SAA-BRB was established, and the comparison against 97 experimental results verified the feasibility of the model. Moreover, the theoretical design 98 formula with restraining ratio requirements to prevent SAA-BRB from global buckling was 99 derived. Finally, extensive FE simulations were performed to validate the proposed design 100 formula. 101 2 Cyclic experiments 102 2.1 Experimental specimens 103 2.1 Experimental specimens 103 Three SAA-BRB specimens were designed to experimentally investigate their hysteretic 104 responses. Fig. 1 shows the construction details of SAA-BRB specimens, which consists of an 105 inner steel core with a uniform cruciform-section and a restraining system as well as spacers. 106 The external restraining system is composed of four steel angles assembled by high-strength 107 bolts (M16). The spacers with holes for the bolts to pass through are installed between the steel 108 angles. The inner core can be divided into yield segment and unrestrained segment composed 109 of a transition segment and a strengthened segment along its longitudinal direction. The 110 unrestrained segment of the core was enhanced at both ends by expanding its cross-sectional 111 size, and it was welded with a flange to prevent it from local buckling. Besides, the stiffened 112 7 plates were set at the end of each steel angle along the longitudinal direction to enhance the 113 integrity of the external restraining member. According to Chinese design code GB50017 (GB 114 2017), the slenderness ratio of the core λc should be greater than 5.07b/t to prevent the torsional 115 buckling failure of SAA-BRB. Additionally, the stoppers (i.e. displacement limitation fastener) 116 were designed and arranged on each side of the mid-span of the core by welding to inhibit the 117 restraining system from sliding longitudinally along the core. 118 plates were set at the end of each steel angle along the longitudinal direction to enhance the 113 integrity of the external restraining member. According to Chinese design code GB50017 (GB 114 2017), the slenderness ratio of the core λc should be greater than 5.07b/t to prevent the torsional 115 buckling failure of SAA-BRB. Additionally, the stoppers (i.e. displacement limitation fastener) 116 were designed and arranged on each side of the mid-span of the core by welding to inhibit the 117 restraining system from sliding longitudinally along the core. 118 Specimens S-1, S-2 and S-3 were similar in geometrical configuration, and the design 119 parameters of each specimen are tabulated in Table 1. For all specimens, SAA-BRB length, l, 120 and restraining system length, l0, were 2400 mm and 2100 mm, respectively. The steel angle 121 width ba of Specimen S-1 was 80 mm, and those of Specimens S-2 and S-3 were 90 mm. Steel 122 angle thickness ta was 6 mm for each specimen. 2.1 Experimental specimens 103 The cruciform-sectional core member width, 123 b, was 100 mm for Specimens S-1 and S-2 while 110 mm for Specimen S-3. Inner core 124 thickness, t, was 22 mm for each specimen. 125 To avoid the axial load sustained by the inner core being transmitted to the restraining 126 system, hard rubbers with a thickness of 1.2 mm were installed between the steel core and four 127 steel angles. The spacer thickness of all specimens was 25 mm, and the spacer widths in the 128 transverse direction of the cross-section and the longitudinal direction were 48 mm and 42 mm, 129 respectively. 130 Flange Lift plate Steel angle Spacer Core Stiffened plate Displacement limitation fastener 42 90 236 326 l0= 2100 har Spacer Hard rubber ly=2100 (Yield segment) lt=100 b1 b 22 43 10 42 48 Displacement limitation fastener l=2400 ls=50 lu=150 (Unrestrained segment) Bolts (M16) 326 326 326 236 90 42 30 30 150 Fig. 1 Construction details of SAA-BRB specimens Flange Lift plate Steel angle Spacer Core Stiffened plate Displacement limitation fastener 42 90 236 326 l0= 2100 har Spacer Hard rubber ly=2100 (Yield segment) lt=100 b1 b 22 43 10 42 48 Displacement limitation fastener l=2400 ls=50 lu=150 (Unrestrained segment) Bolts (M16) 326 326 326 236 90 42 30 30 150 Displacement limitation fastener ly=2100 (Yield segment) Displacement limitation fastener Fig. 1 Construction details of SAA-BRB specimens 8 131 Table 1 Design parameters of SAA-BRB specimens 132 Specimen no. b (mm) b1 (mm) ba×ta (mm) har (mm) g (mm) l0 fyr (MPa) fyc (MPa) ζ S-1 100 200 80×6 25 1 2100 380 288 1.86 S-2 100 200 90×6 35 1 2100 380 288 2.47 S-3 110 220 90×6 35 1 2100 380 288 2.22 2.2 Experimental setup and loading protocol 133 The MTS hydraulic servo testing machine was used for applying cyclic loads. Fig. 2 134 presents the experimental setup consisting of two reaction frames anchored to the strong floor 135 and test specimens loaded via a 600 t actuator with an in-line load cell. At both ends of each 136 specimen, the flange with Q345 steel plate is connected to the cross-shaped connecting plate 137 using high-strength bolts, and the cruciform-shaped connecting plate is also connected to the 138 131 132 131 Table 1 Design parameters of SAA-BRB specimens 132 Specimen no. 2.1 Experimental specimens 103 b (mm) b1 (mm) ba×ta (mm) har (mm) g (mm) l0 fyr (MPa) fyc (MPa) ζ S-1 100 200 80×6 25 1 2100 380 288 1.86 S-2 100 200 90×6 35 1 2100 380 288 2.47 S-3 110 220 90×6 35 1 2100 380 288 2.22 2.2 Experimental setup and loading protocol 133 131 132 Table 1 Design parameters of SAA-BRB specimens The MTS hydraulic servo testing machine was used for applying cyclic loads. Fig. 2 134 presents the experimental setup consisting of two reaction frames anchored to the strong floor 135 and test specimens loaded via a 600 t actuator with an in-line load cell. At both ends of each 136 specimen, the flange with Q345 steel plate is connected to the cross-shaped connecting plate 137 using high-strength bolts, and the cruciform-shaped connecting plate is also connected to the 138 actuator and the reaction frame with the connection of bolts. 139 The MTS hydraulic servo testing machine was used for applying cyclic loads. Fig. 2 134 presents the experimental setup consisting of two reaction frames anchored to the strong floor 135 and test specimens loaded via a 600 t actuator with an in-line load cell. At both ends of each 136 specimen, the flange with Q345 steel plate is connected to the cross-shaped connecting plate 137 using high-strength bolts, and the cruciform-shaped connecting plate is also connected to the 138 actuator and the reaction frame with the connection of bolts. 139 Actuator (600t) Strong floor Reaction frame Cross-shaped connecting plate Reaction frame Axial displacement meter Fig. 2 Experimental setup As shown in Fig. 3, H and L respectively denote the height and length of the frame 140 connected with BRB, α the angle between the BRB and the frame beam, thus the length of the 141 BRB l can be expressed as l= 2 2 H L  . For BRB, the length of the yield segment of the core is 142 ly, thus the ratio of the core yield segment length to BRB length is denoted as φ= ly / l. According 143 to the Chinese code GB50010 (GB 2010) for seismic design of buildings, the maximum drift 144 angle of the frame structure is 1/50. Hence, the maximum axial deformation of the inner core 145 can be expressed as ∆ly=Hcosα/50. 2.1 Experimental specimens 103 3 Calculation of the maximum axial strain requirements Prior to the standard loading process, the axial compressive load of half of the yield load 153 calculated from the FE model was preloaded to check whether the experimental devices and 154 measuring instruments were reliable. After that, the standard loading process, in which three 155 loading cycles with an axial strain of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, and 2.00%, were 156 performed successively to the specimens. The axial displacement loading protocol of the 157 standard loading process for each specimen is tabulated in Table 2. According to this loading 158 protocol, when the SAA-BRB specimens completed three 2.00% axial strain loadings, the 159 cumulative plastic ductility would exceed the required ductility of 200 times of the yield strain 160 as specified in AISC provisions (AISC 2016). The standard cyclic loading protocol of the axial 161 strain with respect to the cycle number for each specimen is illustrated in Fig. 4. 162 Table 2 Axial displacement loading protocol 163 Load step Cycle number Axial strain Axial displacement (mm) 1 3 ± 0.25% ± 5.0 2 3 ± 0.50% ± 10.0 3 3 ± 0.75% ± 16.0 4 3 ± 1.00% ± 21.0 5 3 ± 1.50% ± 32.0 6 3 ± 2.00% ± 42.0 164 Prior to the standard loading process, the axial compressive load of half of the yield load 153 calculated from the FE model was preloaded to check whether the experimental devices and 154 measuring instruments were reliable. After that, the standard loading process, in which three 155 loading cycles with an axial strain of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, and 2.00%, were 156 performed successively to the specimens. The axial displacement loading protocol of the 157 standard loading process for each specimen is tabulated in Table 2. According to this loading 158 protocol, when the SAA-BRB specimens completed three 2.00% axial strain loadings, the 159 cumulative plastic ductility would exceed the required ductility of 200 times of the yield strain 160 as specified in AISC provisions (AISC 2016). The standard cyclic loading protocol of the axial 161 strain with respect to the cycle number for each specimen is illustrated in Fig. 4. 2.1 Experimental specimens 103 In engineering practice, axial deformation mainly occurs in 146 the yield segment of the core due to the unrestrained segment of the core being enhanced by 147 increasing its sectional size. Therefore, the maximum axial strain requirement [εc] of the BRB 148 can be expressed as Eq. (1) by assuming that the axial deformation entirely occurs in the yield 149 Actuator (600t) Strong floor Reaction frame Cross-shaped connecting plate Reaction frame Axial displacement meter Fig. 2 Experimental setup Actuator (600t) Strong floor Reaction frame Cross-shaped connecting plate Reaction frame Axial displacement meter Fig. 2 Experimental setup Fig. 2 Experimental setup As shown in Fig. 3, H and L respectively denote the height and length of the frame 140 connected with BRB, α the angle between the BRB and the frame beam, thus the length of the 141 BRB l can be expressed as l= 2 2 H L  . For BRB, the length of the yield segment of the core is 142 ly, thus the ratio of the core yield segment length to BRB length is denoted as φ= ly / l. According 143 to the Chinese code GB50010 (GB 2010) for seismic design of buildings, the maximum drift 144 angle of the frame structure is 1/50. Hence, the maximum axial deformation of the inner core 145 can be expressed as ∆ly=Hcosα/50. In engineering practice, axial deformation mainly occurs in 146 the yield segment of the core due to the unrestrained segment of the core being enhanced by 147 increasing its sectional size. Therefore, the maximum axial strain requirement [εc] of the BRB 148 can be expressed as Eq. (1) by assuming that the axial deformation entirely occurs in the yield 149 9 9 segment of the core. 0 segment of the core. 150 y c y cos / 50 sin cos [ ] / sin 50 l H l H            (1) (1) Assuming that α=45º and φ=0.5, it is calculated as [εc] = 0.02. Therefore, the axial strain of 151 2.0% was determined as the maximum strain in the loading protocol. 152 % was determined as the maximum strain in the loading protocol. Fig. 3 Calculation of the maximum axial strain requirements H L =H/50 l ly ly' l' F  =H/50 Fig. 3 Calculation of the maximum axial strain requirements Fig. 2.1 Experimental specimens 103 162 Table 2 Axial displacement loading protocol 163 Load step Cycle number Axial strain Axial displacement (mm) 1 3 ± 0.25% ± 5.0 2 3 ± 0.50% ± 10.0 3 3 ± 0.75% ± 16.0 4 3 ± 1.00% ± 21.0 5 3 ± 1.50% ± 32.0 6 3 ± 2.00% ± 42.0 164 10 0 3 6 9 12 15 18 21 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Axial strain (%) Cycle number Fig. 4 Cyclic loading protocol 2 3 Measurement point arrangements 165 0 3 6 9 12 15 18 21 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Axial strain (%) Cycle number Fig. 4 Cyclic loading protocol The measuring-point arrangements of strain gauges and displacement meters are 166 presented in Fig. 5. The strain gauges were installed on the steel angles to record the axial strain. 167 There were two measured sections: section 1 and section 2, and each section had eight 168 measuring points. For displacement meters, in addition to two axial displacement meters (as 169 shown in Fig. 2) used to examine the axial deformation, six lateral displacement meters were 170 located near the mid-span bolt sets and at the quarter points of the restraining member along 171 the longitudinal direction. 172 Section 1 Section 2 South North A A Displacement meter West East Top Bottom West 1 2 7 8 4 6 5 3 Sectional view (A-A) of S-2 or S-3 East 0 84 118 202 Top Bottom West 1 2 7 8 4 6 5 3 Sectional view (A-A) of S-1 East 0 74 108 182 Section 1 Section 2 South North A A Displacement meter West East Fig. 5 Measuring-point arrangements Section 1 North A Top Bottom West 1 2 7 8 4 6 5 3 Sectional view (A-A) of S-2 or S-3 East 0 84 118 202 Top Bottom West 1 2 7 8 4 6 5 3 Sectional view (A-A) of S-1 East 0 74 108 182 East East Sectional view (A-A) of S-2 or S-3 Fig. 5 Measuring-point arrangements 2.4 Experimental results 173 2.4 Experimental results 173 2.4.1 Hysteretic curves and skeleton curves 174 It can be seen from Fig. 2.1 Experimental specimens 103 6 that the hysteretic curves of the three SAA-BRB specimens 175 11 maintained stable during the whole cyclic loading process, indicating that SAA-BRB has great 176 energy-dissipating capacity under severe seismic effects. Fig. 7 illustrates the deformation 177 condition corresponding to a core strain of 2.0%. It is found that none of the specimens showed 178 global buckling or local buckling. This is because the restraining ratio of each specimen is 179 greater than the corresponding restraining ratio requirement. 180 maintained stable during the whole cyclic loading process, indicating that SAA-BRB has great 176 energy-dissipating capacity under severe seismic effects. Fig. 7 illustrates the deformation 177 condition corresponding to a core strain of 2.0%. It is found that none of the specimens showed 178 global buckling or local buckling. This is because the restraining ratio of each specimen is 179 greater than the corresponding restraining ratio requirement. 180 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-1 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-2 (a) Specimen S-1 (b) Specimen S-2 -42 -21 0 21 42 -2.0 -1.0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-3 0 (c) Specimen S-3 Fig. 6 Hysteretic curves of specimens 181 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-2 Axial strain (%) -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-1 Axial strain (%) Axial displacement (mm) Axial displacement (mm) 21 0 21 Axial displacement (mm) Axial displacement (mm) p ( ) Axial displacement (mm) (a) Specimen S-1 (b) Specimen S-2 -42 -21 0 21 42 -2.0 -1.0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-3 0 (c) Specimen S-3 Fig. 2.1 Experimental specimens 103 6 Hysteretic curves of specimens (a) Specimen S-1 (b) Specimen S-2 -42 -21 0 21 42 -2.0 -1.0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-3 0 (c) Specimen S-3 (b) Specimen S-2 (a) Specimen S-1 0 1 0 Axial strain (%) 0 (c) Specimen S-3 Fig. 6 Hysteretic curves of specimens Fig. 6 Hysteretic curves of specimens 12 (a) Specimen S-1 (b) Specimen S-2 (c) Specimen S-3 Fig. 7 Deformation condition corresponding to 2.0% axial compression strain (a) Specimen S-1 (b) Specimen S-2 (a) Specimen S-1 (c) Specimen S-3 (c) Specimen S-3 Fig. 7 Deformation condition corresponding to 2.0% axial compression strain Fig. 7 Deformation condition corresponding to 2.0% axial compression strain The skeleton curves of specimens are depicted in Fig. 8. The displacement and load values 184 corresponding to the yield and peak points of all specimens are shown in Table 3. When the 185 specimens reach 2.0% axial strain, the skeleton curves still maintain increasing trends, 186 indicating that the SAA-BRB specimens have a cyclic effect and great ductility under axial 187 cyclic loading. Hence, it is conservative to take the peak point of the specimen as the last point 188 of the skeleton curve to leave enough safety redundancy. In addition, each specimen curve has 189 obvious inflection points, which are determined as the yield points of the specimen. 190 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-1 S-2 S-3 Fig. 8 Skeleton curves of specimens -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) S-1 S-2 S-3 Fig. 8 Skeleton curves of specimens 13 Table 3 Characteristic points and compression strength factors of specimens 191 Specimen no. Loading direction Yield points Peak points β Fy/kN Δy/mm Fmax/kN Δmax/mm S-1 Tension 1212.58 4.77 1862.36 41.66 1.06 Compression -1208.38 -4.93 -1971.56 -41.33 S-2 Tension 1166.98 4.80 1730.37 40.04 1.09 Compression -1129.18 -4.85 -1884.56 -41.91 S-3 Tension 1299.57 4.92 1916.36 40.38 1.08 Compression -1267.77 -5.13 -2063.96 -41.36 2.4.2 Equivalent viscous damping ratio 192 Table 3 Characteristic points and compression strength factors of specimens 191 Fig. 10 illustrates the equivalent viscous damping ratios of all specimens with respect to 193 the hysteretic loop. 2.1 Experimental specimens 103 The energy-dissipating capacity of the specimen can be quantitatively 194 evaluated by the equivalent viscous damping ratio (ξeq), which can be calculated by Eq. (2). It 195 is seen that the equivalent viscous damping ratio of all specimens generally presents an 196 increasing trend with the increase of the hysteretic loops. Specifically, before the specimens 197 yielded (i.e., before the specimens reached 0.25% axial strain), the values of ξeq ranged from 198 0.2 to 0.3. As the inner core gradually consumed energy during the loading process, the values 199 of ξeq increased until they stabilized above 0.4. Moreover, the maximum equivalent viscous 200 damping ratios of the three specimens all exceeded 0.5, demonstrating that all the SAA-BRB 201 specimens exhibited excellent energy-dissipating capacity. 202 Fig. 10 illustrates the equivalent viscous damping ratios of all specimens with respect to 193 the hysteretic loop. The energy-dissipating capacity of the specimen can be quantitatively 194 evaluated by the equivalent viscous damping ratio (ξeq), which can be calculated by Eq. (2). It 195 is seen that the equivalent viscous damping ratio of all specimens generally presents an 196 increasing trend with the increase of the hysteretic loops. Specifically, before the specimens 197 yielded (i.e., before the specimens reached 0.25% axial strain), the values of ξeq ranged from 198 0.2 to 0.3. As the inner core gradually consumed energy during the loading process, the values 199 of ξeq increased until they stabilized above 0.4. Moreover, the maximum equivalent viscous 200 damping ratios of the three specimens all exceeded 0.5, demonstrating that all the SAA-BRB 201 specimens exhibited excellent energy-dissipating capacity. 202 (ABC CDA) eq (OBE ODF) 1 · 2 S S      (2) (2) in which S(ABC+CDA) denotes the enveloping area of the hysteretic loop curve in Fig. 9; and 203 S(OBE+ODF) denotes the area of the triangles OBE and ODF in Fig. 9. 204 in which S(ABC+CDA) denotes the enveloping area of the hysteretic loop curve in Fig. 9; and 203 S(OBE+ODF) denotes the area of the triangles OBE and ODF in Fig. 9. 204 S(OBE+ODF) denotes the area of the triangles OBE and ODF in Fig. 9. 204 Axial force Axial displacement E B A C F O D Fig. 9 Calculation of the equivalent viscous damping ratio Axial force Fig. 2.1 Experimental specimens 103 9 Calculation of the equivalent viscous damping ratio 205 14 14 0 3 6 9 12 15 18 21 0 0.25 0.50 0.75 1.00 1.5 2.0 2.5 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Axial strain (%) ξeq Loop no. S-1 S-2 S-3 Fig. 10 Equivalent viscous damping ratio of specimens 0 3 6 9 12 15 18 21 0 0.25 0.50 0.75 1.00 1.5 2.0 2.5 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Axial strain (%) ξeq Loop no. S-1 S-2 S-3 Fig. 10 Equivalent viscous damping ratio of specimens 2.4.3 Compression strength adjustment factor 206 The asymmetric response under tension and compression of the SAA-BRB can be 207 evaluated by the compression strength adjustment factor β, which is defined by Eq. (3) (Jiang 208 et al. 2017). The larger the β, the more obvious the asymmetric response is. It is calculated that 209 the β values of the three SAA-BRB specimens are 1.06, 1.09, and 1.08, respectively as 210 tabulated in Table 3. ANSI/AISC 341-16 (AISC 2016) specified that the factor β shall exceed 211 unity but be no greater than 1.50. Therefore, it is shown that SAA-BRB has a reasonable 212 hysteretic response under both tension and compression. 213 i i P P tmax cmax   (3) i i P P tmax cmax   (3) (3) (3) in which cmax i P and tmax i P are the loads corresponding to the compressive and tensile peak points 214 of the hysteretic curve respectively 215 in which cmax i P and tmax i P are the loads corresponding to the compressive and tensile peak points 214 in which cmax i P and tmax i P are the loads corresponding to the compressive and tensile peak points 214 f h h i i l 215 of the hysteretic curve, respectively. 215 of the hysteretic curve, respectively. 215 of the hysteretic curve, respectively. 215 2.4.4 Stiffness degradation 216 2.4.4 Stiffness degradation 216 The stiffness degradation properties of all specimens are shown in Fig. 11. The axial 217 stiffness degradation curve can reflect the damage history of BRB under cyclic loads. 218 According to the Chinese code JGJ/T 101-2015 (JGJ 2015), the secant stiffness Ki is used to 219 express the stiffness of the specimen and its calculation is shown in Eq. (4). 2.1 Experimental specimens 103 The horizontal 220 coordinate of Fig.11 is the displacement corresponding to the peak point of each loop in the 221 compressive or tensile directions. It is found that the secant stiffnesses of all specimens are 222 basically identical at the same load step. In addition, although the axial stiffness of three SAA- 223 BRB specimens decreased during the loading process, the rate of stiffness degradation 224 15 gradually slowed down. This shows that the SAA-BRB would not lose its stiffness 225 instantaneously during loading. 226 gradually slowed down. This shows that the SAA-BRB would not lose its stiffness 225 instantaneously during loading. 226 i i i P K    (4) i i i P K    (4) (4) in which +Pi and -Pi are the loads corresponding to tensile and compressive peak points of the 227 hysteretic curve at the i-th cycle, respectively; +∆i and -∆i are the displacements corresponding 228 to tensile and compressive peak points of the hysteretic curve at the i-th cycle, respectively. 229 in which +Pi and -Pi are the loads corresponding to tensile and compressive peak points of the 227 hysteretic curve at the i-th cycle, respectively; +∆i and -∆i are the displacements corresponding 228 to tensile and compressive peak points of the hysteretic curve at the i-th cycle, respectively. 229 in which +Pi and -Pi are the loads corresponding to tensile and compressive peak points of the 227 hysteretic curve at the i-th cycle, respectively; +∆i and -∆i are the displacements corresponding 228 to tensile and compressive peak points of the hysteretic curve at the i-th cycle, respectively. 229 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 0 50 100 150 200 250 300 Axial strain (%) Axial displacement (mm) S-1 S-2 S-3 Ki (kN·m-1) -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 0 50 100 150 200 250 300 Axial strain (%) Axial displacement (mm) S-1 S-2 S-3 Ki (kN·m-1) Fig. 11 Curves of axial secant stiffness degradation Fig. 11 Curves of axial secant stiffness degradation 2.4.5 Longitudinal strain of restraining system 230 Fig. 12 shows the longitudinal strain distribution and development of the external 231 restraining system of the three specimens. 2.1 Experimental specimens 103 This indicates that significant shear deformation exists in the restraining 246 system and the plane-section assumption is inappropriate for the SAA-BRB. This is mainly 247 caused by the discrete layout of the connecting bolts in the restraining system. When the inner 248 core was under compression and developed multi-wave buckling mode, lateral thrust effect 249 existed between the core and restraining system, thus causing friction along their interface. 250 Hence, part of the axial load of the inner core was transmitted to the restraining system. This 251 explains that the measured compressive strain is larger than tensile strain in the two measuring- 252 points which are symmetrical to the midline of the cross-section of each specimen. 253 -800 -600 -400 -200 0 200 400 0 50 100 150 200 Depth of cross-section (mm) Strain (με ) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-1 -400 -200 0 200 400 600 0 50 100 150 200 Strain (με ) Depth of cross-section (mm) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-2 -1000 -800 -600 -400 -200 0 200 400 0 50 100 150 200 Strain (με ) Depth of cross-section (mm) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-3 254 (a) Specimen S-1 (b) Specimen S-2 (c) Specimen S-3 Fig. 12 Distribution and development of longitudinal strains in restraining system 255 -800 -600 -400 -200 0 200 400 0 50 100 150 200 Depth of cross-section (mm) Strain (με ) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-1 254 (a) Specimen S-1 -1000 -800 -600 -400 -200 0 200 400 0 50 100 150 200 Strain (με ) Depth of cross-section (mm) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-3 (c) Specimen S-3 -400 -200 0 200 400 600 0 50 100 150 200 Strain (με ) Depth of cross-section (mm) c=2.00% c=1.50% c=1.00% c=0.75% c=0.50% c=0.25% c=y S-2 (b) Specimen S-2 (a) Specimen S-1 (c) Specimen S-3 (b) Specimen S-2 (c) Specimen S-3 (b) Specimen S-2 Fig. 12 Distribution and development of longitudinal strains in restraining system 3 FE model and validation 256 2.1 Experimental specimens 103 Since the external restraining system was not loaded 232 when the inner core was under tension, the following analysis focuses on the longitudinal strain 233 of the external restraining member for the peak compressive displacement in each loading loop. 234 It is found that the difference between strain data of 1-1 section and 2-2 section is relatively 235 small, hence the data of 2-2 section is selected for strain analysis. Taking the average value of 236 a pair of strain data at the same height as the vertical coordinate and the height of the cross- 237 section as the horizontal coordinate, the distribution of the longitudinal strains on the cross- 238 section is plotted. It can be seen from Fig. 12 that the major bending direction of SAA-BRB 239 specimens is the up and down direction. Moreover, it is found that all strain values were less 240 than the yield strain (i.e., 1670 με for Q345 steel), indicating a good consistency with the stable 241 load-carrying capacities of these specimens during the loading process as shown in Fig. 7. 242 Theoretically, the strain distribution of the cross-section of the external restraining member is 243 16 strictly linear if the restraining system can be regarded as a fully flexural member. However, 244 the sectional strain distribution of each SAA-BRB specimen depicted in Fig.12 shows that the 245 linearity is violated. This indicates that significant shear deformation exists in the restraining 246 system and the plane-section assumption is inappropriate for the SAA-BRB. This is mainly 247 caused by the discrete layout of the connecting bolts in the restraining system. When the inner 248 core was under compression and developed multi-wave buckling mode, lateral thrust effect 249 existed between the core and restraining system, thus causing friction along their interface. 250 Hence, part of the axial load of the inner core was transmitted to the restraining system. This 251 explains that the measured compressive strain is larger than tensile strain in the two measuring- 252 points which are symmetrical to the midline of the cross-section of each specimen. 253 strictly linear if the restraining system can be regarded as a fully flexural member. However, 244 the sectional strain distribution of each SAA-BRB specimen depicted in Fig.12 shows that the 245 linearity is violated. 3 FE model and validation 256 3.1 Establishment of hysteretic model 257 In this section, the hysteretic behavior of the SAA-BRBs is simulated with ABAQUS by 258 establishing a refined FE model. The refined FE model is depicted in Fig. 13. The four-node 259 shell element (S4R) was used to simulate the steel angles, and the eight-node solid element 260 (C3D8R) was used to simulate the inner core, spacer and high-strength bolts. It is pointed out 261 that the high-strength bolts were set as infinitely rigid in the FE model because of their 262 sufficient stiffness during the cyclic loading process of the experiments. Additionally, an air 263 gap with a thickness of 1.0 mm on the surface of the inner core was set to ensure that the axial 264 load transmission between the core and external restraining member was prevented. The 265 specific geometric parameters of the FE model are identical to those of the specimens. 266 In elastoplastic FE analyses, both geometric nonlinearity and material nonlinearity are 267 17 considered. It is known that the material nonlinear constitutive relationship mainly consists of 268 yielding and strengthening criteria. In modelling the hysteretic behavior of SAA-BRBs, the 269 von Mises yielding criterion was considered in the material, and a combined isotropic and 270 kinematic hardening model was selected as the strengthening criterion to consider the cyclic 271 effect shown in the experimental results. The yield stress values of the inner core and steel 272 angles were 288 MPa and 380 MPa, respectively. It should be noted that these values were the 273 same as the corresponding data tabulated in Table 1. Based on the suggestion of Guo et al. 274 (2017c), the parameters described below were well validated by the previous experiments for 275 SAA-BRB. For isotropic hardening, a rate factor of 4.2 and a maximum change in yield stress 276 of Q∞ = 150 MPa were used in the model. Besides, the relevant moduli and rate factors for 277 kinematic hardening were set as C1 =32 GPa, γ1 = 300; C2 = 6 GPa, γ2 = 150; C3 = 0.6 GPa, 278 and γ3 =22. 279 For the contact behavior, the interface between the core and restraining system was 280 simulated by hard contact along the normal direction and friction option along the tangent 281 direction. The hard contact option allowed the interface to separate under tension but not 282 penetrate under compression. 3 FE model and validation 256 The friction coefficient of 0.1 was adopted based on the 283 suggestion of Chou and Chen (2010). In terms of the boundary conditions, both ends of SAA- 284 BRB were modelled as fixed ends since the core was connected with the flange by high- 285 strength bolts. Correspondingly, the rotational degree of freedom of the core at both ends was 286 constrained. Additionally, an initial geometrical imperfection with a magnitude of 1/1000 of 287 the specimen length for the inner core was assigned to the FE model. The loading protocol was 288 the same as that used in the experiment as listed in Table 2, except for the cycle number of each 289 loading level, which was determined to be only one instead of three. 290 18 291 Fig. 13 FE model for hysteretic analysis 292 3.2 Validation of FE model 293 Fig. 13 FE model for hysteretic analysis Fig. 13 FE model for hysteretic analysis Fig. 13 FE model for hysteretic analysis 3.2 Validation of FE model 293 The comparison between the hysteretic curves obtained from FE simulations and 294 experiments are illustrated in Fig. 14. It is seen that the hysteretic curves of the FE models and 295 experiments can fit very well. Moreover, the model in the FE simulation showed the same 296 stable load-carrying performance at the axial strain of 2.0% as the specimen in the experiment. 297 Taking the model T-2 as an example, neither the restraining member nor the inner core was 298 observed to undergo global buckling failure, while full cross-sectional yielding of inner core 299 was exhibited at the axial strain of 2.0%, as depicted in Fig. 15. Therefore, the refined FE model 300 developed by software ABAQUS could precisely predict the hysteretic response of the SAA- 301 BRB specimens under cyclic loads. 302 In addition, the unrestrained segment of the core remained elastic, while yielding had 303 spread in the yield segment of the core. This further verifies the feasible design of unrestrained 304 segment of the core by enlarging their cross-sectional size. Moreover, the maximum stress of 305 the steel angles was far less than its yield stress of 380 MPa, indicating that the restraining 306 system had sufficient flexural stiffness to limit the lateral deformation of the inner core. 3 FE model and validation 256 307 19 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test (a) Specimen S-1 (b) Specimen S-2 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test (c) Specimen S-3 Fig. 14 Comparison of experimental and numerical results of hysteretic curves -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test Axial force (kN) (a) Specimen S-1 (b) Specimen S-2 -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test (c) Specimen S-3 Fig. 14 Comparison of experimental and numerical results of hysteretic curves (a) Specimen S-1 (b) Specimen S-2 Axial strain (%) -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) FE model Test (c) Specimen S-3 (c) Specimen S-3 (c) Specimen S-3 Fig. 14 Comparison of experimental and numerical results of hysteretic curves Fig. 14 Comparison of experimental and numerical results of hysteretic curves (a) Restraining member (b) Inner core Fig. 15 Stress distribution of model T-2 at the axial strain of 2.0% (a) Restraining member (b) Inner core Fig. 15 Stress distribution of model T-2 at the axial strain of 2.0% (a) Restraining member (b) Inner core Fig. 15 Stress distribution of model T-2 at the axial strain of 2.0% Fig. 15 Stress distribution of model T-2 at the axial strain of 2.0% 4 Theoretical analysis for global buckling design 308 4 Theoretical analysis for global buckling design 308 In this section, the formula for restraining ratio requirements of the SAA-BRB is derived. 309 In designing BRB, a restraining ratioζproposed by Fujimoto et al. (1988), reflecting the 310 restraining effect of the external restraining system on the inner core, is usually used to predict 311 the global buckling behavior and to examine the load-carrying performance of BRBs. It is 312 In this section, the formula for restraining ratio requirements of the SAA-BRB is derived. 309 In designing BRB, a restraining ratioζproposed by Fujimoto et al. (1988), reflecting the 310 restraining effect of the external restraining system on the inner core, is usually used to predict 311 the global buckling behavior and to examine the load-carrying performance of BRBs. It is 312 20 known that the restraining ratioζis defined as the ratio of the Euler buckling load Pcr of BRB 313 to the yield load Py of its inner core. The Euler buckling load Pcr of BRB can be considered as 314 the sum of the Euler buckling load Pcr,c of inner core and the Euler buckling load Pcr,r of 315 restraining system (Guo et al. 2016). Since the restraining system remains elastic while the 316 inner core yields during the loading process, leading to the negligible contribution of inner core 317 in calculating the Euler buckling load Pcr of BRB. Therefore, the restraining ratio ζ is 318 commonly simplified as the ratio of the Euler buckling load Pcr,r of the restraining system to 319 the yield load Py of the inner core, as expressed by Eq. (5). Theoretically, as long as the 320 restraining ratio is slightly larger than unity, the cross-sectional strength of inner core can be 321 fully utilized, thus the BRB can maintain stable without global buckling during the whole 322 loading process. However, considering the influence of physical defects such as initial 323 geometric imperfection, residual stress, as well as the gap between the inner core and the 324 restraining system, the restraining ratio is required to be much larger than unity, indicating the 325 calculation of the requirements of the restraining ratio would be relatively complicated. 326 Through a reasonable design, the SAA-BRB will exhibit stable behavior instead of undergoing 327 global buckling before its inner steel core reaches the full cross-sectional yielding load (Tong 328 and Guo 2017). 4 Theoretical analysis for global buckling design 308 Although Tong and Guo (2018) have given the design suggestion about the 329 restraining requirement of SAA-BRB, there is still a lack of a precise design formula to 330 determine the restraining ratio as required. During the cyclic loading process, the external 331 restraining system can be regarded as a flexural member. Therefore, the formula of the critical 332 restraining ratio, i.e., the calculation of restraining ratio requirement of SAA-BRB, can be 333 derived by using the yielding criteria of outmost fiber for the restraining system section as the 334 elastic limit criterion. 335 known that the restraining ratioζis defined as the ratio of the Euler buckling load Pcr of BRB 313 to the yield load Py of its inner core. The Euler buckling load Pcr of BRB can be considered as 314 the sum of the Euler buckling load Pcr,c of inner core and the Euler buckling load Pcr,r of 315 restraining system (Guo et al. 2016). Since the restraining system remains elastic while the 316 inner core yields during the loading process, leading to the negligible contribution of inner core 317 in calculating the Euler buckling load Pcr of BRB. Therefore, the restraining ratio ζ is 318 commonly simplified as the ratio of the Euler buckling load Pcr,r of the restraining system to 319 the yield load Py of the inner core, as expressed by Eq. (5). Theoretically, as long as the 320 restraining ratio is slightly larger than unity, the cross-sectional strength of inner core can be 321 fully utilized, thus the BRB can maintain stable without global buckling during the whole 322 loading process. However, considering the influence of physical defects such as initial 323 geometric imperfection, residual stress, as well as the gap between the inner core and the 324 restraining system, the restraining ratio is required to be much larger than unity, indicating the 325 calculation of the requirements of the restraining ratio would be relatively complicated. 326 Through a reasonable design, the SAA-BRB will exhibit stable behavior instead of undergoing 327 global buckling before its inner steel core reaches the full cross-sectional yielding load (Tong 328 and Guo 2017). Although Tong and Guo (2018) have given the design suggestion about the 329 restraining requirement of SAA-BRB, there is still a lack of a precise design formula to 330 determine the restraining ratio as required. 4 Theoretical analysis for global buckling design 308 During the cyclic loading process, the external 331 restraining system can be regarded as a flexural member. Therefore, the formula of the critical 332 restraining ratio, i.e., the calculation of restraining ratio requirement of SAA-BRB, can be 333 derived by using the yielding criteria of outmost fiber for the restraining system section as the 334 elastic limit criterion. 335 y r, cr P P   (5) y r, cr P P   (5) (5) in which Pcr,r is the Euler buckling load regarding the restraining system as an axial loading 336 member (Guo et al. 2015); Py is the yield load of the inner core. 337 21 21 For an ordinary BRB with an inner steel core encased by a uniform-sectional restraining 338 member, the Euler buckling load of the restraining system can be calculated as 339 For an ordinary BRB with an inner steel core encased by a uniform-sectional restraining 338 member, the Euler buckling load of the restraining system can be calculated as 339 For an ordinary BRB with an inner steel core encased by a uniform-sectional restraining 338 member, the Euler buckling load of the restraining system can be calculated as 339 2 0 cr,0 2 0 EA P    (6) (6) (6) in which E is the Young’s modulus of the restraining member; A0 is the cross-sectional area of 340 the restraining member and λ0 is the slenderness ratio of the restraining member of ordinary 341 BRB, which can be calculated by Eq. (9). 342 in which E is the Young’s modulus of the restraining member; A0 is the cross-sectional area of 340 the restraining member and λ0 is the slenderness ratio of the restraining member of ordinary 341 BRB, which can be calculated by Eq. (9). 342 It is known that the arrangement of discretely distributed bolts in the longitudinal direction 343 would result in a significant shear deformation of the restraining system, thus reducing the 344 Euler buckling load. Hence, a reduction factor ω is introduced to consider the effect of shear 345 deformation, as expressed in Eq. (7). The value of ω is closely related to the geometric 346 dimensions of the assembled BRB and is generally smaller than unity. For the SAA-BRB, Tong 347 and Guo (2018) introduced the derivation of the formula for ω through numerous FE 348 simulations and theoretical analysis, which is expressed by Eq. 4 Theoretical analysis for global buckling design 308 (8). It should be noted that the 349 derivation of reduction factor ω is based on the equivalence of the restraining system into a 350 four-chord battened column. In the equivalent relation, the four angle steels were equivalent to 351 four chord members, and the spacers were equivalent to batten plates (Tong and Guo 2018). 352 (7) 1 - 27 .1 0 55 .0 1 2 0 2 1 2 4. 17 12 1              (8) 1 - 27 .1 0 55 .0 1 2 0 2 1 2 4. 17 12 1              (8) (8) in which λ1 is the slenderness ratio of a single chord member between adjacent batten plates. 353 l in which λ1 is the slenderness ratio of a single chord member between adjacent batten plates. 353 0 0 0 0 l I A  (9) 0 0 0 0 l I A  (9) 0 0 0 0 l I A  (9) (9) in which λ0 is the slenderness ratio regarding the external restraining system as a member with 354 a uniform cross-section; l0 is the length of the external restraining system; I0 is the moment of 355 inertia regarding the external restraining system as a whole about the x-x axis and A0 is the 356 cross-sectional area of the external restraining system. 357 in which λ0 is the slenderness ratio regarding the external restraining system as a member with 354 a uniform cross-section; l0 is the length of the external restraining system; I0 is the moment of 355 inertia regarding the external restraining system as a whole about the x-x axis and A0 is the 356 cross-sectional area of the external restraining system. 357 22 22 1 1 1 1 l I A  (10) (10) in which l1 is the longitudinal spacing between adjacent high-strength bolts, which is equal to 358 the spacing between adjacent batten plates; I1 is the moment of inertia of a single angle about 359 the x1-x1 axis, A1 is the cross-sectional area of a single steel angle. Besides, the x-x axis for 360 calculating the I0 and x1-x1 axis for calculating the I1 are shown in Fig. 16. 361 Top Buckling direction x x Bottom Top East West x1 x1 362 Fig. 4 Theoretical analysis for global buckling design 308 ζ ˃[ζ], the satisfactory design 381 of the SAA-BRB can be achieved by preventing its global buckling. 382 yr yr y 0 [ ] ( ) Wf Wf P v g         (15) yr yr y 0 [ ] ( ) Wf Wf P v g         (15) yr yr y 0 [ ] ( ) Wf Wf P v g         (15) (15) 4 Theoretical analysis for global buckling design 308 16 Axis of x-x and x1-x1 for calculation 363 x1 x1 East Bottom Fig. 16 Axis of x-x and x1-x1 for calculation Fig. 16 Axis of x-x and x1-x1 for calculation When the load reaches the value corresponding to the yield load of the inner steel core, 364 the maximum lateral deformation vmax at the mid-span of the restraining system in the 365 longitudinal direction can be expressed as (Timoshenko et al. 1962) 366 r, cr y 0 max 1 P P g v v    (11) (11) in which v0 (=L/1000) is the amplitude of initial imperfection and g is the gap between the inner 367 steel core and the restraining member. 368 When global buckling occurs, the maximum bending moment Mmax at the mid-span of the 369 restraining system can be expressed as 370 max y max v P M  (12) max y max v P M  (12) max y max v P M  (12) (12) Correspondingly, the maximum stress of the restraining system can be calculated by Eq. 371 Correspondingly, the maximum stress of the restraining system can be calculated by Eq. 371 (13). It should be aware that the plastic section modulus W is multiplied by the reduction factor 372 ω to express the weakening effect of discrete bolt connection. 373 (13). It should be aware that the plastic section modulus W is multiplied by the reduction factor 372 ω to express the weakening effect of discrete bolt connection. 373 23 max max M W    (13) max max M W    (13) (13) Based on the yielding criteria of outmost fiber for the restraining system section, the 374 overall stability requirement of the SAA-BRB subjected to axial compressive load can be 375 expressed as 376 max yr f   (14) max yr f   (14) in which fy,r is the yield stress of the restraining system. 377 By combining Eqs. (5), (7), and (11) – (14), a calculation for a lower limit of restraining 378 ratio [ζ] for SAA-BRB can be expressed as Eq. (15), which is used for global buckling design 379 of SAA-BRB. For convenience of the design by engineers, ζ / [ζ] can be determined as a design 380 coefficient. When the coefficient ζ / [ζ] value exceeds unity, i.e. 5 Parametric study and design formula verification 383 404 This indicates that the full-sectional yielding load of the inner core could be achieved before 405 reaching 2.0% axial strain. Furthermore, taking model M-13 as an example, its ζ / [ζ] value is 406 0.81. It can be seen from Fig. 20 that the model M-13 exhibited global buckling with a single- 407 wave lateral deformation at the load step of 1.5% axial strain, thus severely pinched hysteretic 408 curve was obtained as illustrated in Fig. 18(e). Besides, stress concentrations were observed at 409 the mid-span and both ends of the yield segment of the core, indicating that full cross-sectional 410 yield load of the inner core could not be reached. Therefore, the accuracy of the formula (Eq. 411 (15)) of restraining ratio requirements for global buckling design of SAA-BRBs is validated, 412 which can provide a valuable reference for practical designs. 413 global buckling failure of the FE model would be prevented and the corresponding hysteretic 397 curves of the models maintained stable; when the ζ / [ζ] value is less than unity, the global 398 buckling mode would occur in the FE model. Overall, a critical value (i.e., the value of red line) 399 exists in Fig. 17, below which a stable bearing-capacity would transition to a global buckling 400 mode. Taking model M-3 as an example, its ζ / [ζ] value is 1.40. It is seen from Fig. 18(a) that 401 its hysteretic curve is full and symmetrical, indicating a stable bearing-capacity and satisfactory 402 energy-dissipation capability during the whole loading process. Moreover, it is observed from 403 Fig. 19 that the yielding had spread in the core while the restraining system remained elastic. 404 This indicates that the full-sectional yielding load of the inner core could be achieved before 405 reaching 2.0% axial strain. Furthermore, taking model M-13 as an example, its ζ / [ζ] value is 406 0.81. It can be seen from Fig. 20 that the model M-13 exhibited global buckling with a single- 407 wave lateral deformation at the load step of 1.5% axial strain, thus severely pinched hysteretic 408 curve was obtained as illustrated in Fig. 18(e). Besides, stress concentrations were observed at 409 the mid-span and both ends of the yield segment of the core, indicating that full cross-sectional 410 yield load of the inner core could not be reached. Therefore, the accuracy of the formula (Eq. 5 Parametric study and design formula verification 383 Based on the proposed FE model, 24 FE hysteretic models listed in Table 4 were analyzed 384 to conduct a parametric study and to verify the accuracy of the proposed formula (Eq. (15)) for 385 global buckling design of SAA-BRB. The geometric dimensions are listed in Table 4, and the 386 geometric parameters and material properties of the models are identical with the specimens in 387 the experimental program. These 24 models were divided into 3 groups, and the core sectional 388 area of models in each group were identical, and their corresponding values were 4356 mm2, 389 4796 mm2, and 6566 mm2, respectively. Besides, the loading protocol in the FE extensive 390 analysis was determined according to the standard loading stage shown in Fig. 4, yet each 391 loading step was applied for only one cycle. 392 Fig. 17 shows the relationship between FE results and the coefficient ζ / [ζ]. Fig. 18 393 presents the hysteretic curves of representative FE models. The solid rectangles in Fig.17 394 represent that the models maintained stable while the hollow rectangles corresponded to 395 models subjected to global buckling failure. When the ζ / [ζ] value is greater than unity, the 396 Fig. 17 shows the relationship between FE results and the coefficient ζ / [ζ]. Fig. 18 393 presents the hysteretic curves of representative FE models. The solid rectangles in Fig.17 394 represent that the models maintained stable while the hollow rectangles corresponded to 395 models subjected to global buckling failure. When the ζ / [ζ] value is greater than unity, the 396 24 global buckling failure of the FE model would be prevented and the corresponding hysteretic 397 curves of the models maintained stable; when the ζ / [ζ] value is less than unity, the global 398 buckling mode would occur in the FE model. Overall, a critical value (i.e., the value of red line) 399 exists in Fig. 17, below which a stable bearing-capacity would transition to a global buckling 400 mode. Taking model M-3 as an example, its ζ / [ζ] value is 1.40. It is seen from Fig. 18(a) that 401 its hysteretic curve is full and symmetrical, indicating a stable bearing-capacity and satisfactory 402 energy-dissipation capability during the whole loading process. Moreover, it is observed from 403 Fig. 19 that the yielding had spread in the core while the restraining system remained elastic. 5 Parametric study and design formula verification 383 411 (15)) of restraining ratio requirements for global buckling design of SAA-BRBs is validated, 412 which can provide a valuable reference for practical designs. 413 25 Table 4 Information of hysteretic models and FE results 414 Group no. Hysteretic model no. Notes: FE resultsa represents the failure mode of SAA-BRBs, in which “S” represents “stable”; “G 5 Parametric study and design formula verification 383 17 Verification of restraining ratio requirements -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-3 Ac=4356mm2 l=2300mm -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-5 Global buckling Ac=4356mm2 l=2900mm -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-8 Global buckling Ac=4356mm2 l=3800mm (a) M-3 (ζ=2.88, [ζ]=2.06) (b) M-5 (ζ=1.82, [ζ]=2.15) (c) M-8 (ζ=1.07, [ζ]=2.30) -48 -24 0 24 48 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-10 Ac=4796mm2 l=2600mm -66 -33 0 33 66 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-13 Global buckling Ac=4796mm2 l=3500mm -84 -42 0 42 84 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-16 Global buckling Ac=4796mm2 l=4400mm (d) M-10 (ζ=2.44, [ζ]=2.01) (e) M-13 (ζ=1.69, [ζ]=2.07) (f) M-16 (ζ=1.08, [ζ]=2.18) 1500 2000 2500 3000 3500 4000 0 1.0 2.0 3.0 ζ / [ζ ] Length of core yield segment ly (mm) Restraining ratio (Ac=4356mm2) Stable Global buckling 1500 2000 2500 3000 3500 4000 0 1.0 2.0 3.0 ζ / [ζ ] Length of core yield segment ly (mm) Restraining ratio (Ac=4356mm2) Stable Global buckling Restraining ratio (Ac=4796mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Stable Global buckling (a) Group 1 (Ac=4356 mm2) (b) Group 2 (Ac=4796 mm2) Restraining ratio (Ac=6556mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Global buckling Stable (c) Group 3 (Ac=6556 mm2) Fig. 5 Parametric study and design formula verification 383 Geometrical dimensions ω ζ [ζ] ζ / [ζ] FE resultsa l (mm) ly (mm) b × t (mm) ba × ta (mm) Ac (mm2) 1 M-1 1700 1400 110 × 22 100 × 6 4356 0.548 5.32 1.99 2.68 S M-2 2000 1700 110 × 22 100 × 6 0.580 3.82 2.02 1.89 S M-3 2300 2000 110 × 22 100 × 6 0.606 2.88 2.06 1.40 S M-4 2600 2300 110 × 22 100 × 6 0.627 2.26 2.10 1.07 S M-5 2900 2600 110 × 22 100 × 6 0.645 1.82 2.15 0.85 GB M-6 3200 2900 110 × 22 100 × 6 0.661 1.50 2.20 0.68 GB M-7 3500 3200 110 × 22 100 × 6 0.675 1.25 2.25 0.56 GB M-8 3800 3500 110 × 22 100 × 6 0.687 1.07 2.30 0.46 GB 2 M-9 2300 2000 120 × 22 110 × 7 4796 0.595 3.86 1.94 1.99 S M-10 2600 2300 120 × 22 110 × 7 0.617 3.03 1.97 1.53 S M-11 2900 2600 120 × 22 110 × 7 0.636 2.44 2.01 1.22 S M-12 3200 2900 120 × 22 110 × 7 0.652 2.01 2.04 0.99 GB M-13 3500 3200 120 × 22 110 × 7 0.666 1.69 2.07 0.81 GB M-14 3800 3500 120 × 22 110 × 7 0.679 1.44 2.11 0.68 GB M-15 4100 3800 120 × 22 110 × 7 0.690 1.24 2.15 0.58 GB M-16 4400 4100 120 × 22 110 × 7 0.700 1.08 2.18 0.49 GB 3 M-17 2300 2000 120 × 22 120 × 8 6556 0.585 4.01 1.96 2.04 S M-18 2600 2300 160 × 22 120 × 8 0.607 3.15 2.00 1.58 S M-19 2900 2600 160 × 22 120 × 8 0.626 2.54 2.03 1.25 S M-20 3200 2900 160 × 22 120 × 8 0.643 2.10 2.06 1.02 S M-21 3500 3200 160 × 22 120 × 8 0.657 1.76 2.10 0.84 GB M-22 3800 3500 160 × 22 120 × 8 0.670 1.50 2.14 0.70 GB M-23 4100 3800 160 × 22 120 × 8 0.682 1.29 2.18 0.59 GB M-24 4400 4100 160 × 22 120 × 8 0.692 1.13 2.22 0.51 GB Notes: FE resultsa represents the failure mode of SAA-BRBs, in which “S” represents “stable”; “GB” represents “global buckling”. 5 Parametric study and design formula verification 383 415 414 Table 4 Information of hysteretic models and FE results Geometrical dimensions 26 27 1500 2000 2500 3000 3500 4000 0 1.0 2.0 3.0 ζ / [ζ ] Length of core yield segment ly (mm) Restraining ratio (Ac=4356mm2) Stable Global buckling Restraining ratio (Ac=4796mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Stable Global buckling (a) Group 1 (Ac=4356 mm2) (b) Group 2 (Ac=4796 mm2) Restraining ratio (Ac=6556mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Global buckling Stable (c) Group 3 (Ac=6556 mm2) Fig. 5 Parametric study and design formula verification 383 17 Verification of restraining ratio requirements -2.0 -1.0 0 1.0 2.0 2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) M-3 Ac=4356mm2 l 2300mm -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) M-5 Global buckling Ac=4356mm2 l=2900mm -2.0 -1.0 0 1.0 2.0 2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) M-8 Global buckling Ac=4356mm2 l 3800mm Restraining ratio (Ac=4796mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Stable Global buckling (a) Group 1 (Ac=4356 mm2) (b) Group 2 (Ac=4796 mm2) Restraining ratio (Ac=6556mm2) 2000 3000 4000 5000 0 0.5 1.0 1.5 2.0 2.5 ζ / [ζ ] Length of core yield segment ly (mm) Global buckling Stable (c) Group 3 (A =6556 mm2) (b) Group 2 (Ac=4796 mm2) (b) Group 2 (Ac=4796 mm2) (a) Group 1 (Ac=4356 mm2) Stable (c) Group 3 (Ac=6556 mm2) (c) Group 3 (Ac=6556 mm2) Fig. 17 Verification of restraining ratio requirements -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-3 Ac=4356mm2 l=2300mm -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-5 Global buckling Ac=4356mm2 l=2900mm -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-8 Global buckling Ac=4356mm2 l=3800mm (a) M-3 (ζ=2.88, [ζ]=2.06) (b) M-5 (ζ=1.82, [ζ]=2.15) (c) M-8 (ζ=1.07, [ζ]=2.30) -48 -24 0 24 48 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-10 Ac=4796mm2 l=2600mm -66 -33 0 33 66 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-13 Global buckling Ac=4796mm2 l=3500mm -84 -42 0 42 84 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-16 Global buckling Ac=4796mm2 l=4400mm (d) M-10 (ζ=2.44, [ζ]=2.01) (e) M-13 (ζ=1.69, [ζ]=2.07) (f) M-16 (ζ=1.08, [ζ]=2.18) Fig. 17 Verification of restraining ratio requirements Fig. 5 Parametric study and design formula verification 383 17 Verification of restraining ratio requirements quirements -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-8 Global buckling Ac=4356mm2 l=3800mm (c) M-8 (ζ=1.07, [ζ]=2.30) -84 -42 0 42 84 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-16 Global buckling Ac=4796mm2 l=4400mm (f) M-16 (ζ=1.08, [ζ]=2.18) g -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-3 Ac=4356mm2 l=2300mm (a) M-3 (ζ=2.88, [ζ]=2.06) -48 -24 0 24 48 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-10 Ac=4796mm2 l=2600mm (d) M-10 (ζ=2.44, [ζ]=2.01) g -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-5 Global buckling Ac=4356mm2 l=2900mm (b) M-5 (ζ=1.82, [ζ]=2.15) -66 -33 0 33 66 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-13 Global buckling Ac=4796mm2 l=3500mm (e) M-13 (ζ=1.69, [ζ]=2.07) 1 0 0 1 0 Axial strain (%) -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-8 Global buckling Ac=4356mm2 l=3800mm -42 -21 0 21 42 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-3 Ac=4356mm2 l=2300mm ( ) 3 (ζ 2 88 [ζ] 2 06) -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-5 Global buckling Ac=4356mm2 l=2900mm (b) M-5 (ζ=1 82 [ζ]=2 15) 27 0 27 Axial displacement (mm) Axial displacement (mm) Axial displacement (mm) (b) M-5 (ζ=1.82, [ζ]=2.15) -66 -33 0 33 66 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-13 Global buckling Ac=4796mm2 l=3500mm (e) M-13 (ζ=1.69, [ζ]=2.07) (a) M-3 (ζ=2.88, [ζ]=2.06) Axial strain (%) (b) M-5 (ζ=1.82, [ζ]=2.15) Axial strain (%) (c) M-8 (ζ=1.07, [ζ]=2.30) Axial strain (%) (c) M-8 (ζ=1.07, [ζ]=2.30) -84 -42 0 42 84 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-16 Global buckling Ac=4796mm2 l=4400mm (f) M-16 (ζ=1.08, [ζ]=2.18) (a) M-3 (ζ=2.88, [ζ]=2.06) -48 -24 0 24 48 -2.0 -1.0 0 1.0 2.0 -2000 -1000 0 1000 2000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-10 Ac=4796mm2 l=2600mm (d) M-10 (ζ=2.44, [ζ]=2.01) Axial strain (%) Axial strain (%) Axial strain (%) p ( ) (d) M-10 (ζ=2.44, [ζ]=2.01) (e) M-13 (ζ=1.69, [ζ]=2.07) (f) M-16 (ζ=1.08, [ζ]=2.18) 27 27 -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-19 Ac=6556mm2 l=2900mm -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-22 Global buckling Ac=6556mm2 l=3800mm -78 -39 0 39 78 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-23 Global buckling Ac=6556mm2 l=4100mm (g) M-19 (ζ=2.54, [ζ]=2.03) (h) M-22 (ζ=2.54, [ζ]=2.03) (i) M-23 (ζ=1.29, [ζ]=2.18) Fig. 5 Parametric study and design formula verification 383 18 Hysteretic curves of FE models (a) Whole member (b) Inner core Fig. 19 Stress distribution of the SAA-BRB (model M-3) (a) Whole member (b) Inner core Fig. 20 Stress distribution of the SAA-BRB (model M-13) -72 -36 0 36 72 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-22 Global buckling Ac=6556mm2 l=3800mm (h) M-22 (ζ=2.54, [ζ]=2.03) -54 -27 0 27 54 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-19 Ac=6556mm2 l=2900mm (g) M-19 (ζ=2.54, [ζ]=2.03) -78 -39 0 39 78 -2.0 -1.0 0 1.0 2.0 -3000 -2000 -1000 0 1000 2000 3000 Axial strain (%) Axial force (kN) Axial displacement (mm) M-23 Global buckling Ac=6556mm2 l=4100mm (i) M-23 (ζ=1 29 [ζ]=2 18) -39 0 39 Axial displacement (mm) p (i) M-23 (ζ=1.29, [ζ]=2.18) (h) M-22 (ζ=2.54, [ζ]=2.03) Fig. 18 Hysteretic curves of FE models Fig. 18 Hysteretic curves of FE models g y (a) Whole member (b) Inner core Fig. 19 Stress distribution of the SAA-BRB (model M-3) (a) Whole member (b) Inner core Fig. 20 Stress distribution of the SAA-BRB (model M-13) (a) Whole member (b) Inner core Fig. 19 Stress distribution of the SAA-BRB (model M-3) (a) Whole member (b) Inner core Fig. 19 Stress distribution of the SAA-BRB (model M-3) (a) Whole member (b) Inner core (a) Whole member (b) Inner core Fig. 20 Stress distribution of the SAA-BRB (model M-13) 6 Conclusions 416 6 Conclusions 416 In this study, the hysteretic responses of steel-angles-assembled buckling-restrained brace 417 (SAA-BRB) were investigated experimentally and numerically. A design formula of restraining 418 ratio requirements with high accuracy for predicting the global buckling of SAA-BRB was 419 proposed and verified by parametric FE hysteretic analysis. The following conclusions can be 420 drawn: 421 (1) Through the experiments conducted on three specimens, the hysteretic curves and 422 skeleton curves were obtained. It is found from the experimental results that all specimens 423 maintained stable without global buckling, indicating that a proper-designed SAA-BRB can 424 exhibit satisfactory hysteretic performance under cyclic loads. Based on the analysis of results 425 from the experiments, it is found that the maximum equivalent viscous damping ratios of all 426 In this study, the hysteretic responses of steel-angles-assembled buckling-restrained brace 417 (SAA-BRB) were investigated experimentally and numerically. A design formula of restraining 418 ratio requirements with high accuracy for predicting the global buckling of SAA-BRB was 419 proposed and verified by parametric FE hysteretic analysis. The following conclusions can be 420 drawn: 421 In this study, the hysteretic responses of steel-angles-assembled buckling-restrained brace 417 (SAA-BRB) were investigated experimentally and numerically. A design formula of restraining 418 ratio requirements with high accuracy for predicting the global buckling of SAA-BRB was 419 proposed and verified by parametric FE hysteretic analysis. The following conclusions can be 420 drawn: 421 (1) Through the experiments conducted on three specimens, the hysteretic curves and 422 skeleton curves were obtained. It is found from the experimental results that all specimens 423 maintained stable without global buckling, indicating that a proper-designed SAA-BRB can 424 exhibit satisfactory hysteretic performance under cyclic loads. Based on the analysis of results 425 from the experiments, it is found that the maximum equivalent viscous damping ratios of all 426 (1) Through the experiments conducted on three specimens, the hysteretic curves and 422 skeleton curves were obtained. It is found from the experimental results that all specimens 423 maintained stable without global buckling, indicating that a proper-designed SAA-BRB can 424 exhibit satisfactory hysteretic performance under cyclic loads. Based on the analysis of results 425 from the experiments, it is found that the maximum equivalent viscous damping ratios of all 426 (1) Through the experiments conducted on three specimens, the hysteretic curves and 422 skeleton curves were obtained. 6 Conclusions 416 It is found from the experimental results that all specimens 423 maintained stable without global buckling, indicating that a proper-designed SAA-BRB can 424 exhibit satisfactory hysteretic performance under cyclic loads. Based on the analysis of results 425 from the experiments, it is found that the maximum equivalent viscous damping ratios of all 426 28 specimens are greater than 0.5 and the compression strength adjustment factors are lower than 427 1.50 as specified in AISC. This shows that the SAA-BRB has favorable load-carrying 428 performance and energy-dissipating capacity. In addition, the plane-section assumption is 429 violated for SAA-BRB by analyzing the longitudinal strain distribution of the retraining system. 430 (2) A refined FE model considering geometric nonlinearity and material nonlinearity was 431 established. It is demonstrated that the results from FE analysis were consistent with those 432 obtained from experiments, indicating the validity of the established model in predicting the 433 hysteretic responses of SAA-BRB. 434 specimens are greater than 0.5 and the compression strength adjustment factors are lower than 427 1.50 as specified in AISC. This shows that the SAA-BRB has favorable load-carrying 428 performance and energy-dissipating capacity. In addition, the plane-section assumption is 429 violated for SAA-BRB by analyzing the longitudinal strain distribution of the retraining system. 430 specimens are greater than 0.5 and the compression strength adjustment factors are lower than 427 1.50 as specified in AISC. This shows that the SAA-BRB has favorable load-carrying 428 performance and energy-dissipating capacity. In addition, the plane-section assumption is 429 violated for SAA-BRB by analyzing the longitudinal strain distribution of the retraining system. 430 (2) A refined FE model considering geometric nonlinearity and material nonlinearity was 431 established. It is demonstrated that the results from FE analysis were consistent with those 432 obtained from experiments, indicating the validity of the established model in predicting the 433 hysteretic responses of SAA-BRB. 434 specimens are greater than 0.5 and the compression strength adjustment factors are lower than 427 1.50 as specified in AISC. This shows that the SAA-BRB has favorable load-carrying 428 performance and energy-dissipating capacity. In addition, the plane-section assumption is 429 violated for SAA-BRB by analyzing the longitudinal strain distribution of the retraining system. 430 (2) A refined FE model considering geometric nonlinearity and material nonlinearity was 431 established. References 440 AISC (2016) Seismic provisions for structural steel buildings. AISC, Chicago 441 Ariyaratana C, Fahnestock LA (2011) Evaluation of buckling-restrained braced frame seismic performance 442 considering reserve strength. 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Eng Struct 132:761-777. 529 https://doi.org/10.1016/j.engstruct.2016.11.069 530 Zhao J, Wu B, Ou J (2013) Global stability design method of buckling-restrained braces considering end bending 531 moment transfer: Discussion on pinned connections with collars. Eng Struct 49:947-962. 532 https://doi.org/10.1016/j.engstruct.2012.12.042 533 Declaration 534 The authors declare that they have no known competing financial interests or personal 535 relationships that could have appeared to influence the work reported in this paper. 536 Acknowledgments 537 This study has been supported by research grants from the National Natural Science 538 Foundation of China (52108180) and the Zhejiang Provincial Natural Science Foundation of 539 China (LQ21E080018). 540 Tremblay R, Bolduc P, Neville R, Devall R (2006) Seismic testing and performance of buckling-restrained bracing 524 systems. Can J Civil Eng 33(2):183-198. References 440 https://doi.org/10.1139/L05-103 525 Yoshino T, Karino Y (1971) Experimental study on shear wall with braces: Part 2. In: Summaries of technical 526 papers of annual meeting. Architectural Institute of Japan, pp 403-404 527 Yoshino T, Karino Y (1971) Experimental study on shear wall with braces: Part 2. In: Summaries of technical 526 papers of annual meeting. Architectural Institute of Japan, pp 403-404 527 Yoshino T, Karino Y (1971) Experimental study on shear wall with braces: Part 2. In: Summaries of technical 526 papers of annual meeting. Architectural Institute of Japan, pp 403-404 527 Zhao J, Lin F, Wang Z (2017) Seismic design of buckling-restrained brace welded end connection considering 528 frame action effects: Theoretical, numerical and practical approaches. Eng Struct 132:761-777. 529 https://doi.org/10.1016/j.engstruct.2016.11.069 530 Zhao J, Wu B, Ou J (2013) Global stability design method of buckling-restrained braces considering end bending 531 moment transfer: Discussion on pinned connections with collars. Eng Struct 49:947-962. 532 https://doi.org/10.1016/j.engstruct.2012.12.042 533 Declaration 534 The authors declare that they have no known competing financial interests or personal 535 relationships that could have appeared to influence the work reported in this paper. 536 Acknowledgments 537 Zhao J, Lin F, Wang Z (2017) Seismic design of buckling-restrained brace welded end connection considering 528 frame action effects: Theoretical, numerical and practical approaches. Eng Struct 132:761-777. 529 https://doi.org/10.1016/j.engstruct.2016.11.069 530 Zhao J, Lin F, Wang Z (2017) Seismic design of buckling-restrained brace welded end connection considering 528 frame action effects: Theoretical, numerical and practical approaches. Eng Struct 132:761-777. 529 https://doi.org/10.1016/j.engstruct.2016.11.069 530 Zhao J, Wu B, Ou J (2013) Global stability design method of buckling-restrained braces considering end bending 531 moment transfer: Discussion on pinned connections with collars. Eng Struct 49:947-962. 532 https://doi.org/10.1016/j.engstruct.2012.12.042 533 The authors declare that they have no known competing financial interests or personal 535 relationships that could have appeared to influence the work reported in this paper. 536 A k l d t 537 The authors declare that they have no known competing financial interests or personal 535 relationships that could have appeared to influence the work reported in this paper. 536 This study has been supported by research grants from the National Natural Science 538 Foundation of China (52108180) and the Zhejiang Provincial Natural Science Foundation of 539 China (LQ21E080018). 540 32
https://openalex.org/W4361255191	https://aacr.figshare.com/articles/journal_contribution/Figure_SF2_from_Predicting_Molecular_Phenotypes_from_Histopathology_Images_A_Transcriptome-Wide_Expression_Morphology_Analysis_in_Breast_Cancer/22362536/1/files/39806294.pdf	English	null	Figure SF2 from Predicting Molecular Phenotypes from Histopathology Images: A Transcriptome-Wide Expression–Morphology Analysis in Breast Cancer	null	2,023	cc-by	551	alue < 0.05) from earman coeffici d RNA-seq expr ng based on me alue < 0.05) from earman coeffici d RNA-seq expr ng based on me FGFR4 TMEM45B GPR160 MMP11 BCL2 SLC39A6 ESR1 MLPH FOXA1 CXXC5 PGR MAPT NAT1 BAG1 MYC ACTR3B PHGDH SFRP1 FOXC1 CDH3 EGFR KRT14 KRT17 TYMS CDC6 RRM2 ANLN MKI67 CENPF EXO1 ORC6L BIRC5 UBE2C MYBL2 PTTG1 CCNB1 CEP55 MELK CDC20 KIF2C NDC80 NUF2 UBE2T CCNE1 Subtype ER.status Coef R2 Coef 0 0.2 0.4 0.6 0.8 R2 0 0.1 0.2 0.3 0.4 Original −5 0 5 10 Grade 1 2 3 NA Subtype NA Basal Her2 LumA LumB Normal ER.status 0 1 NA Coef R2 BAG1 NAT1 PGR MAPT SLC39A6 ESR1 BCL2 FOXA1 MLPH CXXC5 MMP11 FGFR4 TMEM45B GPR160 UBE2T NUF2 RRM2 PTTG1 CCNB1 BIRC5 ANLN CEP55 MELK EXO1 KIF2C CDC20 MYBL2 UBE2C ORC6L Grade ER.status Coef 0 0.2 0.4 0.6 0.8 Original −4 −2 0 2 4 Grade 1 2 3 ER.status 0 1 D C alue < 0.05) from earman coefficien d RNA-seq expres ng based on meas Coef R2 C MMP11 BCL2 SLC39A6 ESR1 MLPH FOXA1 CXXC5 PGR MAPT NAT1 BAG1 MYC ACTR3B PHGDH SFRP1 FOXC1 CDH3 EGFR KRT14 KRT17 TYMS CDC6 RRM2 ANLN MKI67 CENPF EXO1 ORC6L BIRC5 UBE2C MYBL2 PTTG1 CCNB1 CEP55 MELK CDC20 KIF2C NDC80 NUF2 UBE2T CCNE1 Coef R2 Coef 0 0.2 0.4 0.6 0.8 R2 0 0.1 0.2 0.3 0.4 Original −5 0 5 10 Grade 1 2 3 NA Subtype NA Basal Her2 LumA LumB Normal ER.status 0 1 NA Coef R2 BAG1 NAT1 PGR MAPT SLC39A6 ESR1 BCL2 FOXA1 MLPH CXXC5 MMP11 FGFR4 TMEM45B GPR160 UBE2T NUF2 RRM2 PTTG1 CCNB1 BIRC5 ANLN CEP55 MELK EXO1 KIF2C CDC20 MYBL2 UBE2C ORC6L CCNE1 TYMS NDC80 CENPF MKI67 CDC6 ACTR3B MYC SFRP1 EGFR FOXC1 CDH3 KRT14 KRT17 PHGDH Grade ER.status Coef Coef 0 0.2 0.4 0.6 0.8 Original −4 −2 0 2 4 Grade 1 2 3 ER.status 0 1 Coef D C Supplementary Figure 2. Heat map showing clusters of patients based on 44 genes (FDR adjusted p-value < 0.05) from th subtype for each patient are indicated at the top of each heatmap. Prediction performance in terms of Spearman coefficient for each gene are displayed on the left side of the heatmap. a. Hierarchical clustering based on measured RNA-seq expressi expression levels with same row and column order as in A, (internal test set). alue < 0.05) from earman coeffici d RNA-seq expr ng based on me C, Hierarchical clustering based on measu Model predicted gene expression levels with same row and column order as in b (external test set). NAT1 BAG1 MYC ACTR3B PHGDH 0 0.2 0.4 0.6 R2 2 3 NA Subtype FOXC1 CDH3 KRT14 KRT17 PHGDH Coef ters of patients based on 44 genes (FDR adjusted p-value < 0.05) from each heatmap. Prediction performance in terms of Spearman coefficien heatmap. a. Hierarchical clustering based on measured RNA-seq expre r as in A, (internal test set). C, Hierarchical clustering based on mea row and column order as in b (external test set). Coef Supplementary Figure 2. Heat map showing clu subtype for each patient are indicated at the top o for each gene are displayed on the left side of the expression levels with same row and column ord Model predicted gene expression levels with same
https://openalex.org/W3171145653	https://iris.unimore.it/bitstream/11380/1253699/2/on-the-turbulent-flow-past-a-realistic-open-cell-metal-foam.pdf	English	null	On the turbulent flow past a realistic open-cell metal foam	Journal of fluid mechanics	2,021	cc-by	17,243	On the turbulent ﬂow past a realistic open-cell metal foam The Reynolds number based on the pore size is Redp = 4000, corresponding to a Taylor-scale Reynolds number based on the pore size is Redp = 4000, corresponding to a Taylor-scale Reynolds number Reλ ≈80. Closer to the foam than two pore diameters, the pressure and turbulent transports of turbulent kinetic energy are non-negligible. In the same region, Reλ undergoes a steep decrease whereas the dissipation coefﬁcient Cϵ increases like Re−1 λ . At larger distances from the porous layer, the classical grid turbulence situation is recovered, where the mean advection of turbulent kinetic energy equals dissipation. This entails a power-law decay of turbulent quantities and characteristic lengths. The decaying exponents of integral, Taylor and Kolmogorov scales are close to one-half, indicating that the turbulence simulated here differs from Saffman turbulence. Analysis of the scaling exponents of structure functions and the decorrelation length of dissipation reveals that small-scale ﬂuctuations are weakly intermittent. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press Key words: isotropic turbulence, homogeneous turbulence https://doi.org/10.1017/jfm.2021.420 Published online by C J. Fluid Mech. (2021), vol. 920, A9, doi:10.1017/jfm.2021.420 J. Fluid Mech. (2021), vol. 920, A9, doi:10.1017/jfm.2021.420 J. Fluid Mech. (2021), vol. 920, A9, doi:10.1017/jfm.2021.420 J. Fluid Mech. (2021), vol. 920, A9, doi:10.1017/jfm.2021.420 © The Author(s), 2021. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 920 A9-1 On the turbulent ﬂow past a realistic open-cell metal foam R. Corsini1, A. Fregni1, M. Spinolo2 and E. Stalio1,† R. Corsini1, A. Fregni1, M. Spinolo2 and E. Stalio1,† 1Dipartimento di Ingegneria “Enzo Ferrari”, Università degli Studi di Modena e Reggio Emilia, via Pietro Vivarelli 10, 41125 Modena, Italy 1Dipartimento di Ingegneria “Enzo Ferrari”, Università degli Studi di Modena e Reggio Emilia, via Pietro Vivarelli 10, 41125 Modena, Italy 2Ferrari S.p.A., via Enzo Ferrari 27, 41053 Maranello, Italy (Received 30 July 2020; revised 19 March 2021; accepted 11 May 2021) (Received 30 July 2020; revised 19 March 2021; accepted 11 May 2021) Turbulence is investigated in the lee of an open-cell metal foam layer. In contrast to canonical grids, metal foams are locally irregular but statistically isotropic. The solid matrix is characterised by two lengths, the ligament thickness df and the pore diameter dp. A direct numerical simulation is conducted on a realistic metal foam geometry for which df /dp = 0.14 and the porous layer thickness is ﬁve times the pore diameter. The Reynolds number based on the pore size is Redp = 4000, corresponding to a Taylor-scale Reynolds number Reλ ≈80. Closer to the foam than two pore diameters, the pressure and turbulent transports of turbulent kinetic energy are non-negligible. In the same region, Reλ undergoes a steep decrease whereas the dissipation coefﬁcient Cϵ increases like Re−1 λ . At larger distances from the porous layer, the classical grid turbulence situation is recovered, where the mean advection of turbulent kinetic energy equals dissipation. This entails a power-law decay of turbulent quantities and characteristic lengths. The decaying exponents of integral, Taylor and Kolmogorov scales are close to one-half, indicating that the turbulence simulated here differs from Saffman turbulence. Analysis of the scaling exponents of structure functions and the decorrelation length of dissipation reveals that small-scale ﬂuctuations are weakly intermittent. Turbulence is investigated in the lee of an open-cell metal foam layer. In contrast to canonical grids, metal foams are locally irregular but statistically isotropic. The solid matrix is characterised by two lengths, the ligament thickness df and the pore diameter dp. A direct numerical simulation is conducted on a realistic metal foam geometry for which df /dp = 0.14 and the porous layer thickness is ﬁve times the pore diameter. 1. Introduction Grid turbulence has been investigated for a long time owing to its good approximation to homogeneous and isotropic turbulence. In the classic ﬂow conﬁguration, a planar grid or † Email address for correspondence: enrico.stalio@unimore.it © The Author(s), 2021. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. 920 A9-1 R. Corsini, A. Fregni, M. Spinolo and E. Stalio screen with uniform mesh size and deﬁnite rod thickness is held in a uniform ﬂuid stream. Downstream of the grid, homogeneity is achieved on cross-ﬂow planes and a degree of isotropy is exhibited. This canonical ﬂow has assumed an essential role in understanding turbulence and has allowed the formulation and testing of theories and models since the early wind tunnel experiments (Simmons & Salter 1934; Taylor 1935). The search for self-preserving or self-similar forms of correlation or spectral functions has led to the theories of homogeneous isotropic turbulence proposed over the past decades and the common prediction that turbulence decays according to a power law (de Karman & Howarth 1938; Kolmogorov 1941; Batchelor 1948; Saffman 1967; George 1992). Despite the fact that these theories agree on the form, the value of the decay exponent n is still debated. The theoretical analyses by de Karman & Howarth (1938), Batchelor (1948) and Saffman (1967) resulted in values for n of 1, 10/7 and 6/5, respectively. A large collection of experimental measurements involving classical grids of various geometries and at different ﬂow regimes has grown up over the years. While the earliest works supported the prediction that n = 1 (Batchelor & Townsend 1948), later experiments by Comte-Bellot & Corrsin (1966) and Mohamed & Larue (1990) corrected the decay exponent, with values falling between 1.1 and 1.4 (Lavoie, Djenidi & Antonia 2007; Kurian & Fransson 2009; Kitamura et al. 2014, and references therein). George (1992) argued that the apparent discrepancies in the measurements are related to an undeﬁned dependence of the ﬂow on the initial conditions. This precludes the existence of a single universal state, at least at ﬁnite Reynolds number. Lavoie et al. 1. Introduction (2007) investigated the effects of the initial conditions on the characteristics of decaying turbulence, and showed that this impact is more marked as the anisotropy and the strength of large-scale periodic motions increase. On the other hand, the improvement of isotropy and of large-scale periodic character reduces the inﬂuence of initial conditions. This suggested that the dependence on initial conditions is associated with departures from ideal homogeneity and isotropy conditions. py Recent research has attempted to reconcile many of the experimental data with Saffman turbulence. According to these analyses, the value n = 6/5 represents a minimum decay rate valid for (strictly) homogeneous turbulence and which could also have deviations because of an inhomogeneous decay. The large-scale turbulent structures are proven to be regulated by the invariance of the Saffman integral. This is physically interpreted as the conservation of linear momentum and is opposed to the invariance of the Loitsyansky integral, on which Batchelor turbulence is based. It is concluded in the work by Krogstad & Davidson (2010) that ‘it seems very likely’ that the turbulence observed is Saffman turbulence, and that it is also possible that different grid geometries, or other ranges of Re, could produce different results. Also Kitamura et al. (2014) report that grid turbulence is of Saffman type for the Reynolds-number range examined in their work, when grid turbulence is generated by a square or a cylindrical grid. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University P https://doi.org/10.1017/jfm.2021.420 Published online by Camb Aside from the existence of a self-preserving solution, Mohamed & Larue (1990) attributed the difﬁculty in ﬁnding a single decay exponent also to inconsistencies in the data analysis method. In particular, the inaccuracy is related to the uncertainty in the virtual origin and the inclusion of data pertaining to the inhomogeneous and anisotropic region in the developed range. For grid turbulence, the turbulence decay is to be evaluated starting from x/M > 40–60, where x is the distance from the grid and M is the mesh width. A class of turbulence-generating grid consisting of fractal geometries has been introduced starting from the work by Hurst & Vassilicos (2007). Since then, fractal 920 A9-2 920 A9-2 920 A9-2 Turbulent ﬂow past a metal foam grids have attracted a lot of interest because of the speciﬁc type of turbulence generated. 1. Introduction A remarkable increase in the Reynolds number based on the Taylor scale with respect to usual passive grids was noticed by Seoud & Vassilicos (2007). Further studies investigating the decaying turbulence downstream of a set of multiscale grids (Krogstad & Davidson 2012) and a square-element fractal grid (Hearst & Lavoie 2014) have revealed that, while the region close to the grids can be characterised by residual inhomogeneity and is grid-dependent, in the far ﬁeld – where development is accomplished – ﬂow characteristics are in accordance with classical grid turbulence measurements. The in-depth study of turbulence generated by fractal grids has revealed a peculiar behaviour of the dissipation coefﬁcient Cϵ = ⟨ϵ⟩L/u3 (here ⟨ϵ⟩is the rate of dissipation.) in a region close to the turbulence-generating grid but in conjunction with energy spectra, which follow the −5/3 slope for a wide range of wavenumbers. This behaviour has been described as a breakdown of the classical dissipation scaling and is observed in wind tunnel experiments of grid-generated turbulence of different geometries (Mazellier & Vassilicos 2010; Isaza, Salazar & Warhaft 2014; Valente & Vassilicos 2014; Mora et al. 2019) and also in direct numerical simulation (DNS) data of decaying turbulence in a periodic box (Goto & Vassilicos 2016). The breakdown consists in a behaviour of Cϵ at the initial stages of the decay that depends upon the inlet Reynolds number ReM and the local Reynolds number Reλ as follows: Cϵ ∼Re1/2 M /Reλ. This implies that L/λ ∼Re1/2 M along the direction of Reλ decay (Valente & Vassilicos 2012). The turbulent ﬂow behind an open-cell metal foam has never been investigated before. Similar to regular grids, the open-cell metal foam geometry is characterised by two main length scales, i.e. the mean pore diameter dp and the mean ligament thickness df . In contrast to regular grids, open cells are arranged randomly in space and their morphology, based on a polyhedral frame, is never exactly repeated. This generates a structure that is highly irregular and anisotropic at the pore scale but statistically isotropic at the macroscale. In addition, the metal foam layer investigated here has a thickness larger than a single ligament or a pore. Moreover, the solidity of metal foams, measured as 1 −ε, where ε represents grid porosity, is very different from the solidities typical of grids employed for grid turbulence, which generally range between 0.30 and 0.45. 2.1. Mathematical formulation and ﬂow conﬁguration 2.1. Mathematical formulation and ﬂow conﬁguration The system of equations solved numerically comprises the mass conservation and the Navier–Stokes equations for incompressible ﬂows: ⎧ ⎪⎪⎪⎨ ⎪⎪⎪⎩ ∂Ui ∂xi = 0, ∂Ui ∂t + ∂UiUj ∂xj = −∂P ∂xi + 1 Redp ∂2Ui ∂xj∂xj , (2.1) (2.1) complemented with appropriate boundary conditions. The subscripts i and j take the values 1, 2 and 3 to denote the streamwise direction, x, and the two cross-ﬂow directions, y and z, respectively. All the variables are made non-dimensional by the velocity at the inlet U∞ and the mean pore diameter of the metal foam dp. The Reynolds number based on the unperturbed velocity and the mean pore diameter is Redp = 4000. complemented with appropriate boundary conditions. The subscripts i and j take the values 1, 2 and 3 to denote the streamwise direction, x, and the two cross-ﬂow directions, y and z, respectively. All the variables are made non-dimensional by the velocity at the inlet U∞ and the mean pore diameter of the metal foam dp. The Reynolds number based on the unperturbed velocity and the mean pore diameter is Redp = 4000. The governing equations (2.1) are solved using the high-order ﬁnite-difference method implemented in Incompact3d (Laizet & Lamballais 2009). Sixth-order compact schemes are used for spatial discretisation (Lele 1992), and time integration is done by the third-order Adams–Bashforth scheme. The velocity ﬁeld is evaluated on a Cartesian grid with uniform spacing along the three directions, and pressure is deﬁned on a staggered grid. Pressure–velocity decoupling is accomplished by a fractional-step method, which determines the divergence-free velocity ﬁeld by solving a Poisson equation. The Poisson problem is tackled in the spectral space by using a modiﬁed wavenumber formalism, which allows for any kind of boundary conditions for the velocity ﬁeld in the physical space. Inﬂow/outﬂow boundary conditions are enforced along the streamwise direction and periodic boundary conditions are set along the cross-ﬂow directions to represent statistical homogeneity. A uniform velocity ﬁeld is prescribed at the inlet (U∞, 0, 0), while at the outlet the velocity is determined by a convection equation: ∂Ui ∂t + c∂Ui ∂x = 0. (2.2) (2.2) https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press The convection velocity c is calculated at each time step as the mean between the maximum and minimum values of the streamwise velocity component at the outlet. 1. Introduction In high-porosity metal foams, this value is typically lower than 0.10 (Calmidi & Mahajan 2000). In this paper, a description is reported of turbulence downstream of a high-porosity open-cell metal foam. After a qualitative introduction to the ﬂow investigated in § 3.1, the degree of homogeneity and isotropy of turbulence is investigated in § 3.2. The power-law behaviour of turbulent kinetic energy ⟨k⟩and its dissipation rate are assessed in § 3.3 and § 3.4, respectively. The streamwise variations of turbulent length scales are considered in § 3.5. In § 3.6 the behaviour of the dissipation rate coefﬁcient is investigated in detail, also in the context of recent research on the topic. In § 3.7 the discussion is about whether or not Saffman turbulence is achieved for the present ﬂow conﬁguration. The multiscale nature of the ﬂow is examined by means of one-dimensional spectra in § 3.8. Then § 3.9 investigates high-order statistics, the role of intermittency and the decorrelation length of dissipation. Budget terms of turbulent kinetic energy and of velocity variances are reported in § 3.10, where the main mechanisms for the transport of energy and ﬂuctuations are described in the vicinity of the solid structure and in the fully developed region. Final remarks are drawn in § 4. 920 A9-3 R. Corsini, A. Fregni, M. Spinolo and E. Stalio 2. Numerical methodology 2. Numerical methodology 2.1. Mathematical formulation and ﬂow conﬁguration 2.1. Mathematical formulation and ﬂow conﬁguration The convection velocity c is calculated at each time step as the mean between the maximum and minimum values of the streamwise velocity component at the outlet. The representation of the intricate metal foam geometry is achieved via an immersed boundary method (IBM) based on direct forcing (Gautier, Laizet & Lamballais 2014). The IBM enforces the no-slip condition at the solid walls while preserving the simplicity of the ﬁnite-difference schemes applied to the Cartesian grid (Laizet & Lamballais 2009). Further details on the numerical methodology employed here are provided in Corsini & Stalio (2020). https://doi.org/10.1017/jfm.2021.420 Published online by Ca A sketch of the computational domain is displayed in ﬁgure 1. The extents of the domain along the streamwise and the two cross-ﬂow directions are Lx = 45dp and Ly = Lz = 11.25dp, respectively. The origin of the coordinate system is located at the centre of the downstream face of the porous matrix; thus x = 0 describes the most upstream cross-ﬂow plane where the ﬂuid is not in contact with the solid phase. The thickness of the metal foam layer in the streamwise direction is 5dp and it spans the whole domain in the cross-ﬂow directions. It is placed at a 5dp distance from the inlet section; this avoids interference between the upstream boundary and the solid matrix. Turbulent ﬂow past a metal foam y x z 11.25dp 11.25dp 5dp 5dp 35dp Figure 1. Schematic representation of the computational domain. Turbulent ﬂow past a metal foam Figure 1. Schematic representation of the computational domain. The computational domain is discretised by nx = 3073 grid nodes in the streamwise direction and ny = nz = 768 in the cross-ﬂow directions. The spatial resolution is sufﬁciently ﬁne to ensure that x = y = z ⩽2η for x ⩾5. Close to the porous layer (0 < x < 5), where dissipation is larger, x = y = z ⩽5η. In the above comparisons, the Kolmogorov microscale η is calculated a posteriori from its deﬁnition. The time step is kept at t = 0.001dp/U∞during the simulation, which in terms of the Kolmogorov time scale τη yields t ⩽0.033τη. This corresponds to a Courant–Friedrichs–Lewy number CFL < 0.3. Statistical quantities are computed by averaging in time and along the homogeneous y and z directions. Gathering of statistics begins after one ﬂow-through time from the start of the simulation. 2.1. Mathematical formulation and ﬂow conﬁguration In order to obtain well-converged statistics, the time interval of collection is T = 225dp/U∞, and the three-dimensional snapshots of the velocity and pressure ﬁeld are sampled at equal time intervals of T = 4.5dp/U∞. 2.2. Metal foam geometry The problem of the computer modelling of an intricate metal foam porous structure has been tackled in different ways. Pore-scale morphology can be reconstructed through X-ray tomography (Piller et al. 2013) or generated mathematically assuming an ideal cell geometry based on a virtual sample of regular polyhedra (Boomsma, Poulikakos & Ventikos 2003). In this study, where geometric periodicity is a key feature of the numerical representation and irregularity of the foam is a requirement, a third approach is adopted: the open-pore cellular structure is generated synthetically through a numerical algorithm, developed by August et al. (2015). Besides the excellent realism and periodicity, one further favourable feature of synthetic metal foams is the possibility to tune their porosity and permeability. Thanks to a diffuse interface representation of the phase-ﬁeld approach (August et al. 2015), the thickness of ligaments can be easily adjusted. Figure 2 shows the details of a couple of cells of the synthetic structure. The synthetic metal foam structure used in the simulation is characterised by the geometrical features listed in table 1. Both dp and df are calculated by spatial averages and thus represent the mean pore diameter and the mean ligament thickness of the metal foam sample. The value of grid porosity set, ε = 0.92, is representative of high-porosity metal foams (Calmidi & Mahajan 2000). Based on typical sizes of open-cell aluminium foams, an inﬂow velocity of U∞= 15 m s−1 is obtained at Redp = 4000, assuming air at standard conditions as the working ﬂuid. Table 1 reports experimental conditions from previous wind tunnel studies on regular planar grids. 920 A9-5 R. Corsini, A. Fregni, M. Spinolo and E. Stalio R. Corsini, A. Fregni, M. Spinolo and E. Stalio Figure 2. Cells of the aluminium foam generated algorithmically (August et al. 2015). Figure 2. Cells of the aluminium foam generated algorithmically (August et al. 2015). A sample of the metal foam geometry with superimposed computational points is displayed in ﬁgure 2 of Corsini & Stalio (2020). Staircase patterns of the immersed boundaries approximate the rounded borders of the solid region. This referenced picture and the computed ratio between average ligament diameter and grid spacing df /x ≈10 suggest that the ligaments are discretised by an adequate number of grid points. 3.1. Instantaneous and mean ﬂows Figure 3(a) shows the streamwise component of the instantaneous velocity ﬁeld in one of the snapshots collected. The uniform free stream of the inﬂow is disrupted by the irregularly arranged ligaments of the solid structure and velocity ﬂuctuations arise within the porous matrix. Vortices of different orientations are shed from the ligaments and a wake is formed. The largest perturbations are observed close to the downstream edge of the porous matrix. The wakes originated by the ligaments develop in a non-uniform fashion and interact at variable lengths. The smaller wakes are seen to disappear after a couple of pore diameters, whereas larger wakes stemming from ligament clumps meet at a further distance from the foam. The larger wakes also last in time, as revealed by the streaks in the time-averaged velocity ﬁeld ⟨U⟩t shown in ﬁgure 3(b). U∗ ∞ (m s−1) 15.0 U∗ ∞ (m s−1) 10.0 10.0 12.0 12.0 12.0 12.0 12.0 8.0 8.0 13.5 10.6 10.4 17.2 10.4 20.0 10.4 20.0 10.5 es of turbulen 10) and Kitam f the grid, res s provided in 3.2. Homogeneity and isotropy The approximation to statistical homogeneity in the cross-ﬂow directions in grid turbulence is known to depend on the grid geometry and the Reynolds number. While, for regular grids, experiments suggest that the ﬂow becomes nearly homogeneous for x/M > 40 (Comte-Bellot & Corrsin 1966; Mohamed & Larue 1990), for fractal grids, homogeneity is usually retrieved further downstream (Hearst & Lavoie 2014) and, for example, Valente & Vassilicos (2011) report x/Meff > 80, where Meff is the effective mesh size. https://doi.org/10.1017/jfm.2021.420 Publish The distribution of ⟨U⟩t in cross-ﬂow planes is shown in ﬁgure 4 for six streamwise positions at increasing distance from the metal foam. Solid lines mark regions where the percentage of variation of ⟨U⟩t relative to U∞has magnitude greater than 10 %, while dashed lines encompass regions of magnitude greater than 5 %. These are seen to gradually shrink along x. While inhomogeneity is in part to be ascribed to the limited size of the sample composed only by the collection of snapshots in time, for x > 20 their extent is 920 A9-6 U∗ ∞ Redp Reλ n η λ Lg (m s−1) 15.0 4000 79–88 1.12 0.009–0.019 0.17–0.34 0.27–0.54 U∗ ∞ ReM Reλ n η∗/M∗ λ∗/M∗ L∗ g/M∗ (m s−1) 10.0 17 000 37–49 1.29 0.010–0.036 0.14–0.43 — 10.0 34 000 61–72 1.25 0.006–0.013 0.10–0.20 0.47–0.96 12.0 760 (10–20) 1.59 0.067–0.385 0.58–2.28 0.95–2.34 12.0 1310 (10–30) 1.52 0.034–0.260 0.47–1.85 0.78–2.03 12.0 1420 (10–35) 1.50 0.031–0.244 0.41–1.88 0.56–1.96 12.0 2030 (10–35) 1.50 0.029–0.197 0.41–1.54 0.61–1.61 12.0 3320 (20–60) 1.45 0.022–0.123 0.43–1.33 0.75–1.47 8.0 19 000 (85–180) 1.27 0.006–0.020 0.40 0.36–0.71 8.0 26 400 (165–170) 1.53 0.004–0.009 0.25–0.34 0.52–0.79 13.5 36 000 71–82 1.13 (0.006–0.014) (0.11–0.29) 0.60–1.13 10.6 6700 27–29 1.18 0.021–0.045 0.24–0.52 0.98–2.03 10.4 9600 47–50 1.15 0.012–0.021 0.22–0.37 0.72–1.25 17.2 16 000 61–67 1.12 0.009–0.018 0.17–0.28 0.78–1.39 10.4 16 000 59–65 1.12 0.006–0.014 0.15–0.23 0.40–0.67 20.0 33 000 67–72 1.10 0.004–0.010 0.11–0.16 0.44–0.75 10.4 16 000 58–64 1.16 0.005–0.010 0.15–0.22 0.46–0.80 20.0 33 000 88–91 1.12 0.003–0.008 0.10–0.16 0.51–1.01 10.5 33 000 99–112 — 0.002–0.005 — 0.29–0.48 ales of turbulence generated by the open-cell metal foam. The experimental results of Comte-Bellot 2010) and Kitamura et al. (2014) on the turbulence generated by regular grids are also included. Here of the grid, respectively. Asterisk ∗denotes quantities expressed in dimensional form. Quantities in ies provided in the same work. 3.2. Homogeneity and isotropy U∗ ∞ (m s−1) 15.0 U∗ ∞ (m s−1) 10.0 10.0 3 12.0 12.0 12.0 12.0 12.0 8.0 8.0 2 13.5 3 10.6 10.4 17.2 10.4 20.0 3 10.4 20.0 3 10.5 3 es of turbulen 010) and Kitam of the grid, res s provided in t R. Corsini, A. Fregni, M. Spinolo and E. Stalio , g , p 5 0 –5 –10 –5 0 10 20 30 35 5 0 –5 3 2 1 0 –1 –10 –5 0 10 20 30 35 3 2 1 0 –1 y y x (a) (b) Figure 3. Instantaneous velocity ﬁeld of the streamwise velocity component (a) and its temporal mean (b) in an x–y plane. 5 0 –5 –10 –5 0 10 20 30 35 3 2 1 0 –1 y (a) Figure 3. Instantaneous velocity ﬁeld of the streamwise velocity component (a) and its temporal mean (b) in an x–y plane. still appreciable. In the x = 30 station, the time-averaged velocity ⟨U⟩t varies between 0.86 and 1.12. The isotropy level of the large scales of motion can be investigated through the ratio of root-mean-square (r.m.s.) velocity ﬂuctuations along orthogonal directions. In the present case, the ﬂuctuations of the x-component of velocity are the largest: in the developed region, indicators urms/wrms and urms/vrms oscillate within the interval (1.5, 1.6) about a mean of 1.55 for urms/vrms and 1.56 for urms/wrms, where the difference is ﬁnally due to the size of the sample employed in the simulations. In previous grid turbulence measurements (Kurian & Fransson 2009; Krogstad & Davidson 2010; Kitamura et al. 2014), the observed isotropy indicators are in general smaller than those measured here. Kitamura et al. (2014), who also collected experimental results by other authors, report urms/wrms < 1.2 in all cases; similar values are also reported in the lee of fractal grids (Hurst & Vassilicos 2007; Gomes-Fernandes, Ganapathisubramani & Vassilicos 2012). More details about large-scale isotropy measures downstream of the present metal foam are reported in Corsini & Stalio (2020). 3.3. Decay of velocity ﬂuctuations Figure 5 displays the streamwise evolution of the variance of the three components of velocity ⟨uiui⟩as well as the turbulent kinetic energy ⟨k⟩. Very close to the foam and for x < 1, velocity ﬂuctuations are observed to remain constant. The negative slope increases gradually in x until ﬂuctuations exhibit a power-law decay that persists until the end of the computational domain. As demonstrated, for example, in Tennekes & Lumley (1972), this is expected in the region where advection and dissipation of the turbulent kinetic energy become the only non-negligible terms in the transport equation of ⟨k⟩; see § 3.10. Power-law parameters are sought in the form Power-law parameters are sought in the form u2 rms U2∞ = A x −x0 dp −n (3.1) u2 rms U2∞ = A x −x0 dp −n (3.1) 920 A9-8 u2 rms U2∞ = A x −x0 dp −n (3.1) 920 A9-8 920 A9-8 Turbulent ﬂow past a metal foam 5 0 –5 5 0 –5 –5 5 0 –5 5 0 –5 5 0 2.0 1.5 1.0 0.5 0 2.0 1.5 1.0 0.5 0 y y z z z Figure 4. Streamwise mean ﬂow ﬁeld ⟨U⟩t in y–z planes extracted at x = 5, 10, 15, 20, 25 and 30 (left to right then top to bottom): solid line, isolines ⟨U⟩t −U∞= ±0.1; and dashed line, isolines ⟨U⟩t −U∞= ±0.05. 5 0 –5 y 5 0 –5 –5 5 0 y z –5 5 0 2.0 1.5 1.0 0.5 0 z –5 5 0 z 5 Figure 4. Streamwise mean ﬂow ﬁeld ⟨U⟩t in y–z planes extracted at x = 5, 10, 15, 20, 25 and 30 (left to right then top to bottom): solid line, isolines ⟨U⟩t −U∞= ±0.1; and dashed line, isolines ⟨U⟩t −U∞= ±0.05. 100 10–1 10–2 10–3 10–2 10–1 100 101 102 x ⟨uiui⟩, ⟨k⟩ Figure 5. Decay of velocity ﬂuctuations: solid line, ⟨uu⟩; dot-dashed line, ⟨vv⟩; dotted line, ⟨ww⟩; and dashed line, ⟨k⟩. 100 10–1 10–2 10–3 10–2 10–1 100 101 102 x ⟨uiui⟩, ⟨k⟩ Figure 5 Decay of velocity ﬂuctuations: solid line ⟨uu⟩; dot dashed line ⟨vv⟩; dotted line ⟨ww⟩; and dashed https://doi.org/10.1017/jfm.2021.420 Published online Figure 5. Decay of velocity ﬂuctuations: solid line, ⟨uu⟩; dot-dashed line, ⟨vv⟩; dotted line, ⟨ww⟩; and dashed line, ⟨k⟩. through a numerical procedure. In (3.1), A is the multiplicative coefﬁcient, x0 is the virtual origin and n is the decay exponent. 3.3. Decay of velocity ﬂuctuations As n is positive, the power law has a vertical asymptote (and a singularity) at the virtual origin x = x0. As the parameters in (3.1) depend greatly upon the interval of sampling data considered, also the interval limits are determined through a numerical procedure. In (3.1), A is the multiplicative coefﬁcient, x0 is the virtual origin and n is the decay exponent. As n is positive, the power law has a vertical asymptote (and a singularity) at the virtual origin x = x0. As the parameters in (3.1) depend greatly upon the interval of sampling data considered, also the interval limits are determined 920 A9-9 R. Corsini, A. Fregni, M. Spinolo and E. Stalio Variable xmin xmax x0 A n u2 rms 7.98 30.0 0.648 0.155 1.12 ⟨k⟩ 6.83 30.0 0.310 0.155 1.14 Table 2. Left boundary of the ﬁtting interval Id, virtual origin, multiplicative coefﬁcient and decay exponent Table 2. Left boundary of the ﬁtting interval Id, virtual origin, multiplicative coefﬁcient and decay exponent of the power laws in the form of equation (3.1) ﬁtting u2 rms and ⟨k⟩. Table 2. Left boundary of the ﬁtting interval Id, virtual origin, multiplicative coefﬁcient and decay exponent of the power laws in the form of equation (3.1) ﬁtting u2 rms and ⟨k⟩. Table 2. Left boundary of the ﬁtting interval Id, virtual origin, multiplicative coefﬁcient and decay exponent of the power laws in the form of equation (3.1) ﬁtting u2 rms and ⟨k⟩. inside the ﬁtting procedure. A similar approach has been applied in Hearst & Lavoie (2014). inside the ﬁtting procedure. A similar approach has been applied in Hearst & Lavoie (2014). In the present work, a developed region Id = [xmin, xmax] is employed, where the right border of the interval is kept ﬁxed to xmax = 30.0, clear of possible – yet not evident – outﬂow condition effects, while xmin is discretely varied in the interval [0.015, 24.7] to seek an xmin coordinate that ensures the best ﬁt. The coordinate xmin will be taken as the start of the developed region. For each selection of a value for xmin, the virtual origin x0 is discretely varied within I0 = [0.015, xmin], as the singularity is not supposed to belong to Id. The intervals of variation of both xmin and x0 are discretised by the same subdivision as the computational mesh. Comte-Bellot & Corrsin (1966) found 1.15 ⩽n ⩽1.29 for regular grids, while according to Mohamed & Larue (1990) n ≈1.3. More recently, Krogstad & Davidson (2010) found n = 1.13 ± 0.02. 3.3. Decay of velocity ﬂuctuations Parameters A and n are determined through a least-squares ﬁt. Deviations between computed data and ﬁtting laws are then calculated as the Euclidean norm of the error divided by the number of data points: e(x0, xmin) = Nd j=1δ2 j 1/2 Nd . (3.2) (3.2) Nd In (3.2), δj represents the difference between computed statistics and the least-squares ﬁtted power-law approximation of u2 rms at the jth point of Id; and Nd is the number of uniformly spaced points in the data ﬁt region Id. The (x0, xmin) couple which ensures the smallest deviation from computed data provides the ﬁnal A and n coefﬁcients. This procedure leads to the results given in table 2 with error distribution as in ﬁgure 6. In the region of parameters investigated, only one minimum is found, which is located far from the boundaries of the region investigated. The dependence on porosity of the power-law exponents obtained is shown to be only weak in the Appendix. In order to check the dependence of the results from the ﬁtting method employed, a procedure from the literature is also applied to the same set of data; this alternative technique is that utilised by Hearst & Lavoie (2014). In the application of the method to the present case, the virtual origin x0 and lower bound xmin are varied within I0 = [0, 4] and [4, 14.7], respectively. For both u2 rms and ⟨k⟩, the process converges after three iterations. Applied to u2 rms, this ﬁtting procedure provides x0 = 0.610 and xmin = 6.78, which yield the estimate for ˜nu = 1.12. In the case of ⟨k⟩, the values obtained are x0 = 0.360, xmin = 5.02 and ˜nk = 1.14. The results obtained by the method proposed by Hearst & Lavoie (2014) are only marginally different from those obtained as described above and reported in table 2. Comte-Bellot & Corrsin (1966) found 1.15 ⩽n ⩽1.29 for regular grids, while according to Mohamed & Larue (1990) n ≈1.3. More recently, Krogstad & Davidson (2010) found n = 1.13 ± 0.02. 920 A9-10 Turbulent ﬂow past a metal foam Turbulent ﬂow past a metal foam Turbulent ﬂow past a metal foam 10 8 6 4 2 0 5 10 15 x0 xmin Figure 6. 3.3. Decay of velocity ﬂuctuations Contour lines of log10(e), where e is the normalised Euclidean norm of the error in (3.2): dashed line, log10(e) = −4.8; dot-dashed line, log10(e) = −6.03; solid line, log10(e) = −6.05; and dotted line, log10(e) ∈[−6.00 −5.00], with lines separated by δ = 0.2. A single minimum is found in the ﬁeld, as indicated by the ∗symbol, which corresponds to x0 = 0.648 and xmin = 7.98. The singularity is not included in the domain, and thus xmin > x0. 10 8 6 4 2 0 5 10 15 x0 xmin 10 0 xmin Figure 6. Contour lines of log10(e), where e is the normalised Euclidean norm of the error in (3.2): dashed line, log10(e) = −4.8; dot-dashed line, log10(e) = −6.03; solid line, log10(e) = −6.05; and dotted line, log10(e) ∈[−6.00 −5.00], with lines separated by δ = 0.2. A single minimum is found in the ﬁeld, as indicated by the ∗symbol, which corresponds to x0 = 0.648 and xmin = 7.98. The singularity is not included in the domain, and thus xmin > x0. Besides the parameters x0, A and n in (3.1), the procedure provides the coordinate xmin of the start of the developed region as the left boundary of the interval Id. All the subsequent ﬁttings in this work will be carried out on Id = [7.98, 30.0]. Besides the parameters x0, A and n in (3.1), the procedure provides the coordinate xmin of the start of the developed region as the left boundary of the interval Id. All the subsequent ﬁttings in this work will be carried out on Id = [7.98, 30.0]. 3.4. Turbulence decay rate As opposed to experimental studies, where rate of dissipation ⟨ϵ⟩needs to be evaluated using the frozen turbulence assumption or isotropy (3.5), in DNS studies ⟨ϵ⟩can be computed directly from its deﬁnition, https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press ⟨ϵ⟩≡ 2 Redp ⟨sijsij⟩, (3.3) (3.3) where sij = (∂ui/∂xj + ∂uj/∂xi)/2 is the ﬂuctuating rate of strain. Figure 7 displays the spatial evolution of ⟨ϵ⟩, together with the least-squares ﬁtted power law in the form ⟨ϵ⟩∼aϵxh. The coefﬁcients that ﬁt the data over Id = [7.98, 30.0] are aϵ = 0.217 and h = −2.20. where sij = (∂ui/∂xj + ∂uj/∂xi)/2 is the ﬂuctuating rate of strain. Figure 7 displays the spatial evolution of ⟨ϵ⟩, together with the least-squares ﬁtted power law in the form ⟨ϵ⟩∼aϵxh. The coefﬁcients that ﬁt the data over Id = [7.98, 30.0] are aϵ = 0.217 and h = −2.20. The scaling of dissipation ⟨ϵ⟩can also be set in relation to the scaling of ⟨k⟩in § 3.3. As also shown quantitatively in § 3.10, in the developed region of a statistically steady, high-Reynolds-number ﬂow, one has https://doi.org/10.1017/jfm.2021.420 Publish ⟨U⟩dk dx = −⟨ϵ⟩. (3.4) (3.4) Equation (3.4) suggests that the decay exponent in this case should equal h = −(nk + 1). In the present study, h = −2.20 and nk = 1.14 are calculated. The percentage difference between −(nk + 1) and h is below 3 %. Equation (3.4) suggests that the decay exponent in this case should equal h = −(nk + 1). In the present study, h = −2.20 and nk = 1.14 are calculated. The percentage difference between −(nk + 1) and h is below 3 %. R. Corsini, A. Fregni, M. Spinolo and E. Stalio 100 10–1 10–2 10–3 10–4 10–5 10–2 100 102 x ⟨ϵ⟩ Figure 7. Dissipation of the turbulent kinetic energy: dashed line, ⟨ϵ⟩as deﬁned in (3.3); dotted line, ⟨ϵ⟩iso from (3.5); and solid line, aϵxh, with aϵ = 0.217 and h = −2.20. 100 10–1 10–2 10–3 10–4 10–5 10–2 100 102 x ⟨ϵ⟩ Figure 7. Dissipation of the turbulent kinetic energy: dashed line, ⟨ϵ⟩as deﬁned in (3.3); dotted line, ⟨ϵ⟩iso from (3.5); and solid line, aϵxh, with aϵ = 0.217 and h = −2.20. Dissipation is compared in ﬁgure 7 to the same quantity computed under the hypothesis of isotropic turbulence: ⟨ϵ⟩iso = 15ν ∂u ∂x 2 . 3.4. Turbulence decay rate (3.5) (3.5) The close similarity between ⟨ϵ⟩and ⟨ϵ⟩iso is only in apparent contradiction with isotropy ratios larger than 1.5 reported in § 3.2. From the demonstration by Taylor (1935), it appears that hypotheses less strict than isotropy are required for equation (3.5) to hold. The weaker set of hypotheses hold true in the present case; see Corsini & Stalio (2020). 3.5. Length scales The length scales examined for the characterisation of the turbulence generated by a metal foam are the Kolmogorov scale η, the Taylor microscale λ and the integral scales. All the length scales are computed directly from their deﬁnitions. Their distribution within the developed region is approximated by a power law of the distance x. The range of variation of each length scale along Id is reported in table 1 together with data from the literature on classical grid turbulence. 3.5.1. Kolmogorov scale The Kolmogorov scale is deﬁned through dissipation ⟨ϵ⟩. It is predicted from the power-law expression for ⟨ϵ⟩(derived from the expression for ⟨k⟩) that η can be represented by a function of the form η ∼aηxs, where s equals −h/4 and ﬁnally s = (nk + 1)/4. The decay exponent for ⟨k⟩computed here, nk = 1.14, gives s = 0.54. The power-law approximation is displayed in ﬁgure 8 together with the Kolmogorov scale, as computed from its deﬁnition: the ﬁtting coefﬁcients are aη = 0.00291 and s = 0.55. Very similar power-law parameters are obtained in this same ﬂow conﬁguration for a porosity ε = 0.97; see the Appendix. Comte-Bellot & Corrsin (1971) provide Kolmogorov-scale measurements in their table 4, which grow like a power law of exponent ˆs = 0.58. 920 A9-12 Turbulent ﬂow past a metal foam 0.025 0.020 0.015 0.010 0.005 0 10 20 30 x η Figure 8. Streamwise distribution of the Kolmogorov scale: dashed line, η as calculated from its deﬁnition; and solid line, aηxs, where aη = 0.00291, s = 0.55. The uniform grid spacing adopted in all directions is xi = 0.0146, which suggests that spatial resolution is high enough to represent the smallest scales of the turbulence. Turbulent ﬂow past a metal foam 0.025 0.020 0.015 0.010 0.005 0 10 20 30 x η Turbulent ﬂow past a metal foam Figure 8. Streamwise distribution of the Kolmogorov scale: dashed line, η as calculated from its deﬁnition; and solid line, aηxs, where aη = 0.00291, s = 0.55. The uniform grid spacing adopted in all directions is xi = 0.0146, which suggests that spatial resolution is high enough to represent the smallest scales of the turbulence. 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 10 20 30 x λ Figure 9. Streamwise distribution of the Taylor microscale: dashed line, λ as calculated from its deﬁnition; and solid line, aλxc, where aλ = 0.0577 and c = 0.52. 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 10 20 30 x λ Figure 9. Streamwise distribution of the Taylor microscale: dashed line, λ as calculated from its deﬁnition; and solid line, aλxc, where aλ = 0.0577 and c = 0.52. 3.5.2. Taylor microscale Figure 9 displays the streamwise distribution of the Taylor microscale, deﬁned by 3.5.2. Taylor microscale Figure 9 displays the streamwise distribution of the Taylor microscale, deﬁned by 3.5.2. 3.5.1. Kolmogorov scale Taylor microscale u2 rms = λ2 ∂u ∂x 2 . (3.6) (3.6) Comte-Bellot & Corrsin (1971) report Taylor-scale values in a few measurement points (see their table 4); their data ﬁt a power law of exponent c = 0.53. It is predicted in the study by George (1992) that the Taylor scale of homogeneous isotropic turbulence increases in time with t1/2 and, for grid turbulence, λ grows as x1/2 in the laboratory frame. More recently, Kurian & Fransson (2009) reported that λ increases approximately like the square-root of the streamwise coordinate. In the present study, ﬁtting a power-law approximation of the form λ ∼aλxc over Id gives aλ = 0.0577 and c = 0.52. 920 A9-13 920 A9-13 R. Corsini, A. Fregni, M. Spinolo and E. Stalio 250 200 150 100 80 50 0 10 20 30 x Reλ Figure 10. Streamwise distribution of Reλ. The horizontal dashed line speciﬁes the value Reλ = 80. Figure 10. Streamwise distribution of Reλ. The horizontal dashed line speciﬁes the value Reλ = 80. Figure 10 shows the behaviour of the turbulent Reynolds number. After a steep decrease in the vicinity of the metal foam, Reλ reaches a plateau where Reλ ≈80. An expression relating the Taylor and the Kolmogorov scales can be obtained for isotropic turbulence through (3.5): λ η = 151/4Re1/2 λ . (3.7) (3.7) Results from the present investigation, and not reported for brevity, show that the difference between (λ/η)2/ √ 15 and Reλ is less than 3 % over the whole computational domain. Integral scales 3.5.3. Integral scales 3.5.3. Integral scales The autocorrelation coefﬁcient is deﬁned as the ratio between the autocorrelation function of separation r = rej and the autocorrelation function for r = 0: ρii(x, r) = Rii(x, r) Rii(x, 0). (3.8) (3.8) The autocorrelation coefﬁcients along y of the streamwise ρ11(x, re2) and the cross-ﬂow ρ22(x, re2) velocity components are reported in ﬁgure 8 of Corsini & Stalio (2020). A distinction is made between the transverse and longitudinal correlations depending on the relative direction of velocity components and separation vector. Transverse correlations built with streamwise velocity ﬂuctuations have longer tails with respect to longitudinal correlations built with spanwise ﬂuctuations. g p Integral scales are deﬁned as integrals over r of autocorrelation coefﬁcients: Lii(x, ej) = 1 Rii(x, 0) ∞ 0 Rii(x, rej) dr. (3.9) https://doi.org/10.1017/jfm.2021.420 Publis (3.9) These depend upon the coordinates along non-homogeneous directions as well as on the direction of separation ej. In practice, since the computational domain has ﬁnite boundaries, the integral scales are calculated here as the distance over which the autocorrelation function decreases from 1 to 1/e, where e is Euler’s number. For the calculation of integral scales, correlations are assumed to decay exponentially. 920 A9 14 These depend upon the coordinates along non-homogeneous directions as well as on the direction of separation ej. In practice, since the computational domain has ﬁnite boundaries, the integral scales are calculated here as the distance over which the autocorrelation function decreases from 1 to 1/e, where e is Euler’s number. For the calculation of integral scales, correlations are assumed to decay exponentially. Turbulent ﬂow past a metal foam 1.0 0.8 0.6 0.4 0.2 0 10 20 30 L x Figure 11. Streamwise distribution of the integral scales: dot-dashed line, streamwise integral scale L; dashed line, longitudinal integral scale Lg; dotted line, transverse integral scale Lt; and solid line, power-law approximations of the different scales. Turbulent ﬂow past a metal foam 1.0 0.8 0.6 0.4 0.2 0 10 20 30 L x Turbulent ﬂow past a metal foam Figure 11. Streamwise distribution of the integral scales: dot-dashed line, streamwise integral scale L; dashed line, longitudinal integral scale Lg; dotted line, transverse integral scale Lt; and solid line, power-law approximations of the different scales. 3.6. Dissipation rate coefﬁcient In high-Reynolds-number turbulent ﬂows away from solid walls, the dissipation rate can be scaled on the integral length scale and velocity ﬂuctuations through an order-one constant: In high-Reynolds-number turbulent ﬂows away from solid walls, the dissipation rate can be scaled on the integral length scale and velocity ﬂuctuations through an order-one constant: Cϵ = ⟨ϵ⟩L u3rms . (3.10) (3.10) Figure 12 shows that, in the present case, after an initial steep increase for x < 2, the dissipation rate coefﬁcient Cϵ based on L ﬂuctuates over Id between 0.45 and 0.50, where its spatial average is ¯Cϵ = 0.483. This value is very close to values reported in Pearson, Krogstad & van de Water (2002) for shear turbulence at different Reλ numbers. With regard to the initial steep increase, in recent years Vassilicos and coworkers have observed that, close to the turbulence-generating grid, there is a region characterised by spectra that closely match the −5/3 power law, in conjunction with an increase in Cϵ. In the hypothesis of ⟨ϵ⟩= ⟨ϵ⟩iso, combining the deﬁnition (3.10) with equation (3.5) leads to Cϵ = 15 L λ 1 Reλ . (3.11) (3.11) As only small variations of the ratio between length scales L/λ are observed for given Reynolds number of the mesh size, Cϵ is seen to increase like Re−1 λ . This behaviour is reported for both fractal and regular grids (Valente & Vassilicos 2012). As only small variations of the ratio between length scales L/λ are observed for given Reynolds number of the mesh size, Cϵ is seen to increase like Re−1 λ . This behaviour is reported for both fractal and regular grids (Valente & Vassilicos 2012). p g g Equation (3.11) is studied for the present case in ﬁgure 13, where the logarithmic plot emphasises the Cϵ(Reλ) behaviour close to the metal foam. Corresponding to the initial steep decrease in Reλ shown in ﬁgure 10, Cϵ is seen to increase like Re−1 λ . In the same region, a well-deﬁned −5/3 energy spectra behaviour is observed over a broader wavenumber range than the fully developed region; see the inset of ﬁgure 15 in § 3.8. The situation described in Valente & Vassilicos (2012) is thus observed in the present case. 3.5.3. Integral scales Variable Power law Multiplicative coefﬁcient Exponent ⟨ϵ⟩(x) aϵxh 0.217 −2.20 η(x) aηxs 0.00291 0.55 λ(x) aλxc 0.0577 0.52 L(x) aLxq 0.143 0.52 Lg(x) — 0.0946 0.51 Lt(x) — 0.0555 0.51 Table 3. Parameters of the power-law functions of the form f(x) ∼axb ﬁtting the turbulent quantities analysed. Variable Power law Multiplicative coefﬁcient Exponent Table 3. Parameters of the power-law functions of the form f(x) ∼axb ﬁtting the turbulent quantities analysed. Integral scales for the present case are based on the streamwise or the cross-ﬂow velocity components. Given the inhomogeneity of the streamwise direction, separation is set in the cross-ﬂow directions e2 and e3. Thus, the integral scale based on the streamwise velocity is always transverse and will be denoted by L. The integral scales based on cross-ﬂow velocity components can be either longitudinal or transverse and are denoted by Lg and Lt, respectively. As statistically L11(x, e2) = L11(x, e3) = L(x), L22(x, e2) = L33(x, e3) = Lg(x) and L33(x, e2) = L22(x, e3) = Lt(x), these equalities have been exploited in the calculation of integral scales. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press Figure 11 displays the integral scales. The power-law approximations L ∼aLxq have the coefﬁcients reported in table 3; the exponents computed are very close to the L ∼ x1/2 behaviour predicted by Wang & George (2002). Also, in the case of higher porosity presented in the Appendix, the power-law exponent is close to 1/2. Notice that L is one order of magnitude smaller than the domain size, the transverse length of which is 11.25dp; this suggests that the imposed lateral periodic boundary conditions can satisfactorily represent cross-ﬂow homogeneity in the present case. The evolution of the Reynolds number based on the integral scale, deﬁned as ReL ≡Lurms/ν, along the x-axis is displayed in ﬁgure 9 of Corsini & Stalio (2020). 920 A9-15 R. Corsini, A. Fregni, M. Spinolo and E. Stalio R. Corsini, A. Fregni, M. Spinolo and E. Stalio 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 30 x Cϵ Figure 12. Streamwise distribution of Cϵ = ⟨ϵ⟩L/u3 rms: solid line, based on the streamwise integral scale L; dashed line, based on the longitudinal integral scale Lg; and dotted line, based on the transverse integral scale Lt. 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 30 x Cϵ Figure 12. 3.5.3. Integral scales Streamwise distribution of Cϵ = ⟨ϵ⟩L/u3 rms: solid line, based on the streamwise integral scale L; dashed line, based on the longitudinal integral scale Lg; and dotted line, based on the transverse integral scale Lt. 3.6. Dissipation rate coefﬁcient 1 The streamwise evolution of Cϵ experiences a transition between the Re−1 λ behaviour and a region where the variations in Cϵ are much smaller; see ﬁgures 12 and 13. This transition is about x = 2. The turbulent kinetic energy budgets reported in § 3.10 indicate Turbulent ﬂow past a metal foam Turbulent ﬂow past a metal foam 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 x = 4 x = 2 x = 10 Cϵ 80 100 150 200 250 Reλ Figure 13. Plot of Cϵ as a function of Reλ. The dashed line follows Re−1 λ . Figure 13. Plot of Cϵ as a function of Reλ. The dashed line follows Re−1 λ . that, downstream of this location, the turbulent transport terms become negligible and the mean advection of ⟨k⟩equals dissipation. On the contrary, for x < 2, this equality does not hold, and the variations of Cϵ suggest a non-equilibrium condition between energy at the large scales and dissipation. It should be noted that the present results conﬁrm the predictions reported by Tennekes & Lumley (1972) in their (3.2.29) and (3.2.30), where transition is expected at streamwise distances from the grid that are much larger than the integral scale. In the present conﬁguration, x = 2 corresponds to x ≈6L. In the theory by Richardson and Kolmogorov, the constancy of Cϵ requires that turbulence is at a high Reynolds number and far from solid walls. While the small variations of Cϵ in the fully developed region can be attributed to the Reynolds number, which is not very high, the steep increase in Cϵ in the vicinity of the porous matrix (x < 2) is to be ascribed to the vicinity of the solid ﬁlaments and ultimately to non-negligible turbulent transport terms in the budget equation of ⟨k⟩. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press 3.7. Is grid turbulence Saffman turbulence? Setting nk = nu = n, (3.13) combined with the deﬁnition of Cϵ in (3.10) and the hypotheses L ∼xq and Cϵ ∼x f leads to the following relation: (see § 3.4). Setting nk = nu = n, (3.13) combined with the deﬁnition of Cϵ in (3.10) and the hypotheses L ∼xq and Cϵ ∼x f leads to the following relation: q = 1 −n 2 + f. (3.14) (3.14) As is apparent from (3.12a,b) and (3.14), grid turbulence generated at n = 6/5 is not of the Saffman type unless Cϵ stays constant during kinetic energy decay (f = 0). As is apparent from (3.12a,b) and (3.14), grid turbulence generated at n = 6/5 is not of the Saffman type unless Cϵ stays constant during kinetic energy decay (f = 0). 3.7. Is grid turbulence Saffman turbulence? In recent articles (Krogstad & Davidson 2010; Kitamura et al. 2014) it was discussed whether grid turbulence can be considered to be of the Saffman type. Both Krogstad & Davidson (2010) and Kitamura et al. (2014) conclude that grid turbulence is Saffman turbulence. The theory by Saffman (1967) describes the decay of homogeneous turbulence as u2 rms = KC2/5t−6/5, L = K′C1/5t2/5, (3.12a,b) (3.12a,b) (3.12a,b) https://doi.org/10.1017/jfm.2021.420 Published o where K and K′ are constants and C is expressed by an invariant integral. Both equations (3.12a,b) have to hold for turbulence to be of the Saffman type. This is sometimes expressed by the requirement that u2 rmsL3 = const. during decay, but the latter is a necessary, not sufﬁcient, condition for Saffman turbulence. where K and K′ are constants and C is expressed by an invariant integral. Both equations (3.12a,b) have to hold for turbulence to be of the Saffman type. This is sometimes expressed by the requirement that u2 rmsL3 = const. during decay, but the latter is a necessary, not sufﬁcient, condition for Saffman turbulence. The exponents reported in tables 2 and 3 suggest that turbulence investigated in the present study is not of the Saffman type. Figure 14 provides graphical conﬁrmation for this conclusion. 920 A9-17 R. Corsini, A. Fregni, M. Spinolo and E. Stalio 5 4 3 2 1 0 10 20 30 (×10–3) x u2 rmsLm Figure 14. Streamwise evolution of the product of powers of the integral scale times powers of velocity ﬂuctuations: solid line, u2 rmsL2; dashed line, u2 rmsL3; and dot-dashed line, u2 rmsL5. Saffman turbulence requires the constancy of u2 rmsL3 = const., not observed here. 5 4 3 2 1 0 10 20 30 (×10–3) x u2 rmsLm x Figure 14. Streamwise evolution of the product of powers of the integral scale times powers of velocity ﬂuctuations: solid line, u2 rmsL2; dashed line, u2 rmsL3; and dot-dashed line, u2 rmsL5. Saffman turbulence requires the constancy of u2 rmsL3 = const., not observed here. In the fully developed region of grid turbulence, as well as in the case investigated here, the advection of turbulent kinetic energy is almost perfectly balanced by dissipation: In the fully developed region of grid turbulence, as well as in the case investigated here, the advection of turbulent kinetic energy is almost perfectly balanced by dissipation: ⟨ϵ⟩∼x−(nk+1) (3.13) (3.13) (see § 3.4). 3.8. Spectral scaling and energy transfer One-dimensional spectra Eii(κj) are obtained from the discrete Fourier transform of the two-point velocity correlation functions along ej, where κj is the jth component of the wavenumber vector. Only j = 2 and j = 3 are used here because the streamwise direction is not homogeneous. Depending upon the pairing between velocity and wavenumber components, three distinct spectra can be calculated: streamwise spectrum Es = E11(κj), longitudinal spectrum Eg = Ejj(κj) and transverse spectrum Et = Eii(κj), where i = 2, j = 3 or i = 3, j = 2. https://doi.org/10.1017/jfm.2021.420 Published While, in general, spectra at different stages of the decay could be expected to scale with different reference quantities, it is observed here that, as factors ηu2 η, λu2 rms and Lu2 rms evolve at very similar rates in the streamwise direction (see table 3), any of those can be used equivalently. Spectra scaled by λu2 rms and evaluated at increasing distance along the x-axis are displayed in ﬁgure 15. While, in general, spectra at different stages of the decay could be expected to scale with different reference quantities, it is observed here that, as factors ηu2 η, λu2 rms and Lu2 rms evolve at very similar rates in the streamwise direction (see table 3), any of those can be used equivalently. Spectra scaled by λu2 rms and evaluated at increasing distance along the x-axis are displayed in ﬁgure 15. Figure 16 compares the three types of spectra (Es, Eg and Et) at a coordinate x = 20. For κ2η > 0.2, the streamwise and transverse spectra almost coincide, which is conﬁrmation 920 A9-18 Turbulent ﬂow past a metal foam 100 10–5 10–10 10–15 10–2 10–1 100 100 10–5 10–2 100 102 101 102 κ2λ Es(κ2)/(u2 rmsλ) Figure 15. Streamwise one-dimensional spectra normalised in Taylor variables at increasing distance from the metal foam, x = 10, 15, 20, 25 and 30. The inset displays the power-law scaling exhibited by the spectrum at x = 2, as indicated by the dashed line with slope −5/3. Turbulent ﬂow past a metal foam 100 10–5 10–10 10–15 10–2 10–1 100 100 10–5 10–2 100 102 101 102 κ2λ Es(κ2)/(u2 rmsλ) Turbulent ﬂow past a metal foam Figure 15. Streamwise one-dimensional spectra normalised in Taylor variables at increasing distance from the metal foam, x = 10, 15, 20, 25 and 30. 3.8. Spectral scaling and energy transfer The inset displays the power-law scaling exhibited by the spectrum at x = 2, as indicated by the dashed line with slope −5/3. 105 100 10–5 10–10 10–3 10–2 10–1 100 101 κ2η Eii(κ2)/(ϵν5)1/4 Figure 16. Comparison of streamwise, longitudinal and transverse spectra with Kolmogorov scaling at x = 20 (Reλ = 81): solid line, Es; dot-dashed line, Eg; dotted line, Et; and dashed line, (κ2η)−5/3. Symbols are longitudinal spectra of grid turbulence experiments from Comte-Bellot & Corrsin (1971): ×, M∗= 50.8 mm (x∗/M∗= 98, Reλ = 65); and +, M∗= 25.4 mm (x∗/M∗= 120, Reλ = 41). Asterisk ∗denotes quantities expressed in dimensional form. 105 100 10–5 10–10 10–3 10–2 10–1 100 101 κ2η Eii(κ2)/(ϵν5)1/4 Figure 16. Comparison of streamwise, longitudinal and transverse spectra with Kolmogorov scaling at x = 20 (Reλ = 81): solid line, Es; dot-dashed line, Eg; dotted line, Et; and dashed line, (κ2η)−5/3. Symbols are longitudinal spectra of grid turbulence experiments from Comte-Bellot & Corrsin (1971): ×, M∗= 50.8 mm (x∗/M∗= 98, Reλ = 65); and +, M∗= 25.4 mm (x∗/M∗= 120, Reλ = 41). Asterisk ∗denotes quantities expressed in dimensional form. that anisotropy is at large scales only; see Mohamed & Larue (1990) and Corsini & Stalio (2020). Only a narrow range in the wavenumber space (0.025 < κ2η < 0.1) is noticed where Es exhibits a −5/3 behaviour. Figure 16 includes the longitudinal spectra E11(κ1) of Comte-Bellot & Corrsin (1971) for two regular grids of different mesh size and Reλ values in the same range as the present case (Reλ = 65 and 41, compared to the present Reλ = 81). Close agreement is observed between measurements in turbulence generated by classical grids and turbulence simulated in the high-porosity metal foam for κ2η > 0.1. The present results match canonical grid turbulence spectra for κ2η > 0.1, the −5/3 law is not observed for wavenumbers κ2η > 0.1, and local isotropy is observed for κ2η > 0.2. That is the range of scales where dissipation becomes non-negligible. The distinction that anisotropy is at large scales only; see Mohamed & Larue (1990) and Corsini & Stalio (2020). Only a narrow range in the wavenumber space (0.025 < κ2η < 0.1) is noticed where Es exhibits a −5/3 behaviour. 3.8. Spectral scaling and energy transfer Figure 16 includes the longitudinal spectra E11(κ1) of Comte-Bellot & Corrsin (1971) for two regular grids of different mesh size and Reλ values in the same range as the present case (Reλ = 65 and 41, compared to the present Reλ = 81). Close agreement is observed between measurements in turbulence generated by classical grids and turbulence simulated in the high-porosity metal foam for κ2η > 0.1. The present results match canonical grid turbulence spectra for κ2η > 0.1, the −5/3 law is not observed for wavenumbers κ2η > 0.1, and local isotropy is observed for κ2η > 0.2. That is the range of scales where dissipation becomes non-negligible. The distinction The present results match canonical grid turbulence spectra for κ2η > 0.1, the −5/3 law is not observed for wavenumbers κ2η > 0.1, and local isotropy is observed for κ2η > 0.2. That is the range of scales where dissipation becomes non-negligible. The distinction R. Corsini, A. Fregni, M. Spinolo and E. Stalio 1.0 0.8 0.6 0.4 0.2 10–3 10–2 10–1 100 103 100 101 50 25 101 0 κη ℓ/η k(κ,∞)/k, ϵ(0,κ)/ϵ Figure 17. Normalised complement of the cumulative kinetic energy and normalised cumulative dissipation against wavenumber κη and wavelength ℓ/η at x = 20: solid line, k(κ,∞)/k; and dashed line, ⟨ϵ⟩(0,κ)/⟨ϵ⟩. 1.0 0.8 0.6 0.4 0.2 10–3 10–2 10–1 100 103 100 101 50 25 101 0 κη ℓ/η k(κ,∞)/k, ϵ(0,κ)/ϵ 17. Normalised complement of the cumulative kinetic energy and normalised cumulative diss nst wavenumber κη and wavelength ℓ/η at x 20: solid line k /k; and dashed line ⟨ϵ⟩ Figure 17. Normalised complement of the cumulative kinetic energy and normalised cumulative dissipation against wavenumber κη and wavelength ℓ/η at x = 20: solid line, k(κ,∞)/k; and dashed line, ⟨ϵ⟩(0,κ)/⟨ϵ⟩. between scales containing the bulk of the energy from those responsible for dissipation is done by considering the energy spectrum E(κ) and the dissipative spectrum D(κ) = 2νκ2E(κ). The turbulence spectrum E(κ) is obtained by between scales containing the bulk of the energy from those responsible for dissipation is done by considering the energy spectrum E(κ) and the dissipative spectrum D(κ) = 2νκ2E(κ). ( ) The turbulence spectrum E(κ) is obtained by The turbulence spectrum E(κ) is obtained by E(κ) = −κ d dκ 1 2Eii(κ), (3.15) (3.15) where the summation convention is applied. 3.8. Spectral scaling and energy transfer The spectrum in (3.15) removes the directional information from both the velocities and the Fourier modes, as E(κ) is given as a function of the wavenumber magnitude κ = \|κ\|. g The kinetic energy cumulated at wavenumbers lower than κ is k(0,κ) = κ 0 E(κ′) dκ′; (3.16) (3.16) the complement to k(0,κ) is indicated by k(κ,∞). The corresponding quantities for the dissipation are obtained in the same fashion using D(κ) and are indicated by ⟨ϵ⟩(0,κ) and ⟨ϵ⟩(κ,∞). In ﬁgure 17 the fraction of cumulative turbulent kinetic energy at wavenumbers higher than κ and the fraction of cumulative dissipation at wavenumbers lower than κ at a distance from the foam x = 20 are depicted as functions of κη and of the corresponding wavelength ℓ/η = 2π/κη. The peak of the energy spectrum occurs at κη ≈0.02 (see ﬁgure 16) but it may be observed that the main fraction of the kinetic energy (k(κ,∞) = 0.1k) is contained in the range of wavenumbers up to κη ≈0.25, one decade further. The peak of dissipation is roughly at κη ≈0.25 (which corresponds to the maximum derivative in ⟨ϵ⟩(0,κ)/⟨ϵ⟩). The contribution to dissipation reaches ⟨ϵ⟩(0,κ) = 0.9⟨ϵ⟩at κη ≈0.7. The bulk of turbulent kinetic energy is contained in motions of length scales ℓ> 25η ≈1 2L; this range could be viewed as the energy-containing range. On the other hand, the dissipation is effective at the length scales 10η < ℓ< 50η. The overlap between k(κ,∞) and ⟨ϵ⟩(0,κ) reveals that energy starts to be dissipated at a length scale where the energy content is non-negligible. 920 A9-20 Turbulent ﬂow past a metal foam 100 10–5 10–10 10–15 10–20 10–6 10–4 ⟨(δug)p⟩ ⟨\|δug\|3⟩ Figure 18. Log–log plot of second-, sixth- and tenth-order longitudinal structure functions versus ⟨\|δug\|3⟩at the position x = 25: , p = 2; □, p = 6; and ⋄, p = 10. The straight lines superimposed onto the curves are the power laws resulting from the ﬁtting procedure in the extended inertial range indicated by the dashed vertical lines. This range starts at 3η and ends at 30η. Turbulent ﬂow past a metal foam 100 10–5 10–10 10–15 10–20 10–6 10–4 ⟨(δug)p⟩ ⟨\|δug\|3⟩ ⟨\|δug\|3⟩ Figure 18. Log–log plot of second-, sixth- and tenth-order longitudinal structure functions versus ⟨\|δug\|3⟩at the position x = 25: , p = 2; □, p = 6; and ⋄, p = 10. 3.8. Spectral scaling and energy transfer The straight lines superimposed onto the curves are the power laws resulting from the ﬁtting procedure in the extended inertial range indicated by the dashed vertical lines. This range starts at 3η and ends at 30η. 3.9. Structure functions 3.9. Structure functions In this section, the local structure of turbulence is investigated at x = 25 by analysing the scaling properties of the structure functions with separation r. The general deﬁnition is given by In this section, the local structure of turbulence is investigated at x = 25 by analysing the scaling properties of the structure functions with separation r. The general deﬁnition is given by ⟨[δui,j(x, r)]p⟩= ⟨[ui(x + ejr, t) −ui(x, t)]p⟩. (3.17) (3.17) Because of turbulence decay along x, two different structure functions are identiﬁed in this work: longitudinal structure functions ⟨(δug)p⟩= ⟨(δuj,j)p⟩and transverse structure functions ⟨(δut)p⟩= ⟨(δui,j)p⟩, where i = 2, j = 3 or i = 3, j = 2. According to the ﬁrst, original theory by Kolmogorov (1941), for high Reynolds numbers when the separation lies in the inertial subrange η ≪r ≪L, the moments of velocity difference ⟨(δug)p⟩take a universal form that depends only on ⟨ϵ⟩through the following scaling property: ⟨(δug)p⟩∼(⟨ϵ⟩r)ζ p g with ζ p g = p/3. (3.18) (3.18) In the reﬁned similarity theory (Kolmogorov 1962), the intermittency effects are taken into account by rewriting expression (3.18) in terms of ϵr, the dissipation averaged over a volume of linear dimension r, and assuming that ϵr has a log-normal distribution, In the reﬁned similarity theory (Kolmogorov 1962), the intermittency effects are taken into account by rewriting expression (3.18) in terms of ϵr, the dissipation averaged over a volume of linear dimension r, and assuming that ϵr has a log-normal distribution, ⟨(δug)p⟩∼⟨ϵp/3 r ⟩rp/3 ∼rζ p g with ζ p g = 1 3p −1 18μp( p −3), (3.19) (3.19) https://doi.org/10.1017/jfm.2021.420 Published on where μ is the exponent of the dissipation autocorrelation function, where μ is the exponent of the dissipation autocorrelation function, ⟨ϵ(x + r)ϵ(x)⟩∼ L r μ , (3.20) (3.20) again for inertial range separations (Monin & Yaglom 1975). Given the Reynolds number Reλ ≈80 of the present case, the study is conducted using the extended self-similarity (ESS) observation by Benzi et al. (1993). The pth moments 920 A9-21 R. Corsini, A. Fregni, M. Spinolo and E. 3.9. Structure functions Stalio Present work Camussi Zhou Boratav Iyer Reλ ≈80 Reλ ≈37 Reλ = 75 Reλ ∼100 Reλ = 1300 Direct ESS ESS ESS ESS Direct p = 2 0.722 ± 0.027 0.684 ± 0.005 0.68 ± 0.006 0.70 0.6931 0.72 ± 0.002 p = 4 1.343 ± 0.052 1.296 ± 0.008 1.29 ± 0.008 1.32 1.2818 1.30 ± 0.005 p = 6 1.852 ± 0.076 1.829 ± 0.032 1.81 ± 0.029 1.86 1.7804 1.78 ± 0.010 p = 8 2.241 ± 0.128 2.303 ± 0.068 — 2.34 2.2123 2.18 ± 0.021 p = 10 2.507 ± 0.272 2.748 ± 0.126 — — 2.6000 — p = 12 2.679 ± 0.509 3.187 ± 0.208 — — 2.9614 — Table 4. Scaling exponents for the longitudinal structure functions of even orders up to p = 12 calculated using the direct and the ESS methods. Comparison of the present work with the results from Camussi et al. (1996), Zhou & Antonia (2000), Boratav & Pelz (1997) and Iyer et al. (2017). Table 4. Scaling exponents for the longitudinal structure functions of even orders up to p = 12 calculated using the direct and the ESS methods. Comparison of the present work with the results from Camussi et al. (1996), Zhou & Antonia (2000), Boratav & Pelz (1997) and Iyer et al. (2017). of velocity differences ⟨(δug)p⟩calculated at moderate Reynolds number are characterised by the same scaling exponents as in the high-Reynolds-number case when ⟨(δug)p⟩are treated as functions of ⟨\|δug\|3⟩. The resulting exponents are indicated by ξp g = ζ p g /ζ 3 g to discriminate them from those evaluated directly, ζ p g . The quality of the ESS scaling is displayed in ﬁgure 18 and in table 4. Errors are calculated by summing the uncertainty obtained from the estimate of scaling in the two spatial directions y and z and by modifying the scaling range over which ξp g is evaluated in the interval [3, 30]η. Data from the literature reported for comparison in table 4 are at Reλ comparable to the present, i.e. 3.9. Structure functions In the same plot there are the results from different intermittency models assuming μ = 0.08: ◦, ξp g ; ∗, ξp t ; solid line, KM41 model; dashed line, KM62 model; dot-dashed line, β model; and dotted line, She–Leveque model. (b) Behaviours of the intermittency exponent along x computed according to two different methods: solid line, based on ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 ∼(L/r)μ; and dotted line, μ = 2 −ξ6 g . Turbulent ﬂow past a metal foam 0.4 0.3 0.2 0.1 0 10 20 30 x μ (b) 8 6 4 2 0 5 10 15 20 p ξp g, ξp t (a) Figure 19. (a) Scaling exponents ξp g and ξp t computed with the ESS method versus p for orders up to 20 (x = 25). In the same plot there are the results from different intermittency models assuming μ = 0.08: ◦, ξp g ; ∗, ξp t ; solid line, KM41 model; dashed line, KM62 model; dot-dashed line, β model; and dotted line, She–Leveque model. (b) Behaviours of the intermittency exponent along x computed according to two different methods: solid line, based on ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 ∼(L/r)μ; and dotted line, μ = 2 −ξ6 g . Figure 19. (a) Scaling exponents ξp g and ξp t computed with the ESS method versus p for orders up to 20 (x = 25). In the same plot there are the results from different intermittency models assuming μ = 0.08: ◦, ξp g ; ∗, ξp t ; solid line, KM41 model; dashed line, KM62 model; dot-dashed line, β model; and dotted line, She–Leveque model. (b) Behaviours of the intermittency exponent along x computed according to two different methods: solid line, based on ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 ∼(L/r)μ; and dotted line, μ = 2 −ξ6 g . ( ) p y ξp g ; ∗, ξp t ; solid line, KM41 model; dashed line, KM62 model; dot-dashed line, β model; She–Leveque model. (b) Behaviours of the intermittency exponent along x computed according methods: solid line, based on ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 ∼(L/r)μ; and dotted line, μ = 2 −ξ6 g . 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 100 101 r/η r˜/η ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 Figure 20. Dissipation correlation coefﬁcient at x = 25: solid line, ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 as calculated from its deﬁnition; and dot-dashed line, ad(r/η)d, where ad = 1.07 and d = −0.082. 3.9. Structure functions of order ∼102, except for those by Iyer, Sreenivasan & Yeung (2017), which are obtained at Reλ ≈1300 and a high degree of isotropy.p p Figure 19(a) shows scaling exponents ξp g and ξp t computed using the ESS method, compared to the original predictions by Kolmogorov (1941, KM41) extended to p > 3, the reﬁned similarity hypotheses of Kolmogorov (1962, KM62), the intermittency β model by Frisch, Sulem & Nelkin (1978) and the model by She & Leveque (1994). The transverse scaling exponents ξp t in ﬁgure 19 are obtained by making ⟨(δut)p⟩depend on the magnitude of transverse velocity differences for p = 3 rather than the longitudinal, thus following the ESS method (Camussi et al. 1996; Grossmann, Lohse & Reeh 1997). The reﬁned similarity hypotheses are found to hold, but there is a visible tendency of ξp t to deviate from the longitudinal scaling for p ⩾4. This is to be ascribed to the Reynolds number and the residual anisotropy of the ﬂow, as argued by Iyer et al. (2017). Figure 19(b) displays the evolution of the intermittency exponent μ along the streamwise direction; the quantity deﬁned in (3.20) is also employed in the β-model by Frisch et al. (1978). The μ exponent is calculated directly from the slope of the correlation coefﬁcient obtained by dividing the autocorrelation of dissipation by ⟨ϵ⟩2 for separations in the inertial range; see ﬁgure 20. A second possible way to obtain μ is by using the expression relating the longitudinal sixth-order moment to the autocorrelation of dissipation (Frisch et al. 1978): ⟨(δug)6⟩ r2 ∼⟨ϵ(x + r)ϵ(x)⟩. (3.21) (3.21) The two quantities are reported in ﬁgure 19(b). As the reference value reported in many studies for the intermittency exponent is μ = 0.25 ± 0.05, which is deemed to be valid for high-Reynolds-number ﬂows (Pope 2000), the values calculated here suggest 920 A9-22 Turbulent ﬂow past a metal foam 8 0.4 0.3 0.2 0.1 0 10 20 30 6 4 2 0 5 10 15 20 p x μ ξp g, ξp t (a) (b) Figure 19. (a) Scaling exponents ξp g and ξp t computed with the ESS method versus p for orders up to 20 (x = 25). 3.9. Structure functions The dashed lines delimit the interval used for the power-law ﬁtting procedure, 25η < r < 75η, and the dotted lines demarcate the decorrelation scale ˜r. 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 100 101 r/η r˜/η ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press Figure 20. Dissipation correlation coefﬁcient at x = 25: solid line, ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 as calculated from its deﬁnition; and dot-dashed line, ad(r/η)d, where ad = 1.07 and d = −0.082. The dashed lines delimit the interval used for the power-law ﬁtting procedure, 25η < r < 75η, and the dotted lines demarcate the decorrelation scale ˜r. that the effect generated by intermittent structures on small-scale turbulence in the moderate-Reynolds-number range is less intense than at high Reynolds numbers. It is noticed that the method based on sixth-order structure functions, (3.21), displays a decay in the x-direction that is not expected nor recovered in the calculation of μ directly from the deﬁnition (3.20). https://doi.org/10.1017/jfm.2021.420 Publishe The decorrelation scale ˜r is deﬁned as the length of decorrelation of the instantaneous dissipation ϵ, i.e. the separation scale r where ⟨ϵ(x + r)ϵ(x)⟩/⟨ϵ⟩2 becomes unitary with a 1 % error. In ﬁgure 21 the development of ˜r along x is compared to that of the integral scale L. The decorrelation scale can be considered as a very large length scale, which 920 A9-23 R. Corsini, A. Fregni, M. Spinolo and E. Stalio 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 5 10 15 20 x 25 30 35 r˜, L Figure 21. Streamwise evolution of the decorrelation scale in comparison with the integral scale: solid line, ˜r; and dashed line, L. For x = 25, ˜r/η = 135 and L/η = 45. 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 5 10 15 20 x 25 30 35 r˜, L Figure 21. Streamwise evolution of the decorrelation scale in comparison with the integral scale: solid line, ˜r; and dashed line, L. For x = 25, ˜r/η = 135 and L/η = 45. in homogeneous isotropic turbulence depends on the Reynolds number and also the intermittency characteristics of the ﬂow. in homogeneous isotropic turbulence depends on the Reynolds number and also the intermittency characteristics of the ﬂow. 3.10. Turbulent energy budgets The equation governing the transport of ⟨k⟩in a statistically steady case can be written in symbols as −A = Tp + Tt + Dv + P −⟨˜ϵ⟩, (3.22) (3.22) where A is the contribution by mean advection and Tp, Tt and Dv represent pressure, turbulent and diffusive transports; P and ⟨˜ϵ⟩stand for the production and pseudo-dissipation rate of turbulent kinetic energy, deﬁned as where A is the contribution by mean advection and Tp, Tt and Dv represent pressure, turbulent and diffusive transports; P and ⟨˜ϵ⟩stand for the production and pseudo-dissipation rate of turbulent kinetic energy, deﬁned as ⟨˜ϵ⟩= 1 Redp ∂ui ∂xj ∂ui ∂xj . (3.23) (3.23) As the difference ⟨ϵ⟩−⟨˜ϵ⟩is seldom important (Pope 2000), in this section ⟨ϵ⟩ As the difference ⟨ϵ⟩ ⟨ϵ⟩is seldom important (Pope 2000), in this section ⟨ϵ⟩is used. In grid turbulence cases, where mean velocity gradients are zero and cross-ﬂow directions y and z are homogeneous, the production term vanishes: P = −⟨uiuj⟩∂⟨Ui⟩ ∂xj = 0. (3.24) (3.24) In addition, for the Reynolds number computed here, the molecular contribution to transport is negligible. The resulting expression for the budget equation (3.22) thus becomes In addition, for the Reynolds number computed here, the molecular contribution to transport is negligible. The resulting expression for the budget equation (3.22) thus becomes ⟨U⟩d⟨k⟩ dx −A = −d⟨up⟩ dx Tp −d⟨uk⟩ dx Tt −⟨˜ϵ⟩, (3.25) (3.25) where the single terms are distributed along the x-axis as depicted in ﬁgure 22. T i b id iﬁd i ﬁ 22 ﬁld i h where the single terms are distributed along the x-axis as depicted in ﬁgure 22. Two regions can be identiﬁed in ﬁgure 22: a near-ﬁeld region where the three conservative terms redistribute the kinetic energy along x, and a far-ﬁeld region where 920 A9-24 Turbulent ﬂow past a metal foam 0.4 0.2 –0.2 10–2 10–1 10–2 100 100 101 102 103 100 102 0 0.4 0.3 0.2 0.1 –0.1 0 2 (×10–3) 1 0 –1 –2 10 20 30 x x x∗/df (a) (b) A, Tp, Tt, –⟨ϵ˜⟩ ⟨U⟩⟨k⟩, ⟨up⟩, ⟨uk⟩ Figure 22. Turbulent kinetic energy along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, A; dot-dashed line, Tp; dotted line, Tt; and dashed line, −⟨˜ϵ⟩. Panel (b) presents turbulent kinetic energy ﬂuxes: solid line, ⟨U⟩⟨k⟩; dot-dashed line, ⟨up⟩; and dotted line, ⟨uk⟩. 3.10. Turbulent energy budgets The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. 0.4 0.2 –0.2 10–2 10–1 100 101 102 103 100 102 0 2 (×10–3) 1 0 –1 –2 10 20 30 x x∗/df (a) A, Tp, Tt, –⟨ϵ˜⟩ 10–2 100 0.4 0.3 0.2 0.1 –0.1 0 x (b) ⟨U⟩⟨k⟩, ⟨up⟩, ⟨uk⟩ (a) x Figure 22. Turbulent kinetic energy along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, A; dot-dashed line, Tp; dotted line, Tt; and dashed line, −⟨˜ϵ⟩. Panel (b) presents turbulent kinetic energy ﬂuxes: solid line, ⟨U⟩⟨k⟩; dot-dashed line, ⟨up⟩; and dotted line, ⟨uk⟩. The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. pressure and turbulent transports become negligible. Thus dissipation is solely balanced by turbulent kinetic energy advection; see ﬁgure 22(a) and its inset. A possible downstream boundary for the near-ﬁeld region can be set at x = 2, where the turbulent and pressure transports become 100 times smaller than advection and dissipation. The near-ﬁeld region can be subdivided into two subregions. By considering the ligament diameter df as the length scale, a region very close to the metal foam is identiﬁed for x∗/df < 0.5, where turbulent transport is larger than the advective one. This region is characterised by the sustainment of turbulence by means of streamwise ﬂuctuations, which is counter-balanced by dissipation, as pressure and advective transports account for negligible shares. The second subregion extends for 0.5 < x∗/df < 14, where turbulent kinetic energy is provided through pressure and advective mechanisms while dissipation and turbulent transport drain ⟨k⟩; see ﬁgure 22(a). p g Data gathered by Norberg (1998) indicate that the vortex formation length ℓf for the present Reynolds number based on the mean diameter of the ﬁlaments, Redf = 560, lies in the range 1.5df < ℓf < 2df ; see Bloor, Gerrard & Lighthill (1966) for other possible deﬁnitions of the vortex formation length. Figure 22(a) displays that this range matches the streamwise location where all the transport terms in (3.25) peak. Therefore, the present data suggest that kinetic energy is originated in the second part of the near-ﬁeld region, where transport mechanisms show local peaks. 3.10. Turbulent energy budgets Turbulent kinetic energy ⟨k⟩is drained from here by the turbulent transport, which transfers turbulent ﬂuctuations upstream, as indicated by the negative sign of the turbulent ﬂux (see ﬁgure 22b), feeding the region very close to the metal foam. On the other hand, pressure and advective mechanisms are found to provide turbulent kinetic energy in the entire region 0.5 < x∗/df < 14, and the signs of the relative ﬂuxes indicate that velocity ﬂuctuations are transferred downstream. At the coordinate x∗/df = 14 (x = 2), ﬂuxes ⟨up⟩and ⟨uk⟩become constant in the streamwise direction, leading to the budget reported in (3.4) for x > 2, which is typical in developed decaying ﬂows; see ﬁgure 22(a) and its inset, which reports the budget terms in the Id region. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge Univ https://doi.org/10.1017/jfm.2021.420 Published onlin g In order to separately assess the behaviour of streamwise and cross-ﬂow velocity ﬂuctuations, the transport equations for velocity variances are analysed. The budget of 920 A9-25 R. Corsini, A. Fregni, M. Spinolo and E. Stalio streamwise velocity variance reads streamwise velocity variance reads ⟨U⟩d⟨u2⟩ dx −Au = −d⟨u3⟩ dx T u t −2d⟨up⟩ dx T u p + 2 p ∂u ∂x Su −⟨ ϵu⟩, (3.26) (3.26) where, from left to right, the ﬁrst three terms, respectively, represent the advective, turbulent and pressure transports of ⟨u2⟩, ⟨p∂u/∂x⟩is the pressure strain term and ϵu stands for the u-variance pseudo-dissipation rate, ϵu = 2((∂u/∂xj) (∂u/∂xj))/Redp. The diffusive transport is again neglected with respect to the other terms. Equations similar to (3.26) can be derived for the transverse velocity components, v and w. As the ﬂow is isotropic on y–z planes, statistics for the transverse velocity component are obtained by averaging results along y and z. For conciseness, the transverse velocity component is indicated by v and its direction by y, ⟨U⟩d⟨v2⟩ dx −Av = −d⟨uv2⟩ dx T v t + 2 p ∂v ∂y Sv −⟨ ϵv⟩, (3.27) (3.27) Sv where the terms have the same interpretation as in (3.26), and the pseudo-dissipation rate of the v-variance is computed as ϵv = 2((∂v/∂xj) (∂v/∂xj))/Redp. Note that, with respect to equation (3.25), budgets of velocity component variances include the pressure strain terms. 3.10. Turbulent energy budgets Another difference with respect to the ⟨u2⟩terms is the positive – yet not that intense – turbulent ﬂux very close 920 A9-26 Turbulent ﬂow past a metal foam 0.4 0.6 0.2 –0.2 –0.4 0 (a) 0.4 0.5 0.3 0.2 0.1 –0.1 –0.2 0 (b) 10–2 10–1 10–2 100 100 101 102 103 100 102 x x x∗/df 2 (×10–3) 1 0 –1 –2 10 20 30 Au, Tt u, Tp u, Su, –⟨ϵu⟩ ⟨U⟩⟨u2⟩, ⟨up⟩, ⟨u3⟩ Figure 23. Variance of streamwise velocity component along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, Au; dotted line, T u t ; dot-dashed line, T u p ; solid line with circles, Su; and dashed line, −⟨ ϵu⟩. Panel (b) presents ⟨u2⟩ﬂuxes: solid line, ⟨U⟩⟨u2⟩; dot-dashed line, ⟨up⟩; and dotted line, ⟨u3⟩. The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. 0.4 0.5 0.3 0.2 0.1 –0.1 –0.2 0 (b) 10–2 100 x ⟨U⟩⟨u2⟩, ⟨up⟩, ⟨u3⟩ 0.4 0.6 0.2 –0.2 –0.4 0 (a) 10–2 10–1 100 101 102 103 100 102 x x∗/df 2 (×10–3) 1 0 –1 –2 10 20 30 Au, Tt u, Tp u, Su, –⟨ϵu⟩ x Figure 23. Variance of streamwise velocity component along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, Au; dotted line, T u t ; dot-dashed line, T u p ; solid line with circles, Su; and dashed line, −⟨ ϵu⟩. Panel (b) presents ⟨u2⟩ﬂuxes: solid line, ⟨U⟩⟨u2⟩; dot-dashed line, ⟨up⟩; and dotted line, ⟨u3⟩. The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. 0.1 –0.1 –0.2 –0.3 0 10–1 100 101 102 103 x∗/df 10–2 100 102 x 10–2 100 x 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 –0.02 (b) (a) (×10–3) 1 0 –1 –2 10 20 30 Av, Tt v, Sv, –⟨ϵv⟩ ⟨U⟩⟨v2⟩, ⟨uv2⟩ Figure 24. Variance of cross-ﬂow velocity component along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, Av; dotted line, T v t ; solid line with circles, Sv; and dashed line, −⟨ ϵv⟩. Panel (b) presents ⟨v2⟩ﬂuxes: solid line, ⟨U⟩⟨v2⟩; and dotted line, ⟨uv2⟩. The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. 3.10. Turbulent energy budgets 10–2 100 x 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0 –0.02 (b) ⟨U⟩⟨v2⟩, ⟨uv2⟩ 0.1 –0.1 –0.2 –0.3 0 10–1 100 101 102 103 x∗/df 10–2 100 102 x (a) (×10–3) 1 0 –1 –2 10 20 30 Av, Tt v, Sv, –⟨ϵv⟩ x Figure 24. Variance of cross-ﬂow velocity component along the streamwise direction in logarithmic plots. In panel (a) budget terms are reported: solid line, Av; dotted line, T v t ; solid line with circles, Sv; and dashed line, −⟨ ϵv⟩. Panel (b) presents ⟨v2⟩ﬂuxes: solid line, ⟨U⟩⟨v2⟩; and dotted line, ⟨uv2⟩. The inset in (a) displays an enlargement of the budget terms in the range Id in linear scale. to the metal foam, indicating that cross-ﬂow ﬂuctuations are more correlated with positive u-ﬂuctuations in this region (see ﬁgure 24b). 3.10. Turbulent energy budgets As will be shown in the following, the role of these terms is to redistribute kinetic energy among the velocity components and thus it is responsible for the ‘return to isotropy’ in homogeneous turbulence. Such a phenomenon has been studied for several decades as it is involved in second-order turbulence models; see, for example, the works by Rotta (1951) and Lumley & Newman (1977). Pressure strain terms are not included in (3.25) as their sum Su + 2Sv vanishes because of incompressibility. p y Figures 23 and 24 show the velocity variance budgets and ﬂuxes along the streamwise coordinate. It appears that transport mechanisms are more intense in the ⟨u2⟩budget with respect to ⟨v2⟩, while dissipations ⟨ ϵu⟩and ⟨ ϵv⟩are almost equal. The behaviour of the pressure strain terms reveals that turbulent energy is drained from the streamwise velocity ﬂuctuations, as Su represents a sink term in the ⟨u2⟩budget, and provided to cross-ﬂow velocity ﬂuctuations, where Sv acts as a source; see ﬁgures 23(a) and 24(a). The role of Su and Sv does not change along the whole streamwise extension of the domain, as shown by the insets in ﬁgures 23(a) and 24(a). This occurs because, as reported in § 3.2, ﬂow anisotropy is conserved in the ﬂow domain considered. In the decaying region Id, the pressure strain terms maintain an intensity comparable to the advective and dissipation terms, suggesting that the isotropic condition would have been reached in a longer computational domain. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press The proﬁles in ﬁgures 23 and 24 show that the u-variance terms behave very similarly to the budgets and ﬂuxes of turbulent kinetic energy reported in ﬁgure 22. In particular, the peaks of turbulent, advective and pressure transports are located at the same distance from the metal foam, comparable to the vortex formation length. This suggests that velocity ﬂuctuations are more intensely triggered along the streamwise direction with respect to the transverse ones. On the other hand, the v-variance terms peak slightly downstream with respect to terms in the ⟨u2⟩and ⟨k⟩budgets, and the negative peak of turbulent transport is not very intense (see ﬁgure 24a), but it drains enough energy to sustain cross-ﬂow ﬂuctuations in the region just downstream of the metal foam. 4. Conclusions An analysis is presented of the turbulent ﬂow behind a synthetic metal foam layer of thickness equal to ﬁve times the mean pore diameter. Unlike classical grid turbulence geometries, the metal foam ligaments are variably oriented, unevenly spaced and in general less ordered. Similar to classical grids, metal foams are mainly characterised by two length scales i.e. the pore diameter and the ligament thickness. The analysis encompasses one single Reynolds number Redp = 4000 and ε = 0.92 but the effect of a different porosity (ε = 0.97) on selected quantities is addressed in the Appendix. https://doi.org/10.1017/jfm.2021.420 Published on The analysis of the turbulent kinetic energy budget suggests that turbulence is triggered in the region close to the metal foam, approximately at a distance equal to the vortex formation length indicated by Norberg (1998), x∗/df ≈2. In the near-ﬁeld region, x < 2, 920 A9-27 920 A9-27 920 A9-27 R. Corsini, A. Fregni, M. Spinolo and E. Stalio turbulent kinetic energy is distributed by transport mechanisms; the turbulent transport moves ⟨k⟩upstream, sustaining ﬂuctuations in close proximity to the metal foam, while pressure and advective transports provide velocity ﬂuctuations to larger x-coordinates. At x = 2, pressure and turbulent transports are found to be negligible with respect to the other terms, entailing for x > 2 the expected behaviour of grid turbulence, where viscous dissipation is balanced by the mean advection of ⟨k⟩. Budgets of streamwise and cross-ﬂow velocity variances indicate that ﬂuctuations along x are the most intensely triggered and pressure strain terms act to redistribute turbulent energy towards the isotropic condition, which however is not observed due to the limited domain extension. In that same near-ﬁeld region, Reλ decreases steeply and the dissipation rate coefﬁcient Cϵ approximates Cϵ ∝Re−1 λ while L/λ remains almost constant. These observations, in conjunction with the typical −5/3 slope in the turbulent power spectra, represent a typical behaviour already observed in the literature (Valente & Vassilicos 2012). Given that this occurs very close to the porous matrix, where the hypotheses for the Richardson cascade are not veriﬁed, a considerable variation in Cϵ is not interpreted here as an anomalous behaviour. The developed region is deﬁned here as the region where urms decays following a power law and starts at xmin = 7.98. 4. Conclusions Besides ⟨k⟩, a number of relevant quantities, like the integral length scale, the Taylor microscale and the Kolmogorov scale of the ﬂow, are observed to follow a power-law behaviour. The exponents obtained from a least-squares ﬁt are in many cases very close to values predicted and measured in classical grid turbulence experiments. Given nk = 1.14 and q = 0.52 (where nk and q are the exponents of the power laws for ⟨k⟩ and L), the constancy of u2 rmsL3 does not hold in the developed region simulated here, and thus the turbulence does not follow the theory by Saffman. y y Structure functions of different orders are calculated at a ﬁxed position x = 25 in the fully developed region. The extended self-similarity is used to calculate the scaling exponents. The results are interpreted in the light of the reﬁned similarity hypothesis. The intermittency exponent μ computed directly from its deﬁnition is seen to behave uniformly within the computational domain. It reveals that intermittency is not very intense at the moderate Reynolds number of this study. The decorrelation length of dissipation ˜r is larger than the integral scale. It depends on the Reynolds number as well as the intermittency characteristics of the ﬂow. Acknowledgements. The authors would like to thank S. Chibbaro and A. Cimarelli for fruitful discussions and suggestions. Funding. The authors acknowledge the CINECA award under the ISCRA initiative, for the availability of high-performance computing resources and support. Declaration of interests. The authors report no conﬂict of interest. Author ORCIDs. R. Corsini https://orcid.org/0000-0001-9342-0773; A. Fregni https://orcid.org/0000-0002-1810-3281; E. Stalio https://orcid.org/0000-0001-6983-4204. Appendix. A glance at the eﬀect of porosity The aim of this appendix is to verify the extent to which turbulent ﬂow statistics in the lee of an open-cell metal foam layer are affected by porosity. The results obtained for a foam of porosity ε = 0.97 are compared here against the ε = 0.92 case, as investigated in the main 920 A9-28 Turbulent ﬂow past a metal foam 100 10–1 10–2 10–3 10–2 10–1 100 101 102 x ⟨uiui⟩, ⟨k⟩ Figure 25. Streamwise evolution of velocity ﬂuctuations and turbulent kinetic energy for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, ⟨uu⟩; dot-dashed line, ⟨vv⟩; dotted line, ⟨ww⟩; and dashed line, ⟨k⟩. The lines with circles mark the case with higher porosity ε = 0.97. Turbulent ﬂow past a metal foam Turbulent ﬂow past a metal foam 100 10–1 10–2 10–3 10–2 10–1 100 101 102 x ⟨uiui⟩, ⟨k⟩ x Figure 25. Streamwise evolution of velocity ﬂuctuations and turbulent kinetic energy for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, ⟨uu⟩; dot-dashed line, ⟨vv⟩; dotted line, ⟨ww⟩; and dashed line, ⟨k⟩. The lines with circles mark the case with higher porosity ε = 0.97. body of the paper. The two foams have equal pore size but different ligament thickness, i.e. the foam with larger porosity is characterised by a thinner ligament, d′ f < df . The comparison is conducted by means of DNS performed with the same numerical parameters as outlined in § 2 except for the computational grid, which consists of nx = 2049 and ny = nz = 512 grid nodes. y The porous matrix of higher porosity generates a turbulent ﬁeld where ﬂuctuations are less intense in all the spatial directions. Application of the ﬁtting procedure outlined in § 3.3 on the high-porosity foam provides decay exponents for u2 rms and ⟨k⟩of n′ u = 1.05 and n′ k = 1.08, respectively. The results obtained on the coarse grid for the ε = 0.92 case yield nu = 1.12 and nk = 1.13; see table 2 for comparisons against results on the ﬁne mesh. The power-law decay of the turbulent kinetic energy begins more upstream than in the ε = 0.92 case; see ﬁgure 25. Appendix. A glance at the eﬀect of porosity Inside the developed region, the Kolmogorov and the longitudinal integral length scales evaluated for ε = 0.97 behave according to power laws with growing rates estimated by the exponents s′ = 0.54 and q′ = 0.47, respectively (see table 3 for notation). The lower-porosity case exhibits decay exponents along the x-direction of s = 0.56 and q = 0.51 (see table 3 for comparisons against results on the ﬁne mesh) but the Kolmogorov and the integral scales are different in magnitude, as displayed by ﬁgure 26(a) and (b). Associated with the smaller kinetic energy content of the ﬂow and a smaller dissipation rate observed at ε = 0.97, the turbulence induced by the higher-porosity foam is characterised by a larger Kolmogorov scale and a smaller integral scale. https://doi.org/10.1017/jfm.2021.420 Published online by Cambridge University Press Figure 27 displays the effects of porosity on the terms of the turbulent kinetic energy budget. The transport mechanisms of ⟨k⟩for ε = 0.97 are the same as described in § 3.10 for ε = 0.92 but are characterised by an upstream shift of the transport peaks with respect to that case. This shift is consistent with the reduction of the vortex formation length associated with a smaller ligament thickness, as indicated by Norberg (1998) and reported in § 3.10. In summary, while the results obtained for ε = 0.92 show small quantitative differences with respect to ε = 0.97, no signiﬁcant discrepancies are observed between the two cases. An upstream shift in the peaks of ⟨k⟩transports characterises the higher-porosity case, 920 A9-29 R. Corsini, A. Fregni, M. Spinolo and E. Stalio 0.02 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 30 0.01 0 10 20 30 x x L η (a) (b) Figure 26. Comparison between the streamwise distributions of the Kolmogorov scale (a) and the longitudinal integral scale (b) for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, length scales for ε = 0.92; and solid line with circles, length scales for ε = 0.97. 0.02 0.01 0 10 20 30 x η (a) 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 30 x L (b) Figure 26. Appendix. A glance at the eﬀect of porosity Comparison between the streamwise distributions of the Kolmogorov scale (a) and the longitudinal integral scale (b) for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, length scales for ε = 0.92; and solid line with circles, length scales for ε = 0.97. 0.5 0.4 0.3 0.2 0.1 –0.1 –0.2 –0.3 10–2 10–1 100 x 101 102 0 A, Tp, Tt, –⟨ϵ˜⟩ Figure 27. Comparison between the turbulent kinetic energy budget terms for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, A; dot-dashed line, Tp; dotted line, Tt; and dashed line, −⟨˜ϵ⟩. The lines with circles mark the case with higher porosity ε = 0.97. 0.5 0.4 0.3 0.2 0.1 –0.1 –0.2 –0.3 10–2 10–1 100 x 101 102 0 A, Tp, Tt, –⟨ϵ˜⟩ Figure 27. Comparison between the turbulent kinetic energy budget terms for the ε = 0.97 and ε = 0.92 cases on the coarse computational grid: solid line, A; dot-dashed line, Tp; dotted line, Tt; and dashed line, −⟨˜ϵ⟩. The lines with circles mark the case with higher porosity ε = 0.97. which in turn implies a reduced vortex formation length and the achievement of power-law decays at shorter distances from the porous matrix. The size of such displacement is apparently very small. Turbulent ﬂow past a metal foam BLOOR, M.S., GERRARD, J.H. & LIGHTHILL, M.J. 1966 Measurements on turbulent vortices in wake. Proc. R. Soc. Lond. A 294 (1438), 319–342. BOOMSMA, K., POULIKAKOS, D. & VENTIKOS, Y. 2003 Simulations of ﬂow through open cell metal foams using an idealized periodic cell structure. Intl J. Heat Fluid Flow 24, 825–834. 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https://openalex.org/W2029870425	https://europepmc.org/articles/pmc4396131?pdf=render	English	null	Experience sharing, emotional reciprocity, and turn-taking	Frontiers in psychology	2,015	cc-by	5,467	Keywords: turn-taking, emotion, experience sharing, emotional contagion, conversation analysis Keywords: turn-taking, emotion, experience sharing, emotional contagion, conversation analysis Keywords: turn-taking, emotion, experience sharing, emotional contagion, conversation analysis Reviewed by: Beatrice S. Reed, University of York, UK Alexa Hepburn, Loughborough University, UK Experience sharing, emotional reciprocity, and turn-taking Melisa Stevanovic* and Anssi Peräkylä In this perspective article, we consider the relationship between experience sharing and turn-taking. There is much evidence suggesting that human social interaction is perme- ated by two temporal organizations: (1) the sequential framework of turn-taking and (2) the concurrent framework of emotional reciprocity. From this perspective, we introduce two alternative hypotheses about how the relationship between experience sharing and turn-taking could be viewed. According to the first hypothesis, the home environment of experience sharing is in the concurrent framework of emotional reciprocity, while the motivation to share experiences is in tension with the sequential framework of turn-taking. According to the second hypothesis, people’s inclination to coordinate their actions in terms of turn-taking is motivated precisely by their propensity to share experiences. We consider theoretical and empirical ideas in favor of both of these hypotheses and discuss their implications for future research. Sequentiality and Concurrency in Human Social Interaction In recent years, there has been an increasing number of conversation analytic studies explicating the social organization of, and the highly ordered interactional tasks performed by, emotional expressions in social interaction (e.g., Peräkylä and Sorjonen, 2012). This perspective paper seeks to further this line of research by considering how expressions in the service of what we call experience sharing are embedded in the most primordial temporal organizations of interaction. Correspondence: Melisa Stevanovic, Finnish Centre of Excellence in Intersubjectivity in Interaction, Department of Social Research, University of Helsinki, P.O.Box 4, 00014 University of Helsinki, Finland melisa.stevanovic@helsinki.fi Many naturally occurring interactions call for individuals to coordinate their actions in terms of turn-taking. This happens especially in the context of language use: the principle of one participant talking at a time (Sacks et al., 1974) allows humans to communicate complex thoughts and intentions. In conversation, social actions (e.g., proposals, offers, and invitations) and their responses (e.g., acceptances and rejections) are organized in terms of successive turns at talk. As pointed out by Schegloff (1988, pp. 98–99), turn-taking enables humans to pursue stable trajectories of action and responsive action. This arrangement will be referred to as the sequential framework of turn- taking. It denotes not only the temporal but also the conditional relationship between participants’ interactional moves. Specialty section: This article was submitted to Language Sciences, a section of the journal Frontiers in Psychology Received: 19 November 2014 Accepted: 30 March 2015 Published: 14 April 2015 In addition to the sequential framework of turn-taking, human interactive conduct is permeated by another temporo-conditional arrangement—something that we call the concurrent framework of emotional reciprocity. The literature on “emotional contagion” suggests that humans have an automatic tendency to mimick other people’s non-verbal emotional expressions, which affects the emotional experience of the mimicking person (Hatfield et al., 1993, 1994; Dimberg, 2007). This happens as a result of afferent feedback generated by elementary motor mimicry, which produces a simultaneous emotional match independently of people’s cognitive abilities to understand what is PERSPECTIVE published: 14 April 2015 doi: 10.3389/fpsyg.2015.00450 PERSPECTIVE published: 14 April 2015 doi: 10.3389/fpsyg.2015.00450 Edited by: Edited by: Kobin H. Kendrick, Max Planck Institute for Psycholinguistics, Netherlands Citation: Stevanovic M and Peräkylä A (2015) Experience sharing, emotional reciprocity, and turn-taking. Front. Psychol. 6:450. doi: 10.3389/fpsyg.2015.00450 April 2015 \| Volume 6 \| Article 450 1 Frontiers in Psychology \| www.frontiersin.org Experience sharing, emotional reciprocity, and turn-taking Stevanovic and Peräkylä going on in the mind of the other (Carr et al., 2003; Leslie et al., 2004; Barresi and Moore, 2008; Decety and Meyer, 2008). From this perspective, two participants’ interactional moves are, by definition, connected by a causal relationship (one participant produces an expression first and the other acts in response to him/her), but the actions/expressions take place in a shared time. Overlap of expressions is regular. Regarding the opportunity for expression, the participants are positioned symmetrically, which is reflected in the frequent concurrency of the participants’ inter- actional moves. sharing of affective stances in the form of concurrent agreeing assessments. Their example involves two conversationalists prais- ing something a mutual friend has baked. One of them says: “Jeff made an asparagus pie, it was so good.” In overlap with the first speaker’s “so,” the co-interactant launches an assessment: “I love it.” Hence, it appears that, in the moments of experience sharing, the concurrent framework of emotional reciprocity colonizes the organization of spoken interaction, leading to the momentary relaxation of turn-taking rules. Also studies within the domain of mother-infant interaction have shown that positive affective expressions tend to coincide with simultaneous vocalizations (Stern et al., 1975; Beebe et al., 1979). There are different motives that lead humans to interact with each other. In addition to their instrumental communicative goals, humans are also motivated to share experiences about events and things in the environment with their significant others and to become “swept along” by them (Feinman, 1982; Striano and Rochat, 1999; Tomasello, 1999; Hobson and Hobson, 2008; Rochat et al., 2009). This motivation has even been regarded as the force that has driven the evolution of language (Locke, 1996, 2002; Dunbar, 1997). But how does experience sharing relate to the sequential framework of turn-taking, where language use reg- ularly takes place? Could it be that experience sharing is “at home” in the concurrent framework of emotional reciprocity, while being in tension with turn-taking? Or, do humans cast their experi- ence sharing into the system of turn-taking precisely because it supports experience sharing? Hypothesis I: Experience Sharing is in Tension with the Sequential Framework of Turn-Taking During the most part of the first speaker’s (B’s) telling (lines 1–6) both participants are looking down with straight faces (Frame 1). At the end of the telling, however, they establish mutual gaze and the teller (B) adopts a smiling face (Frame 2). After a gap (line 7), the recipient (A) reciprocates the smile and raises her brows (Frame 3), simultaneously producing an ani- mated verbal response of “ritualized disbelief” (Heritage, 1984, p. 339; line 8). While the interaction has thus far abided to the sequential framework of turn-taking, now, as soon as the mutual smile has been established, the participants’ facial conduct gets detached from the sequential organization of turn-taking as the concurrent framework of emotional reciprocity breaks through. During a moment of “heightened emotive involvement” (Selting, 1994), they maintain their smiles and mutual gaze over a num- ber of turns conveying ostensibly different actions (assessment, line 10; joking advice, line 11; Frame 4). The temporality of the mutual smile goes beyond the turn-taking organization. After this, the participants, however, break their mutual gaze (Frame 5), which is followed by their smiles becoming less intensive. One participant (A) adopts a straight face at the onset of her ques- tion (lines 13–14), while the other (B) does the same during her answer to the question (line 15; Frame 6). Thereby, they indicate that the heightened moment of experience sharing is over. While the primary modality of expression surpassing the Our first hypothesis suggests that the home environment of experience sharing is in the concurrent framework of emotional reciprocity, while there is a tension between experience sharing and the sequential framework of turn-taking. The first pieces of support for this hypothesis come from the developmental psychological research literature, which suggests there to be automatic resonance processes that allow humans, right from the outset, to bridge their own and others’ experiences. With reference to neonatal imitation, Meltzoff and Brooks (2001) have argued that, in reproducing the behavior of others, infants automatically perceive others as “like me” and thus begin to develop a sense of social connectedness, mutual recognition, and shared experience. In other words, experience sharing has been suggested to emerge “in the guise of emotional contagion” (Brinck, 2008). Another type of support for our first hypothesis comes from adult interaction, and is provided by the temporal organization of the instances of language use associated with experience sharing. Citation: In what is to come, we will discuss these two alternative hypotheses one after another, with the aim of paving the way for future empirical research on the topic. Besides overlapping talk, there are also other ways in which experience sharing, as it were, “surpasses” turn-taking. Face is central here. Speakers may use their facial displays to mark a transition from affectively neutral talk to emotional experience sharing in the middle of their ongoing turns at talk (cf. Iwasaki, 2011). Likewise, a recipient may display an emotional stance toward an actional or a topical element in a speaker’s ongoing turn at talk, thereby inviting the speaker to redirect her utterance production (Kaukomaa et al., in press). Detailed considerations of parallel uses of words and facial displays in moments of experience sharing are thus particularly intriguing. From this perspective, let us consider Example 1 (taken from Peräkylä and Ruusuvuori, 2006), where one participant’s telling (about a dress code that a mutual friend working in a newspaper needed to conform to) is followed by a shared amusement of both of the participants. Here, the onset and the completion of the smiles take place with one party doing the first move and the other party following. However, between these sequentially organized boundary moves, the participants maintain simultaneous smiles, embodying the sharing of experiences over a lengthy period of time. Frontiers in Psychology \| www.frontiersin.org Hypothesis I: Experience Sharing is in Tension with the Sequential Framework of Turn-Taking While language use may be anchored in the organization of turn- taking (see e.g., Schegloff, 1996, 2006), still, there is much work suggesting that the moments of experience sharing may be excep- tional in this respect (Coates, 1994; Lerner, 2002; Pillet-Shore, 2012; Vatanen, 2014). In conversation analysis, a classic example is provided by Goodwin and Goodwin (1987), who described the April 2015 \| Volume 6 \| Article 450 Frontiers in Psychology \| www.frontiersin.org 2 Experience sharing, emotional reciprocity, and turn-taking Stevanovic and Peräkylä Frontiers in Psychology \| www.frontiersin.org turn-taking rules in Example 1 was facial expression, and the organization of talk followed turn-taking rules, the overlapping talk (lines 10–11) during the experience sharing should also be acknowledged. with turn-taking, this seems not to be the case for instrumental communication. In her study on university subcommittee meet- ings, Edelsky (1981) observed that those participants who oth- erwise made frequent use of overlapping talk still abided to the canonical turn-taking system when their talk was oriented toward the official business of the meeting. The same phenomenon seems to apply also for those emotional expressions that are used to carry out different kinds of instrumental communicative tasks. For example, Heath (1989) showed that, when patients in medical consultations tried to legitimize medical attention to their ailment through cries of pain, these expressions of suffering abided to the organization of turn-taking. The idea of experience sharing being in tension with the sequential temporality of turn-taking reverberates with certain recent suggestions presented in the conversation analytic litera- ture: Heritage (2011) has described the epistemic dilemmas asso- ciated with the “empathic moments in interaction” and Enfield (2011) has suggested that it is turn-taking with its inherent asymmetries that helps to account for the existence of such dilemmas. But even if experience sharing would be in tension April 2015 \| Volume 6 \| Article 450 Frontiers in Psychology \| www.frontiersin.org 3 Experience sharing, emotional reciprocity, and turn-taking Stevanovic and Peräkylä In the subsequent development of the human infant, the expe- riences of contingent emotional reciprocity get more nuanced. Already from 2 months on, infants and their caretakers start to look and listen to each other more carefully; producing vocal, facial and gestural responses elicited by the expressed feelings and interests of their interaction partners (Spitz and Wolf, 1946; Trevarthen, 1979; Stern, 1985; Cohn and Elmore, 1988; Rochat and Passos-Ferreira, 2008). During these monitoring processes, the infants gradually develop expectations for how the inter- action is likely to proceed; for example, they learn to expect that, following an emotional bid on their part, be it via a smile, gaze, or frown, the other will respond in return (Sagi and Hoff- man, 1976; Meltzoff and Moore, 1977; Wolff, 1987; Sroufe, 1996; Rochat, 2001). Compared to the automatic resonance processes, the reliance on social expectations is risky but, when successful, likely to result in a powerful experience of shared emotion (Rochat et al., 2009). The points detailed above suggest that the home environ- ment for experience sharing might be in the concurrent frame- work of emotional reciprocity, while experience sharing (unlike instrumental communicative goals) may call for the participants to depart from the sequential framework of turn-taking. Hypothesis II: The Sequential Framework of Turn-Taking Serves Experience Sharing While our first hypothesis questions the relevance of turn-taking for experience sharing, our second hypothesis represents just the opposite view: it suggests that the sequential framework of turn- taking is not in tension with experience sharing but, instead, serves it. Previously, we highlighted the significance of the automatic resonance processes for experience sharing. However, it has been pointed out that the mere reproduction of other people’s behav- iors represents a closed loop system: it reflects what is already out there (Rochat and Passos-Ferreira, 2008). So, for people really to relate and share each other’s experiences more fully, these automatic resonance processes need to be embedded in an open system of contingent emotional reciprocity. It can thus be argued that contingent emotional reciprocity, and thus also expe- rience sharing, can be facilitated by the sequential framework of turn-taking. From this perspective, let us consider Example 2, where an 11-month-old girl, Nea, prompts a prominent instance of expe- rience sharing. First, she looks at her parents, assuring their attention. Thereafter, she puts a funny grimace on her face, thus prompting her parents’ to laugh heartfeltly. Finally, she joins in the laughter, expressing a high level of positive arousal. The sharing of experience is organized in successive turns. This turn- taking organization of sharing builds on the child’s capacity to anticipate her significant others’ reactions to her behavior, while there is a genuine possibility that the parents will not behave as expected. In human ontogeny, the first instantiations of contingent emo- tional reciprocity appear in the context of alternation between approach and withdrawal tendencies (Beebe and Stern, 1977; Hietanen et al., 2008). Infants have been shown occasionally visu- ally to disengage from their interactive partners and then return to the engagement. Often, this happens in a highly coordinated fashion—that is, when one partner moves from a less engaged phase to a more engaged phase, or vice versa, the other partner responds with a corresponding change in the same direction. Still, these changes are likely to take place within certain time- lags (Cohn and Tronick, 1987). This gives the interaction a sense of one person making a bid of engagement and another person responding to that bid in a positive way. Example 2 demonstrates how a turn-taking structure, where expressions are organized as distinct temporal units that follow each other, provides for experiences of contingent emotional reciprocity. Hypothesis II: The Sequential Framework of Turn-Taking Serves Experience Sharing By making a grimace, Nea, took on trust that her parents would recognize what she was up to; in their “next turn” (laughter), the parents displayed that they indeed did; and, the correctness of the parents’ interpretation was confimed by Nea, through her final laughter. Here, turn-taking enables the partic- ipants to establish meaningful linkages between their behaviors. While each behavior occupies a forward-looking status, raising expectations for what is to happen next (on conditional relevance, Consequences for the Study of Turn-Taking see Schegloff, 2007, p. 20), each response to such behaviors occu- pies a backward-looking status as something that was invoked by what just occurred (on the next-turn proof procedure, see Sacks et al., 1974, pp. 728–729). It is thus the dynamic inter- play between expectations and their overflowing satisfaction that provides a scaffolding for a possible escalation of emotion dis- plays (each participant endorsing the affective aspect of their co-participants’ turns), which may generate particularly intensive instances of experience sharing. Also the conversation analytic research on emotional expression provides support for our second hypothesis. This line of research has shown that the partici- pants may refrain from the immediate reciprocation of contagious emotional expressions, such as laughter and crying (Hepburn and Potter, 2012; Shaw et al., 2013). Instead, these expressions seem to be regulated by the turn-by-turn sequential organization of interaction. For example, the recipients of complaint stories (Couper-Kuhlen, 2012) and news deliveries (Maynard and Freese, 2012) have been shown to produce their emotional responses at the completion of the news delivery or narrative, rather than immediately after the tellers’ emotional displays. Likewise, sur- prise tokens such as wow, gosh, oh my good, ooh, phew have been described as interactionally organized performances, inter- actional achievements, instead of automatic emotional eruptions (Wilkinson and Kitzinger, 2006; see also Heath et al., 2012). It could be precisely the sensitivity of emotional expression to the sequential framework of turn-taking that enables the participants to display their willingness to grasp—as fully as possible—the particular experience that their co-participants are about to share. We have now considered two alternative hypotheses about how the relationship between experience sharing and turn-taking could be viewed. What consequences would these imply for the study of turn-taking? According to our first hypothesis, the home environment of experience sharing is in the concurrent framework of emotional reciprocity, while there is a tension between experience sharing and the sequential framework of turn-taking. If this hypothe- sis is valid, then there must be evidence that, not only does a lesser amount of experience sharing lead to a greater amount of turn-taking, and vice versa, but also, that turn-taking obstructs experience sharing. Furthermore, if turn-taking is there to facili- tate instrumental communication (instead of experience sharing), then we would expect that there would be a tension between overlapping vocalizations and effective instrumental communi- cation. Consequences for the Study of Turn-Taking While there is some evidence for the tension between turn-taking and experience sharing (Enfield, 2011; Heritage, 2011; Vatanen, 2014), the potentially problematic combination of over- lapping vocalizations and effective instrumental communication has rather been taken for granted than really unpacked through empirical investigation (cf. Stevanovic and Frick, 2014). One further challenge from the perspective of this hypothesis would be to account for the occurrences of overlapping talk serving instrumental purposes. According to the second hypothesis, turn-taking is in the ser- vice of experience sharing. In allowing the participants to engage in increasingly complex forms of joint action, and, hence, in ever more exciting shared experiences, there seems to be no tension between turn-taking and experience sharing. From this perspec- tive, there seems to be a developmental continuity between the early infant-caretaker interactions governed by emotional reci- procity and the later, more complex forms of social interaction. If this hypothesis is valid, then there must be evidence that the instances of experience sharing cast in the sequential framework of turn-taking can, in principle, reach at least the same level of intensity as those occurring in the context of overlapping talk. Moreover, one would need to show that occurrences of experience sharing and instrumental goal-pursuit would be relatively evenly distributed between the instances of overlapping talk and talk abiding to the norms of turn-taking. Finally, we may turn the table around and ask whether experi- ence sharing can also serve turn-taking. This possibility appears relevant with reference to the Goffmanian idea of the ubiquitous insecurity of people in social interaction—a theme that runs all through his work (see e.g., Goffman, 1955; Rawls, 1987). In social interaction, by every turn that a person takes, s/he claims being worthy of other’s attention and calls for others to recognize this claim (Goffman, 1955, pp. 9–10), while there is always a possibil- ity that this claim will not be recognized by others (Stevanovic, 2015; Peräkylä, in press). The emotionally secure framework of experience sharing with significant others, however, provides an embarrassment-free site for the practicing of making such claims and for the acquisition of the more asymmetric responsibilities that the sequential framework of turn-taking enforces the partic- ipants to assume. No matter which of the above hypothese is more valid than the other, the relationship between experience sharing and turn- taking is something worth further investigation—something that this paper has sought to highlight. April 2015 \| Volume 6 \| Article 450 4 Frontiers in Psychology \| www.frontiersin.org Experience sharing, emotional reciprocity, and turn-taking Stevanovic and Peräkylä Consequences for the Study of Turn-Taking In sum, the coordination of actions in terms of turn-taking enables forms of experience sharing that the concurrent organi- zation could not afford. This suggests that one motivation under- lying the human propensity to coordinate their actions in terms of turn-taking could be the human propensity to share experiences. Even more, there might be a bidirectional linkage of enhancement between these two propensities. Acknowledgments This work was supported by the Academy of Finland (Decision No. 274735). Beebe, B., and Stern, D. 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The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Striano, T., and Rochat, P. (1999). Developmental link between dyadic and triadic social competence in infancy. Br. J. Dev. Psychol. 17, 551–562. doi: 10.1348/ 026151099165474 Tomasello, M. (1999). The Cultural Origins of Human Cognition. Cambridge, MA: Harvard University Press. April 2015 \| Volume 6 \| Article 450 Frontiers in Psychology \| www.frontiersin.org 7
https://openalex.org/W4239597597	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0235456&type=printable	English	null	Correction: Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches	PloS one	2,020	cc-by	269	PLOS ONE PLOS ONE OPEN ACCESS Citation: The PLOS ONE Staff (2020) Correction: Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches. PLoS ONE 15(6): e0235456. https://doi.org/10.1371/journal. 0235456 CORRECTION Correction: Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches The PLOS ONE Staff The first author’s initials appear incorrectly in the citation. The publisher apologizes for the error. The correct citation is: Nutor JJ, Duodu PA, Agbadi P, Duah HO, Oladimeji KE, Gondwe KW (2020) Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches. PLoS ONE 15(6): e0234034. https://doi.org/10.1371/journal.pone.0234034. Reference 1. John Nutor J, Duodu PA, Agbadi P, Duah HO, Oladimeji KE, Gondwe KW (2020) Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches. PLoS ONE 15(6): e0234034. https://doi.org/10.1371/journal.pone.0234034 PMID: 32497145 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1. John Nutor J, Duodu PA, Agbadi P, Duah HO, Oladimeji KE, Gondwe KW (2020) Predictors of high HIV+ prevalence in Mozambique: A complex samples logistic regression modeling and spatial mapping approaches. PLoS ONE 15(6): e0234034. https://doi.org/10.1371/journal.pone.0234034 PMID: 32497145 Published: June 25, 2020 Copyright: © 2020 The PLOS ONE Staff. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235456 June 25, 2020
https://openalex.org/W2996800570	https://europepmc.org/articles/pmc6938482?pdf=render	English	null	Risk of yellow fever virus importation into the United States from Brazil, outbreak years 2016–2017 and 2017–2018	Scientific reports	2,019	cc-by	10,376	Risk of yellow fever virus importation into the United States from Brazil, outbreak years 2016–2017 and 2017–2018 OPEN Ilaria Dorigatti 1, Stephanie Morrison2,3, Christl A. Donnelly1,4, Tini Garske1, Sarah Bowden2,3 &ArdathGrills2 Southeast Brazil has experienced two large yellow fever (YF) outbreaks since 2016. While the 2016–2017 outbreak mainly affected the states of Espírito Santo and Minas Gerais, the 2017–2018 YF outbreak primarily involved the states of Minas Gerais, São Paulo, and Rio de Janeiro, the latter two of which are highly populated and popular destinations for international travelers. This analysis quantifies the risk of YF virus (YFV) infected travelers arriving in the United States via air travel from Brazil, including both incoming Brazilian travelers and returning US travelers. We assumed that US travelers were subject to the same daily risk of YF infection as Brazilian residents. During both YF outbreaks in Southeast Brazil, three international airports—Miami, New York-John F. Kennedy, and Orlando—had the highest risk of receiving a traveler infected with YFV. Most of the risk was observed among incoming Brazilian travelers. Overall, we found low risk of YFV introduction into the United States during the 2016–2017 and 2017–2018 outbreaks. Decision makers can use these results to employ the most efficient and least restrictive actions and interventions. Yellow fever (YF) is an acute, vector-borne, vaccine-preventable disease caused by the YF virus (YFV) that circu- lates enzootically in parts of South America and sub-Saharan Africa1–3. YF is usually maintained in a sylvatic cycle involving mosquitoes and nonhuman primates4, but spillovers into the human population are frequent5,6. For decades, fewer than 100 human cases were reported annually in Brazil5. However, in 2016–2017 and 2017–2018, two large YF outbreaks caused, respectively, 7847 and 1,376 confirmed human cases (as of October 4, 2018)8 in the Southeast Region of Brazil. This region comprises the states of Espírito Santo, Minas Gerais, São Paulo, and Rio de Janeiro. Historically, yellow fever vaccination had previously only been recommended in Minas Gerais and western São Paulo states in this region; yellow fever vaccination was not recommended for eastern São Paulo state and the other two states because they were deemed not at risk of YFV transmission3. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 Risk of yellow fever virus importation into the United States from Brazil, outbreak years 2016–2017 and 2017–2018 OPEN Baseline risk of YFV importation into the United States in 2016–2017. Mean and 95% confidence interval of the estimated total number of YFV importations (IS A W , S, comprising incoming Brazilian travelers, returning US travelers and international travelers) entering the United States at the specified ports of entry (A) and final destination airports (B) during the 2016–2017 YF outbreak. Only US airports with an upper 95% confidence limit exceeding 0.5 over all Brazilian states in Southeast Brazil (regional estimate) are shown. These estimates were obtained assuming 70% vaccination coverage of US and international travelers to Brazil. Uncertainty derives from sampling 10,000 stochastic realizations of the length of stay, incubation period, infectious period, number of asymptomatic or mild infections for each severe YFV case and vaccine efficacy from their respective distributions. SE Brazil = Southeast Brazil. Analyses of global travel patterns have demonstrated that high connectivity is associated with increasing risks of disease emergence10,12,13. Mathematical models have been used in the past to quantify the risk of infection for travelers14–16, evaluate the probability of autochthonous transmission upon importation17,18 and estimate the risk of importation when incidence (the number of new cases in time) is available19. In a previous study, we devel- oped a mathematical model to quantify the number of YFV importations introduced at the country level from the cumulative number of cases reported during the 2015–2016 YF outbreak in Brazil and found that the United States was at risk of YFV importation20. However, targeted public health prevention and preparedness planning require a more detailed analysis of where, within the United States, YFV-infected travelers are most likely to arrive. This targeting is particularly relevant because proof of yellow fever vaccination is not required to enter the United States when coming from countries at risk of YFV transmission2. However, the Centers for Disease Control and Prevention (CDC) recommends that travelers get yellow fever vaccination before traveling to affected areas of Brazil21. Additionally, the United States is currently experiencing a limited availability of yellow fever vac- cine caused by an interruption in the supply of the only US licensed vaccine (YF-Vax) since July 2017 and a time- line for production of more vaccine has not been established. Risk of yellow fever virus importation into the United States from Brazil, outbreak years 2016–2017 and 2017–2018 OPEN y Particularly concerning is that, during the 2017–2018 YF outbreak, multiple cases occurred among unvac- cinated travelers who became infected with YFV while travelling in Southeast Brazil and subsequently returned to countries in South America and Europe (Argentina (3), Chile (3), Netherlands (1), France (1), Romania (1), Switzerland (1), and United Kingdom (1)) where they developed severe clinical disease leading, in some cases, to death9. These incidents raise concern about the risk of seeding local YFV transmission outside Brazil into countries that have competent vector mosquito populations, including the United States. As of January 2019, the United States has not reported any YFV importation from Brazil. Recent estimates suggest that the United States is the country that received the highest number of air travelers arriving in cities suitable for yellow fever transmission from YFV endemic areas worldwide10. Analyses of the 2016 air passenger volume data show that approximately 60% of the air travelers arriving in US cities suitable for yellow fever transmission from YFV endemic areas came from Southeast Brazil11. The scale of the Brazilian 2016–2017 and 2017–2018 YF outbreaks combined with the extensive interconnectedness between Southeast Brazil and the United States created concern for the importation of YFV-infected travelers2. 1MRC Centre for Global Infectious Disease Analysis, School of Public Health, Imperial College London, London, United Kingdom. 2Centers for Disease Control and Prevention, Atlanta, Georgia, USA. 3Eagle Medical Services, LLC, San Antonio, Texas, USA. 4Department of Statistics, University of Oxford, Oxford, United Kingdom. email: i.dorigatti@imperial.ac.uk Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 1. Baseline risk of YFV importation into the United States in 2016–2017. Mean and 95% confidence interval of the estimated total number of YFV importations (IS A W , S, comprising incoming Brazilian travelers, returning US travelers and international travelers) entering the United States at the specified ports of entry (A) and final destination airports (B) during the 2016–2017 YF outbreak. Only US airports with an upper 95% confidence limit exceeding 0.5 over all Brazilian states in Southeast Brazil (regional estimate) are shown. These estimates were obtained assuming 70% vaccination coverage of US and international travelers to Brazil. Uncertainty derives from sampling 10,000 stochastic realizations of the length of stay, incubation period, infectious period, number of asymptomatic or mild infections for each severe YFV case and vaccine efficacy from their respective distributions. SE Brazil = Southeast Brazil. Figure 1. Risk of yellow fever virus importation into the United States from Brazil, outbreak years 2016–2017 and 2017–2018 OPEN Although the manufacturer, Sanofi-Pasteur, gained FDA approval to import and distribute its Stamaril yellow fever vaccine in the United States under an expanded access Investigational New Drug protocol22, only 260 clinics across the United States have been approved to administer Stamaril (down from approximately 4,000 sites that usually distribute YF-Vax)22. ( pp y y ) In this analysis, we estimate the risk of human YFV importation across all US airports with international travel from Southeast Brazil (n = 304 airports). We evaluate this risk during the 2016–2017 and 2017–2018 outbreaks in Southeast Brazil. We also explore how this risk changes with vaccination coverage. Results Mean and 95% confidence interval of the estimated total number of YFV importations (IS A W , S, comprising incoming Brazilian travelers, returning US travelers and international travelers) entering the United States at the specified ports of entry (A) and final destination airports (B) during the 2017–2018 YF outbreak. Only US airports with an upper 95% confidence limit exceeding 0.5 over all Brazilian states in Southeast Brazil (regional estimate) are shown. These estimates were obtained assuming 70% vaccination coverage of US and international travelers to Brazil. Uncertainty derives from sampling 10,000 stochastic realizations of the length of stay, incubation period, infectious period, number of asymptomatic or mild infections for each severe YFV case and vaccine efficacy from their respective distributions. SE Brazil = Southeast Brazil. of risk as opposed to regional-level homogeneity of risk of infection is generally associated with lower central estimates of the risk of YFV importation. We performed sensitivity and counterfactual analyses to test the impact of hypothetical vaccination coverage scenarios on the risk of YFV-infected travelers arriving at US airports dur- ing both YF outbreaks in 2016–2017 and 2017–2018. All regional- and state-level estimates of the total num- ber of YFV importations (comprising Brazilian, US and international resident travelers) obtained for first US ports of entry and final destination airports in the main, sensitivity and counterfactual analyses are presented in Supplementary Tables S2–S7. of risk as opposed to regional-level homogeneity of risk of infection is generally associated with lower central estimates of the risk of YFV importation. We performed sensitivity and counterfactual analyses to test the impact of hypothetical vaccination coverage scenarios on the risk of YFV-infected travelers arriving at US airports dur- ing both YF outbreaks in 2016–2017 and 2017–2018. All regional- and state-level estimates of the total num- ber of YFV importations (comprising Brazilian, US and international resident travelers) obtained for first US ports of entry and final destination airports in the main, sensitivity and counterfactual analyses are presented in Supplementary Tables S2–S7. Sensitivity analysis. Varying the assumed vaccination coverage of US and international travelers. In a sensitivity analysis, we tested the effect of extreme vaccination coverages of US and international travelers to Brazil on YFV importations during both outbreak years. Supplementary Figs. S7–S10 and S11–S14 show the estimated number of US and international travelers and total YFV importations (comprising Brazilian, US and international travelers) obtained under the extreme scenarios of no (i.e. Results We estimated that within the United States, the Miami-MIA, New York-JFK, and Orlando-MCO international airports were at risk of receiving at least 1 YFV-infected traveler during the 2016–2017 and 2017–2018 YF out- breaks in Southeast Brazil (<1 YFV infected traveler per 100,000 travelers). This analysis included all US first points of entry (n = 90) and all US final destination airports (n = 304) with international travel volume from airports in Southeast Brazil (n = 21). Our results approximate the importation risk because we assumed that US travelers were at the same risk of YFV infection as Brazilian residents, as well as regional homogeneity in the risk of infection and in the probability of travel. Figures 1 and 2 show the mean number of YFV importations and its 95% confidence interval. Only US airports with an upper 95% confidence limit exceeding 0.5 over all Brazil’s Southeast states are shown. Specific numbers of importations among arriving Brazilian and returning US and international travelers are presented separately in the Supplementary Information (Supplementary Figs. S1–S4). They show that under the baseline assumption that 70% of US and international (non-US and non-Brazilian) travelers were vaccinated against YFV, Brazilian travelers primarily drove the risk of YFV importation at US airports during the 2016–2017 and 2017–2018 outbreaks in Southeast Brazil. Assuming state-level homogeneity Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 2. Baseline risk of YFV importation into the United States in 2017–2018. Mean and 95% confidence interval of the estimated total number of YFV importations (IS A W , S, comprising incoming Brazilian travelers, returning US travelers and international travelers) entering the United States at the specified ports of entry (A) and final destination airports (B) during the 2017–2018 YF outbreak. Only US airports with an upper 95% confidence limit exceeding 0.5 over all Brazilian states in Southeast Brazil (regional estimate) are shown. These estimates were obtained assuming 70% vaccination coverage of US and international travelers to Brazil. Uncertainty derives from sampling 10,000 stochastic realizations of the length of stay, incubation period, infectious period, number of asymptomatic or mild infections for each severe YFV case and vaccine efficacy from their respective distributions. SE Brazil = Southeast Brazil. Figure 2. Baseline risk of YFV importation into the United States in 2017–2018. Discussionh This study used an established risk-modeling framework to inform potential policy decisions and actions. Results showed Miami-MIA in 2016–2017, along with New York-JFK, and Orlando-MCO airports in 2017–2018 to be at risk of receiving at least one YFV-infected traveler both as US first ports of entry and final destination airports, with the risk varying with departure state, outbreak year, vaccine coverage, and traveler type. These results are driven by the strong connectivity between Southeast Brazil and Miami-MIA, New York-JFK, and Orlando-MCO airports, which respectively acted as US first port of entry for 29% (30%), 18% (16%) and 13% (14%) of the total number of travelers entering the United States from Southeast Brazil in 2016 (2017). In terms of final destination, Miami-MIA, New York-JFK, and Orlando-MCO airports respectively received 18% (20%), 16% (15%) and 15% (17%) of the travel volume from Southeast Brazil in 2016 (2017).i Overall, we found good agreement between the regional- and state-level risk of YFV importations at US first ports of entry and final destination airports. In a sensitivity analysis, under the assumption of no vaccination of US and international travelers, we found Boston-BOS to be at risk of YFV importation in 2016–2017 as final destination airport but not as first port of entry (Supplementary Fig. S6) and Atlanta-ATL to be at risk of YFV importation as first port of entry but not as final destination airport in 2017–2018 (Supplementary Fig. S10). These results reflect the extent to which Boston-BOS mainly acts as final destination airport for travels started in Southeast Brazil through connections via other US ports of entry (of the travel volume from Southeast Brazil to Boston-BOS in 2016 and 2017, 15% entered the US at the airport and 0.4% was for transit), while Atlanta-ATL acts as a main port of entry and transit airport for the connection between Southeast Brazil and the United States (of the travel volume from Southeast Brazil to Atlanta-ATL in 2016 and 2017, 95% entered the US at the airport and 83% was for transit). Miami-MIA, New York-JFK, and Orlando-MCO airports lie within the estimated potential range of Aedes mosquitoes in the United States23. A recent modelling study24 has demonstrated that high degrees of consensus and low uncertainty exist around the presence of Ae. aegypti in Miami and Orlando and of Ae. albopictus in New York, which makes these locations suitable to potential local YF transmission upon introduction. Results US and international travelers contribute little to the overall risk of YFV importation in presence of full yellow fever vaccination coverage of US and inter- national travelers to Brazil (Supplementary Figs. S7 and S11). remain at risk for YFV importation (Supplementary Figs. S8 and S12). US and international travelers contribute little to the overall risk of YFV importation in presence of full yellow fever vaccination coverage of US and inter- national travelers to Brazil (Supplementary Figs. S7 and S11). Counterfactual analysis. Varying the assumed vaccination coverage of the Brazilian populations. We conducted a counterfactual analysis to assess the impact of the vaccination campaigns conducted during the 2016–2017 and 2017–2018 outbreaks and explored the extent to which higher or lower vaccine coverage among residents of Brazil’s Southeast states would have affected the risk of YFV spread. In this analysis we estimated the counterfactual cumulative number of confirmed YF cases having assumed proportionality between the changes in the case counts and susceptibility to YFV. We then used the counterfactual number of confirmed YF cases to estimate the risk of importation due to Brazilian travelers and returning US and international (non-US and non-Brazilian) travelers (for details see Supplementary Section 4). pp y We found that vaccination campaigns in Brazil during the 2016–2017 YF outbreak reduced the risk of YFV importation that the United States would have faced without them (Supplementary Fig. S16). Our estimates suggest that in the absence of the 2016–2017 vaccination campaign, in addition to Miami-MIA airport iden- tified in the main analysis, the New York-JFK, Orlando-MCO and Atlanta-ATL, international airports would also have also been at risk of YFV importation (Supplementary Fig. S16). In the absence of the 2017–2018 YF vaccination campaign, we found a significant increase in the risk of YFV importation into the Miami-MIA and Orlando-MCO international airports from the state of São Paulo (Supplementary Fig. S17), although the overall risk of YFV importation from Southeast Brazil did not substantially differ from the risk estimated in the main analysis (Fig. 1). Our results also suggest that in the absence of substantial increases in vaccination coverage (especially in Minas Gerais) during the 2016–2017 YFV outbreak, Miami-MIA airport would have remained at risk of YFV importation (Supplementary Figs. S13–S15). This highlights the importance of outbreak containment at the source, e.g. through preventive vaccination campaigns. The results obtained in this counterfactual analysis only account for the direct effects of vaccination. Results 0%) and complete (i.e., 100%) vaccination coverage of US and international travelers to Brazil during the 2016–2017 and 2017–2018 YF outbreaks respec- tively. Under the extreme scenario of no vaccination of US and international travelers to Brazil, we found that the regional-level risk of YFV importation into US ports of entry increases to an average of 1.51 (95% CI: 0.66, 2.94) in Miami-MIA, 0.89 (95% CI: 0.37, 1.79) in New York-JFK and 0.70 (95% CI: 0.30, 1.38) in Orlando-MCO during 2016–2017 (Supplementary Fig. S5 and Table S2) and to 2.30 (95% CI: 1.00, 4.45) in Miami-MIA, 1.39 (95% CI: 0.57, 2.76) in New York-JFK, 1.06 (95% CI: 0.48, 2.01) in Orlando-MCO and 0.58 (95% CI: 0.24, 1.16) in Atlanta-ATL during 2017–2018 (Supplementary Fig. S9 and Table S6). In terms of final destination, the same risk increases to an average of 0.88 (95% CI: 0.39, 1.65) in Miami-MIA, 0.76 (95% CI: 0.31, 1.51) in New York-JFK and 0.71 (95% CI: 0.32, 1.33) in Orlando-MCO during 2016–2017 (Supplementary Fig. S5 and Table S4) and 1.47 (95% CI: 0.65, 2.80) in Miami-MIA, 1.21 (95% CI: 0.49, 2.38) in New York-JFK and 1.23 (95% CI: 0.57, 2.34) in Orlando-MCO during 2017–2018 (Supplementary Fig. S9 and Table S7). We attribute this increase to a larger number of returning US and international travelers with YFV infection (Supplementary Figs. S5 and S9) com- pared to the estimate obtained in the main analysis (Supplementary Figs. S2 and S4). In the absence of vaccina- tion, the risk of importation from returning US and international (non-US and non-Brazilian) travelers becomes comparable to the risk posed by Brazilian travelers (Supplementary Figs. S1 and S3). We found that in the absence of YFV vaccination among US and international travelers visiting Brazil, the New York-JFK, Orlando-MCO and Boston-BOS international airports in 2016–2017 and the Fort Lauderdale-FLL and Atlanta-ATL international airports in 2017–2018 would also have been at risk of receiving at least 1 YFV-infected traveler (Supplementary Figs. S6 and S10). g ) On the other hand, even under the hypothetical scenario that all US travelers to Brazil were immunized against YFV, all airports identified as at risk of YFV importation in our main analysis (i.e., the Miami-MIA in 2016–2017, together with the New York-JFK, and Orlando-MCO international airports in 2017–2018) would still Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ remain at risk for YFV importation (Supplementary Figs. S8 and S12). Results Accounting for the indirect effects of vaccination (herd immunity) would reduce the resulting estimates of the risks of YF importation. Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ While we recognize that there are likely heterogeneities in force of infection over time and space, as well as differences in individual behavior, it is difficult to quantify these in the absence of more detailed information. Transmission risk for vector-borne dis- eases is multifaceted, and fully estimating that risk would require, at minimum, information on vector presence or environmental suitability, vector abundance or density, vector biting rates, vector competence, prevalence of the pathogen in the vector population, fine-scale human density and movement, and individual behavior. Data on these variables are either unavailable or not available at a granular level to be able to properly parameterize transmission risk over time, space, and behavior. p We assumed homogeneity in the probability of traveling and the risk of infection. We assumed that US and international travelers visiting Brazil were at the same risk of YF infection as Brazilian residents due to lack of information on US and international travelers’ final destinations, activities and behavior which are all factors that could inform any differential risk of YF infection of visitors relative to the resident population. We worked with travel volume between airports, which are generally in urban areas, while most YFV transmission to humans has occurred in rural areas. Lack of data on the extent to which travelers arriving in urban areas visit rural areas dur- ing their stay may lead to overestimating the absolute or per capita risk of YFV importation. Monath et al. (2002) cites several factors that contribute to risk of acquiring infection, such as immunization status, geographic loca- tion, season of travel, and recreational activities30. However, the lack of data at the appropriate spatial and tempo- ral scale limits our ability to differentiate between populations and their activities. Tatem et al. explains that such data are scattered and recommends the development of a database that combines population attributes with spa- tial and temporal information32. The granularity of the available data allowed us to explore the effect of homog- enizing the risk of YF infection at the regional (Supplementary Section 1.2) versus the state (Supplementary Section 1.3) level. While increasing heterogeneity to the state level is associated with lower central estimates of the risk of YFV importation, we found that homogenizing exposure at different spatial scales generally produced comparable results. The results obtained in this analysis were obtained at the finest spatial resolution allowed by the available data. www.nature.com/scientificreports/ outbreaks. Importantly, onward transmission following introduction is a chance event. As demonstrated in29, the estimated levels of seeding produce high (>80%) probabilities of stochastic extinction across a wide range of transmission settings. outbreaks. Importantly, onward transmission following introduction is a chance event. As demonstrated in29, the estimated levels of seeding produce high (>80%) probabilities of stochastic extinction across a wide range of t i i tti g In the main analysis we assumed 70% vaccination coverage of US and international (non-US and non-Brazilian) travelers to Brazil and found that Brazilian travelers primarily drove the risk of importation. This result is driven by the relative proportion of Brazilian travelers (58% in 2016 and 62.5% in 2017) versus US travel- ers (37% in 2016 and 34% in 2017) and international (non-US and non-Brazilian) travelers (5% in 2016 and 3.5% in 2017) entering the United States and by the 70% vaccination coverage of US and international (non-US and non-Brazilian) travelers assumed in the main analysis. Sensitivity analysis on the assumed vaccination coverage of US and international (non-US and non-Brazilian) travelers shows that in the extreme hypothesis of absence of vaccination, the contribution of returning US and international (non-US and non-Brazilian) travelers to the risk of YFV importation increases to similar levels to the ones posed by Brazilian travelers (Fig. S2 vs S5 and Fig. S4 vs S9). The fact that during recent and previous outbreaks, travel-related cases occurred among return- ing travelers9,21,30,31, as opposed to residents of the source country, suggests that the actual vaccination coverage among US and international travelers may have been lower than what we have assumed in our main analysis and that international travelers may be less likely to report symptoms or seek healthcare outside their home country. Another potential reason is that returning US and international resident travelers could be at greater risk of infection than Brazilian residents, either due to increased exposure or increased susceptibility (e.g. due to lack of cross-protective immunity elicited by previous infections with other flaviviruses). A key assumption we make in this model is that the force of infection—which we define as the probability of being bitten by a YFV-infected mosquito and becoming infected with YFV—is the same for both Brazilian residents and US and international resident travelers to Brazil and differs only by the length of stay or exposure. Discussionh The discovery of YFV in field-collected Ae. albopictus in Southeastern Brazil in 2017, along with the demonstrated potential of YFV adaptation to Ae. albopictusto25 imply that autochthonous YF transmission could potentially extend to temperate regions of the United States. However, strong climatic seasonality affecting entomological traits and mosquito population dynamics26, along with asynchrony in the climatic patterns between the United States and Southeast Brazil, could limit the extent to which local YFV transmission upon introduction can occur. p In the main analysis we found that the average YFV importation risk in Miami-MIA was 0.92 traveler (95% CI: 0.43, 1.77) in 2016–2017 and 1.4 traveler (95% CI: 066, 2.65) in 2017–2018; in New York-JFK the average risk was 0.49 traveler (95% CI: 0.23, 0.94) in 2016–2017 and 0.78 traveler (95% CI: 0.36, 1.45) in 2017–2018; in Orlando-MCO the average risk of YFV importation was 0.42 (95% CI: 0.20, 0.80) in 2016–2017 and 0.69 traveler (95% CI: 0.32, 1.33) in 2017–2018 (Figs. 1, 2 and Supplementary Tables S2 and S6). These results are consistent with previous work by Dorigatti et al.20. During the 2016–2017 and 2017–2018 outbreaks, there were no reports of YFV-infected traveler arriving into the United States. Because of the large proportion of asymptomatic infec- tions typically observed in YF outbreaks27,28, implying that a YFV-infected traveler could arrive without being detected because of the higher probability of being mild or asymptomatic than symptomatic, our estimates are in agreement with no reports of YFV importations into the United States during the 2016–2017 and 2017–2018 Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ Methods In this study, we sought to quantify the risk of YFV importation into US airports (both first ports of entry and final destination airports, separately) during the 2016–2017 and 2017–2018 YF outbreaks in Brazil’s Southeast states (i.e., Espírito Santo, Minas Gerais, Rio de Janeiro, and São Paulo). To do this, we extended the risk model framework developed by Dorigatti et al.20 to explicitly include importations from Brazilian residents (Brazilian travelers), US residents (US travelers) and international (non-Brazilian and non-US) residents (international travelers) who travelled from Southeast Brazil to the United States during the 2016–2017 and 2017–2018 YFV outbreaks in Brazil. We estimated traveler residency using OAG Aviation Worldwide Ltd.’s11 country of sale infor- mation (full description in Supplementary Information Section 1.1). Table 1 summarizes the notation, defini- tions, values and data sources used and Fig. 3 provides an illustration of the risk model adopted in the analysis.i , g p p y We defined importations from Brazilian travelers as Brazilian residents infected with YFV during the 2016– 2017 or 2017–2018 outbreak who traveled to the United States during the incubation or infectious period (i.e. between the time of infection and the end of the period during which human blood is infectious). Similarly, we defined importations from US residents as travelers living in the United States who visited Southeast Brazil during the 2016–2017 or 2017–2018 outbreak, became infected with YFV during their stay, and returned to the United States during the incubation or infectious period. We defined importations from international (i.e. non-Brazilian and non-US) residents as international travelers who visited Southeast Brazil during the 2016–2017 or 2017–2018 outbreak, became infected with YFV during their stay, and then traveled to the United States during the incuba- tion or infectious period. Together, these comprise the total number of YFV importations likely to arrive in the United States. In this analysis, residency was defined by the country of ticket sale. Brazilian resident travelers (hereinafter referred to as Brazilian travelers) were defined as travelers from Southeast Brazil to the United States who purchased their ticket in Brazil. Similarly, US and international resident travelers (hereinafter referred to as US and international travelers) were defined as travelers from Southeast Brazil to the United States who pur- chased their ticket in other countries than Brazil. www.nature.com/scientificreports/ The availability of data a finer spatial resolution and the inclusion of higher degrees of hetero- geneity in the model might decrease the risk of importation, but this is uncertain. g y g p We assumed the same length of stay in Brazil for US travelers and non-US travelers entering the United States from Southeast Brazil (i.e. visiting the United States following a visit to Southeast Brazil). This assumption could have overestimated the risk of YFV importation posed by South American travelers, who on average spend 9 nights in Brazil33, and underestimated the risk posed by European travelers, who on average stay for 23 nights33 compared to the average 19 days spent in Brazil by US travelers34,35. We also assumed the same vaccination cov- erage of US and international (non-US and non-Brazilian) travelers. However, the limited travel volume of inter- national (non-US and non-Brazilian) residents entering the United States from Southeast Brazil (5% and 3.5% of the total travel volume in 2016 and 2017, respectively) implies that non-US and non-Brazilian residents do not significantly contribute to the risk of YFV importation in the United States and that our estimates are robust to changes in the assumed length of stay and vaccination coverage of non-US and non-Brazilian travelers.ii g g y g Our definition of time window (the number of days between the time of symptom onset of the first and last confirmed YFV cases) assumes no asymmetry in the temporal distribution of asymptomatic or mild YF infections compared to severe cases. Undetected or unreported transmission occurring before (or after) the detection of the first (or last) confirmed case would increase the risk of importation.i ii Limitations of the data used in this analysis, specifically passenger volume and vaccination coverage data, also warrant discussion. The estimates generated in this study are based on detailed aggregated data on the volume of passengers who traveled between any Brazilian airport and any US airport at a monthly temporal resolution during 2016 and 2017. While we estimated the risk of YFV importation both by first US port of entry and final destination airport, by construction these data do not capture the actual origin or destination of multicity travel itineraries. In the absence of individual-level travel information, we assumed that the origins and destinations reported in the dataset represented the actual origins and destinations of the travel itineraries. Conclusions During the 2016–2017 YF outbreak in Southeast Brazil Miami-MIA, together with New York-JFK, and Orlando-MCO international airports in 2017–2018, were estimated to be at risk of YFV importation. Under the baseline scenario of 70% vaccination coverage of US and international travelers, we found that arriving YFV-infected Brazilian travelers drove the overall risk of YFV importation into the United States. This implies that outbreak containment at the source, e.g. through preventive or catch-up vaccination campaigns, is key to limit the international spread of YFV. This is particularly important as the temporary limited availability of YFV vaccine in the United States necessitates a consideration of alternative strategies, such as the targeted use of full or fractional YF vaccine doses.h This analysis informed the CDC’s Division of Global Migration and Quarantine – which strives to prevent the introduction, transmission, and spread of communicable disease using the most effective, innovative, efficient, and least restrictive actions – on the risk of YFV importation faced by US airports during the 2016–2017 and 2017–2018 YF outbreaks in Southeast Brazil. Any response needs to be commensurate with the level of risk. Because the risk of YFV importation obtained in this study yielded a relatively low risk of YFV importation and local YFV transmission following introduction, no further action among those potentially considered to be implemented (e.g. travel restriction or proof of vaccination at entry) was taken. The results presented in this study and the identification of Miami-MIA, New York-JFK, and Orlando-MCO as the main US airports at risk of YFV importation reflect the travel volume between the United States and Southeast Brazil and the scale of the YF out- breaks observed in 2016–2017 and 2017–2018. In future outbreaks, it will be necessary to reassess the risk of YFV importation and identify the new locations at risk. Similarly, to assess the risk of importation during a highly focal outbreak, like the one observed in Ilha Grande, detailed travel information would need to be included. However, models with higher complexity do not always yield greater explanatory power18,38. g p y y y g p y p Understanding the risk of importation through mathematical models can aid public health decision-making related to vaccination policies and other control strategies. When the ultimate objective is to prevent local YFV transmission within the United States, decision makers can consider targeted courses of action at specific ports of entry, particularly when resources are constrained. www.nature.com/scientificreports/ Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ This analysis accounted for YFV vaccination coverage estimates both among US and international travelers and in Brazil and evaluated its influence on the risk of YFV importation in the United States. For the United States, the only available vaccination coverage estimate was at the national level, which does not account for the heterogeneity in vaccination coverage likely existing across the United States. In the absence of information on the actual municipalities targeted by vaccination campaigns in Brazil in 2016–2017, we calculated the overall expected vaccination coverage in each state in Southeast Brazil. This calculation approximates the actual vacci- nation coverage within each state and does not capture the heterogeneity among municipalities. While a recent systematic review provides estimates on YFV vaccination coverage from 1970 to 201636, it has its limitations. The population estimates used in the study do not appear to be consistent with the UN population estimates that were taken as a reference37 and do not account for the most recent outbreaks in Brazil. Methods To obtain a high spatial resolution of YFV importation risk, our estimates were calculated based on air travel volume from each of the four Southeast Brazilian states to all US first points of entry and final destination airports. Therefore, the total number of YFV importations (IS A W , S) was calcu- lated as the sum of the number of YFV importations due to Brazilian travelers from Brazilian state S to US airport A during time window WS (ES A W , S) and the number of YFV importations due to US and international travelers from Brazilian state S to US airport A during time window WS (RS A W , S): Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ Symbol Definition Value Source CS,Ws Cumulative number of confirmed YFV cases reported in Brazilian state S during time window WS ES (2016–2017): 260 ES (2017–2018): 6 MG (2016–2017): 487 MG (2017–2018): 477 RJ (2016–2017): 17 RJ (2017–2018): 188 SP (2016–2017): 20 SP (2017–2018): 455 5,7 WS Number of days between the time of symptom onset of the first and last confirmed YFV cases in Brazilian state S ES (2016–2017): 116 days ES (2017–2018): 83 days MG (2016–2017): 122 days MG (2017–2018): 83 days RJ (2016–2017): 80 days RJ (2017–2018): 83 days SP (2016–2017): 124 days SP (2017–2018): 83 days 5,7 popS Population size of Brazilian state S ES: 3,973,697 MG: 20,997,560 RJ: 16,635,996 SP: 44,749,699 39 pV Vaccination coverage of US travelers to Brazil 70% 41 L Average length of stay of US visitors to Brazil Empirical distribution with mean 19 days 34,35 vcS Average vaccination coverage of municipalities in Brazilian state S during the 2016–2017 YF outbreak 25.00% (<50%, Table 3 in9) 62.45% (50%–74.9%, Table 3 in9) 84.95% (75%–94.9%, Table 3 in9) 97.50% (≥95%, Table 3 in9) 7 MvcS Number of municipalities in Brazilian state S with average vaccination coverage vcS during the 2016–2017 YF outbreak See Table 3 in9 7 popS M , Average population size of municipalities in Brazilian state S ES: 50,944.83 MG: 24,616.13 RJ: 180,826.00 SP: 69,379.38 39 popS V , 2018 Number of subjects vaccinated, as of April 10, 2018, during the YFV vaccination campaign started on January 25, 2018, in Brazilian state S SP: 6,405,872 RJ: 1,497,840 5 TS A WS , Number of Brazilian travelers from airports in Brazilian state S to US airport A during time window WS (i.e. Methods air passengers with Brazil as country of ticket sale) Cannot disclose (third-party data) 11 TA S WS , Number of US and international travelers from airports in Brazilian state S to US airport A during time window WS (i.e. air passengers with country of ticket sale other than Brazil) Cannot disclose (third-party data) 11 α Number of asymptomatic or mild YFV cases for each severe YFV case Lognormal distribution with mean 7 and variance 13 28 TE Incubation period of YFV (days) Lognormal distribution with mean 4.6 and variance 2.7 40 TI Infectious period of YFV (days) Normal distribution with mean 4.5 and variance 0.6 4,40 VE YFV vaccine efficacy Uniform distribution between 90% and 99% 42 Table 1. Summary of notation, data, and sources used. “ES” denotes Espírito Santo, “MG” denotes Minas G i “SP” d t Sã P l d “RJ” d t Ri d J i V l t li t d i thi t bl l t d i Table 1. Summary of notation, data, and sources used. “ES” denotes Espírito Santo, “MG” denotes Minas Gerais, “SP” denotes São Paulo, and “RJ” denotes Rio de Janeiro. Values not listed in this table are located in the Supplementary Information. We are unable to share air passenger data because they are proprietary. Gerais, “SP” denotes São Paulo, and “RJ” denotes Rio de Janeiro. Values not listed in this table are located in the upplementary Information. We are unable to share air passenger data because they are proprietary. = + I E R , (1) S A W S A W S A W , , , S S S (1) We defined the time window W as the number of days between the time of symptom onset of the first and last confirmed YFV cases in each Brazilian state for each outbreak (Table 1). Calculating importations from Brazilian travelers. Methods The total number of importations (IS A W , S, left) is the sum of importations caused by incoming Brazilian travelers (ES A W , S, top middle) and importations caused by US and international travelers (RS A W , S, bottom middle). Each of these components can be further broken down into three model terms (right). = p T pop , (4) S A W S A W S , , S S = p T pop , (4) S A W S A W S , , S S = p T pop , (4) S A W S A S , , S S (4) where TS A W , S is the number of Brazilian resident air passengers (i.e. passengers who purchased their ticket in Brazil) traveling from airports in Brazilian state S to US airport A during time window WS 1,2 and popS is the population size of Brazilian state S39. Lastly, we calculated the probability that a YFV-infected traveler is incubating or infec- tious during time window WS as: =    +    p min T T W , 1 , (5) i W E I S S (5) where TE is the human incubation period40 and TI is the human infectious period4,40 of YFV. where TE is the human incubation period40 and TI is the human infectious period4,40 of YFV. Calculating importations from US and international (non-Brazilian and non-US) travelers. The number of YFV-infected US and international (non-Brazilian and non-US) travelers from Brazil was calculated as: λ = ⋅ ⋅  R T p , (6) S A W A S W S i W , , S S S (6) where TA S W , S is the number of US and international (non-Brazilian and non-US) resident traveling to US airport A from Brazilian state S during time window WS with no YFV vaccine-induced immunity; λS is the per capita risk of YFV infection in Brazilian state S for US and international travelers during time window WS; and pi WS is the probability that a YFV infected US or international traveler flies to the United States during the incubation or infectious period (identical to Eq. 5, above). Methods The number of YFV infected Brazilian travelers arriving at US airports was calculated as: = ⋅ ⋅ ˆ E C p p , (2) S A W S W S A W i W , , , S S S S (2) where ˆCS W , S is the estimated total number of YFV cases in Brazilian state S during time window WS; pS A W , S is the probability of traveling from Brazilian state S to US airport A during time window WS; and pi WS is the probability that a YFV infected traveler is either incubating or infectious during time window WS. We assumed that all con- firmed cases were severe and estimated the cumulative number of YFV cases in a given Brazilian state as: α = + ˆC C C , (3) S W S W S W , , , s S S α = + ˆC C C , S W S W S W , , , s S S (3) where α is the number of asymptomatic or mild YFV cases for each severe (i.e., reported) YFV case28. We calcu- ated the probability of traveling from Brazilian state S to US airport A as: Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ Figure 3. Branching tree illustrating the hierarchical nature of our risk model. The total number of importations (IS A W , S, left) is the sum of importations caused by incoming Brazilian travelers (ES A W , S, top middle) and importations caused by US and international travelers (RS A W , S, bottom middle). Each of these components can be further broken down into three model terms (right). Figure 3. Branching tree illustrating the hierarchical nature of our risk model. The total number of importations (IS A W , S, left) is the sum of importations caused by incoming Brazilian travelers (ES A W , S, top middle) and importations caused by US and international travelers (RS A W , S, bottom middle). Each of these components can be further broken down into three model terms (right). Figure 3. Branching tree illustrating the hierarchical nature of our risk model. Methods pop pop M vc VE (9) S v M S M vc S , , S (9) The term popS V , for the 2017–2018 YF outbreak was calculated as above, having added the number of sub- jects vaccinated during the YFV vaccination campaign started on January 25, 20185 (popS V , 2018): The term popS V , for the 2017–2018 YF outbreak was calculated as above, having added the number of sub- jects vaccinated during the YFV vaccination campaign started on January 25, 20185 (popS V , 2018): ∑ =    ⋅ ⋅ ⋅   + . pop pop M vc VE pop (10) S v M S M vc S S V , , , S 2018 (10) Variability in the length of stay (L), incubation periods (TE), infectious periods (TI), number of asymptomatic or mild YFV cases for each severe YFV case (α) and vaccine efficacy (VE) was accounted for by sampling 10,000 times from their respective distributions (Table 1). The risk estimates are directly proportional to the number of asymptomatic or mild YFV cases for each severe YFV case (α), to the probability that a YFV-infected traveler is incubating or infectious during the epidemic time window (pi WS) and to the vaccination coverage of US and inter- national travelers (pV). The results of the analysis are reported in terms of mean and 95% confidence interval (CI) of the number of incoming Brazilian travelers (ES A W , S),US and international travelers (RS A W , S), and total number of YFV importations (IS A W , S) likely to arrive at individual US airports (Figs. 1 and 2).h S A , The total number of YFV importations from Southeast Brazil into the United States was estimated in two ways: (i) by applying the framework developed for Brazilian state S to the cumulative number of confirmed cases, population figures, and joint travel volume from Espírito Santo, Minas Gerais, São Paulo, and Rio de Janeiro (we refer to this estimate as regional-level estimate, see Supplementary Information, Methods Section 1.2 for details) and (ii) by summing the total number of YFV importations obtained for Espírito Santo, Minas Gerais, São Paulo, and Rio de Janeiro (we refer to this estimate as state-level estimate as detailed in Supplementary Information, Methods Section 1.3). Methods We can further decompose the number of US and international trav- elers without YFV vaccine-induced immunity to: = ⋅ − ⋅ T T p VE (1 ), (7) A S W A S W V , , S S (7) where TA S W , S is the number of US and international resident air passengers (i.e. passengers who purchased their ticket in countries other than Brazil) entering US airport A from Brazilian state S during time window WS 11,25; pV is the YFV vaccination coverage of US travelers to Brazil41; and VE is the YFV vaccine efficacy42. We assumed that US and international (non-Brazilian and non-US) travelers visiting Brazil were at the same risk of exposure to YFV as the population of Brazil. Therefore, the Brazilian state-specific per capita risk of YFV infection was calcu- lated as: λ = ⋅ − ⋅ ˆC L pop pop W ( ) , (8) S S W S S V s , , S (8) Scientific Reports \| (2019) 9:20420 \| https://doi.org/10.1038/s41598-019-56521-9 www.nature.com/scientificreports/ where ˆCS W , S is the estimated total number of YFV cases in Brazilian state S during time window WS 5,7; L is the length of stay of US travelers in Brazil34,35; popS is the population size of Brazilian state S39; and popS V , is the num- ber of individuals with YFV vaccine-induced immunity in Brazilian state S. We assumed that international (non-Brazilian and non-US) travelers had the same vaccination coverage and length of stay of US travelers. The distribution of the length of stay was obtained by fitting the frequency of the number of nights US residents spent in South America reported in35. The term popS V , for the 2016–2017 YF outbreak was given by the sum of the average population size of municipalities in Brazilian state S28 (pop ) S M , , multiplied by the number of municipal- ities in Brazilian state S (M ) vcS with average vaccination coverage (vc ) S during the 2016–2017 YF outbreak7 and the YFV vaccine efficacy VE: ∑ = ⋅ ⋅ ⋅ . Methods We also performed multiple sensitivity analyses to explore the impact of uncertainty of vaccination coverage on the risk of YFV importation among both US and international residents and residents of Brazil traveling to the United States (Supplementary Information, Sensitivity Analysis section 3 and Counterfactual Analysis section 4). References 1. Barnett, E. D. Yellow fever: epidemiology and prevention. Clin. Infect. Dis. 44(6), 850–6 (2007). 1. Barnett, E. D. Yellow fever: epidemiology and prevention. Clin. Infect. Dis. 44(6), 850–6 (2007). 1. Barnett, E. D. Yellow fever: epidemiology and prevention. Clin. Infect. 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Acknowledgementsh g This work is jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 programme supported by the European Union. Ilaria Dorigatti, Christl A. Donnelly and Tini Garske also acknowledge research funding from an Imperial College Junior Research Fellowship, a Sir Henry Dale Fellowship funded by Wellcome Trust and the Royal Society [grant 213494/Z/18/Z] and the National Institute for Health Research. I.D., S.M. and A.G. conceived the study. I.D. and S.M. analyzed the data and drafted the manuscript. All authors interpreted the results, reviewed and approved the manuscript. D., S.M. and A.G. conceived the study. I.D. and S.M. analyzed the data and drafted the manuscript. All authors nterpreted the results, reviewed and approved the manuscript. 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https://openalex.org/W3132126450	https://www.researchsquare.com/article/rs-193345/v1.pdf?c=1631888911000	English	null	Recurrent Brain Metastases: The Role of Resection of in the Era of Molecular Medicine	Research Square (Research Square)	2,021	cc-by	3,818	Nadine Hessler Nadine Hessler University Hospital Cologne: Uniklinik Koln Stephanie T Jünger (  stephanie.juenger@uk-koeln.de ) University Hospital Cologne: Uniklinik Koln Anna-Katharina Meissner University Hospital Cologne: Uniklinik Koln Martin kocher University Hospital Cologne: Uniklinik Koln Roland H Goldbrunner University Hospital Cologne: Uniklinik Koln Stefan Josef Grau University Hospital Cologne https://orcid.org/0000-0002-9 Purpose To evaluate the efficacy of surgical resection for pretreated, recurrent brain metastases (BM) in the era of molecular oncologic medicine. Results In total, 107 patients with different primary tumor entities and individual previous treatment for BM were included. Primary tumors comprised non-small cell lung cancer (NSCLC) (37.4%), breast cancer (19.6%), melanoma (13.1%), gastro-intestinal cancer (10.3%) and other, rare entities (19.6%). The number of previous treatments of BM ranged from one to four; these comprised: resection only, focal or whole brain radiotherapy, brachytherapy and radiosurgery. BM-related symptoms were present in 73.8% of the patients. Median pre-operative Karnofsky Performance Score (KPS) was 70% (range 40–100) which was improved to 80% (range 0-100) after surgery. The complication rate was 26.2% and two patients died during the perioperative period. Postoperative local radio-oncologic and/or systemic therapy regimens were applied in 67 (62.6%) patients. Median postoperative EFS and OS were 7.1 (95%CI 5.8–8.2) and 11.1 (95%CI 8.4–13.6) months, respectively. The clinical status (postoperative KPS ≥ 70 (HR 0.27 95%CI 0.16–0.46; p < 0.001) remained the only independent factor for survival in multivariate analysis. Conclusion Surgical resection of recurrent BM may improve the clinical status and thus OS, but is associated with a high complication rate; thus, careful patient selection is crucial. Patients and Methods: In a retrospective single center study, patients were analyzed who had undergone surgical resection of recurrent BM between 2007 and 2019. Intracranial event-free survival (EFS) and overall survival (OS) were evaluated by Kaplan-Maier and Cox regression analysis. Research Article Research Article Keywords: recurrent brain metastasis, radio-oncological treatment, overall survival Posted Date: February 17th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-193345/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/12 Page 1/12 Page 1/12 Selection of study population For this retrospective, monocentric cohort study, we queried our database for patients who had undergone resection of previously treated, recurrent BM in our department between 2007 and 2019. The following parameters were identified: demographic/baseline characteristics (gender, age at time of diagnosis and at time of surgery of the recurrent lesions), tumor characteristics (type of primary tumor, local and systemic tumor status, number and location of recurrent BM, time to recurrence since initial cancer diagnosis, time to recurrence since initial diagnosis of BM), therapeutic interventions (previous treatment, types of adjuvant therapy, number of previous recurrences), clinical status (neurological symptoms, pre- and postoperative Karnofsky-Performance-Scale (KPS)), and outcome measures (surgery-related complications, time to further recurrence after surgery). Data were retrieved from the electronic hospital database and paper charts. The study was approved by the local ethical committee (reference number: 18–089). Indication for surgery Indication for surgery was based on suspected recurrent BM detected by magnetic resonance imaging (MRI) or, if required, amino acid positron emission tomography (PET). All decisions were made within an interdisciplinary institutional tumor board comprising board-certified neurosurgeons, neuro-oncologists, medical oncologists, neuro-radiologists, neuropathologists, and palliative care physicians. In general, criteria for (re-)operation were accessibility and size of the lesion allowing safe resection, good clinical condition, adjuvant treatment options (re-irradiation, chemotherapy, or molecular therapy), necessity for obtaining tissue diagnosis, rapid progression leading to neurological complications, or no remaining treatment options other than surgery. Histopathological diagnosis was made by the local Departments of Introduction Due to rising medical standards and novel therapeutic regimens, the number of patients with brain metastases (BM) is increasing [1–4]. Although BM are considered, in principle, a fatal event for oncological patients, treatment paradigms are changing and affected patients are nowadays frequently treated with repeated non-invasive therapeutic procedures such as radiotherapy and systemic oncological treatments. While the role of neurosurgical resection of primary and symptomatic BM is clearly defined [5, Page 2/12 Page 2/12 6], the application of surgery for recurrent BM, especially after previous multimodal treatments, remains an individual decision [7], particularly since underlying studies [8, 9] are scarce. 6], the application of surgery for recurrent BM, especially after previous multimodal treatments, remains an individual decision [7], particularly since underlying studies [8, 9] are scarce. In the light of the available novel comprehensive oncological treatment regimens, including several types of focused radiotherapy and targeted medical treatments that generally lead to an increased overall survival (OS), the role of surgery in the context of relapse, especially for symptomatic BM, needs to be clearly defined. Surgical treatment and follow-up The extent of resection was assessed by early postoperative MRI performed within 48 hours after surgery and classified as gross total resection when no residual contrast-enhancing tissue was visible on T1- weighted imaging. Any residual contrast enhancement was defined as subtotal resection. Clinical and radiological follow-up was performed in three-monthly intervals. Intracranial failure was defined as newly developing contrast enhancement in brain MR imaging. Page 3/12 Complications were classified according to the Common Terminology Criteria of Adverse Events (CTCAE) by the National Cancer Institute (NCI) [10, 11] (see Table 3). Statistical analysis For descriptive statistics, continuous values are given in median and range, ordinal and categorical variables are stated in numbers and percentages. Post-surgical survival time was calculated from the date of surgery to date of death or last follow-up; patients alive at the time of their last follow up were censored. Event-free survival (EFS) was assumed in the case of no intracranial relapse. Predictive variables for both endpoints were identified by univariate and multivariate analysis. For categorical variables, the log-rank test was used to identify covariates with an influence on EFS and OS and visualized in Kaplan-Meier plots. For continuous variables, Hazard ratios were calculated using Cox regression. P-values < 0.05 were considered statistically significant. Variables with a significant impact were included in a multivariate Cox regression model. All statistical analyses were performed using SPSS Statistics Version 25 (IBM, Armonk, NY, USA). Previous treatment and clinical status at ti Previous cerebral treatment comprised one or more local and/or systemic therapies including surgery, whole brain radiation therapy (WBRT), focal/partial brain radiation therapy (fRT), stereotactic radiosurgery (sRS) and brachytherapy (BT). The number and detailed information on previous treatment modalities were recorded (Table 2). At the time of resection, 79 (73.8%) patients suffered from BM-related symptoms including vertigo, hemiparesis, cognitive impairment, epilepsy, and headache. The median preoperative Karnofsky performance scale (KPS) was 70 (range 40–100). Previous treatment and clinical status at time of recurrence Previous treatment and clinical status at time of recurrence Baseline parameters and demographics The study included 107 patients with a median age of 61 (range 26–83) years at the time of operation. Forty-three patients (40.2%) were male. Primary tumor entities comprised non-small cell lung cancer (NSCLC) (37.4%), breast cancer (19.6%), melanoma (13.1%), gastro-intestinal tumor (GIT) (10.3%) and other, rare entities (19.6%). At the time of BM relapse, extracranial metastases were present in 61 (57.0%) patients. Detailed demographic and clinical data are displayed in Table 1. Survival In 51 patients (47.7%), a cerebral treatment failure was detected, resulting in a median EFS of 7.1 (95%CI 5.8–8.2) months. None of the factors analyzed influenced EFS. In 51 patients (47.7%), a cerebral treatment failure was detected, resulting in a median EFS of 7.1 (95%CI 5.8–8.2) months. None of the factors analyzed influenced EFS. At the time of analysis, 73 (68.2%) patients had died. Median OS time was 11.1 (95%CI 8.4–13.6) months. Three patients (2.8%) died within the first 30 days after surgery, two from surgical complications. In the remaining cohort, the causes of death were systemic disease progression in 12 patients (11.2%), cerebral progression in 37 patients (34.6%) and other causes in two patients (1.9%). In the remaining patients, the cause of death was unspecified. In univariate analysis, a pre- and postoperative KPS ≥ 70 (p = 0.002 and p < 0.001) and neurological symptoms caused by BM (p = 0.036) were prognostic for survival, while all other parameters (age, primary, number of BM, location, previous treatment, application and type of local treatment, ongoing systemic treatment, extracranial status) showed no significant impact. In multivariate analysis only the postoperative clinical status (HR 0.207 95%CI 0.0816–0.3436; p < 0.001) remained independent. Surgical treatment, complications, and adjuvant treatment At time of surgery 80 (74.8%) patients suffered from a single recurrent BM, 19 patients (17.8%) from oligo- (2–3) BM and eight patients (7.5%) from multiple (≥ 4) BM. Resection of the target lesion was complete (gross total resection) in 78 (72.9%) patients. Surgery was performed in all patients under general anesthesia with the aid of neuro-navigation, ultrasound, and intra-operative monitoring, if required. Surgery improved the Karnofsky performance scale to a median of 80 (0-100). After resection, adjuvant local treatment was administered in 67 patients (62.6%), comprising WBRT (n = 5), fRT (n = 49), Page 4/12 Page 4/12 stereotactic radiosurgery (n = 11), or a combination of the latter two (n = 2). Medical treatment was initiated or continued in 37 (34.6%) patients (Table 2). Surgery-related complications occurred in 28 patients (26.2%) with two patients dying during the acute phase. Details on postsurgical complications and their grading are displayed in detail in Table 3. Discussion Possible subsequent development of resistance may leave few remaining therapeutic approaches. In cases of extensive pretreatment by radiotherapy, resection might therefore be the only local treatment option left. As the cerebral progression partly reflects treatment failure of previous irradiation, the negligible impact of postoperative radiotherapeutic measures on either EFS or OS in this present study is not surprising. The major argument for surgery in this patient cohort may be seen in the clinical improvement which is, in line with the current literature, the strongest predictor for further survival after recurrent BM treatment [4, 8, 16]. The major argument for surgery in this patient cohort may be seen in the clinical improvement which is, in line with the current literature, the strongest predictor for further survival after recurrent BM treatment [4, 8, 16]. As opposed to the clinical improvement mentioned above, the postoperative complication rate was high and included a critical number of life-threatening complications. This is in contrast to other studies reporting on resection in the setting of initial BM diagnosis, where neurosurgery was usually well tolerated and proved to be feasible and safe [8, 14–16, 18, 19]. As opposed to the clinical improvement mentioned above, the postoperative complication rate was high and included a critical number of life-threatening complications. This is in contrast to other studies reporting on resection in the setting of initial BM diagnosis, where neurosurgery was usually well tolerated and proved to be feasible and safe [8, 14–16, 18, 19]. The high incidence of complications may be explained by the general condition of oncological patients. The underlying malignancy and/or multiple varied (systemic) pre-treatments would impair wound healing and hemostasis, and increase cardio-pulmonary complications [4, 20]. Furthermore, patient age was described as independently correlating with clinical outcome, since comorbidities are more common in elderly patients [4, 21, 22]. In this context, the indication for re-resection of BM must be based upon multidisciplinary consent that takes into account the patients´ general condition, the possible (and probable) clinical benefit, and the availability of further treatments. Conclusion Surgical resection of recurrent BM may improve patients´ clinical status and possibly indirectly prolong survival but carries a high risk for surgery-related complications. Thus, careful patient selection in a multidisciplinary comprehensive setting is mandatory. Discussion Due to the availability of effective systemic therapies and closer surveillance during follow-up, the number of patients diagnosed with recurrent BM is increasing [1–4]. However, the inevitable question of how to treat these patients adequately after cerebral progression still remains unsolved, especially for patients maintaining a good clinical condition over a longer period of time before BM recurrence [4]. Most studies with respect to treatment of recurrent BM focus on a single treatment option such as (re-) radiosurgery or re-irradiation [12]. Surgery is well established as a first-line treatment for larger and symptomatic BM. However, the role of surgery for pretreated, recurrent BM is not yet defined, and only scarce data, originating from the pre- molecular era, are available. Only a few studies have reported on the feasibility of (re-)surgery in patients with single or multiple recurrent BM [8, 9, 13, 14]. They included narrowly defined patient cohorts previously treated by either surgery [8, 15] or sRS [14, 16], and reported median survival rates after resection of between 7.5 and 11.5 months. With 11.1 months the survival rate in the present study was within the range of the previously reported data. The high rate of fatal cerebral progression in this series compared to previous studies may be due to the fact that besides surgery, most therapeutic options are used, leading to a lack of salvage treatment in the case of further cerebral progression. As surgical resection may result in rapid symptom release by reducing the mass effect, the subsequent improvement in the patient’s clinical condition, possibly in Page 5/12 combination with a re-evaluation of the tumor’s molecular status, may represent the major benefit of surgery. Since a fair clinical status is a prerequisite for radio-oncological and a tailored adjuvant treatment, this may positively influence the outcome, as observed before [17]. However, this benefit could not be observed with statistical significance for the patients in the present study. combination with a re-evaluation of the tumor’s molecular status, may represent the major benefit of surgery. Since a fair clinical status is a prerequisite for radio-oncological and a tailored adjuvant treatment, this may positively influence the outcome, as observed before [17]. However, this benefit could not be observed with statistical significance for the patients in the present study. Probably, the specific condition of this study’s population offers an explanation since it comprises patients who had already undergone extensive oncological treatment. Abbreviations BM: brain metastases Disclosure All authors declare no conflict of interests. BM: brain metastases BM: brain metastases BT: brachytherapy BT: brachytherapy BT: brachytherapy Page 6/12 CTCAE: Common Terminology Criteria of Adverse Events CTx: chemotherapy KPS: Karnofsky performance scale MRI: Magnetic resonance imaging NCI: National Cancer Institute NSCLC: non-small cell lung cancer OS: overall survival PET: positron emission tomography RCC: renal cell cancer SCLC: small cell lung cancer sRS: stereotactic radiosurgery TT: targeted therapy WBRT: whole brain radiation therapy Code availability: n/a Authors' contributions: N.H, S.T.J.: collected the data and wrote the paper, A-K.M. collected the data, M.K. performed the analysis, R.G. & S.G. conceived the project, designed the analysis. Authors' contributions: N.H, S.T.J.: collected the data and wrote the paper, A-K.M. collected the data, M.K. performed the analysis, R.G. & S.G. conceived the project, designed the analysis. Acknowledgement: Funding: no funding was received Page 7/12 Conflicts of interest/Competing interests: all authors declare no conflict of interests Ethics approval:The study was approved by the local ethical committee (reference number: 18-089) Consent to participate: n/a Consent for publication: n/a Availability of data and material: data are available upon reasonable request Code availability: n/a Authors' contributions: N.H, S.T.J.: collected the data and wrote the paper, A-K.M. collected the data, M. performed the analysis, R.G. & S.G. conceived the project, designed the analysis. https://doi.org/10.3171/2009.4.JNS09246 10. Atkinson TM, Ryan SJ, Bennett AV et al (2016) The association between clinician-based common terminology criteria for adverse events (CTCAE) and patient-reported outcomes (PRO): a systematic review. Support Care Cancer 24:3669–3676. https://doi.org/10.1007/s00520-016-3297-9 11. 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Kano H, Kondziolka D, Zorro O et al (2009) The results of resection after stereotactic radiosurgery for brain metastases: Clinical article. JNS 111:825–831. https://doi.org/10.3171/2009.4.JNS09246 17. Schödel P, Jünger ST, Wittersheim M et al (2020) Surgical resection of symptomatic brain metastases improves the clinical status and facilitates further treatment. Cancer Med 9:7503–7510. https://doi.org/10.1002/cam4.3402 18. Kamp MA, Dibué M, Niemann L et al (2012) Proof of principle: supramarginal resection of cerebral metastases in eloquent brain areas. Acta Neurochir (Wien) 154:1981–1986. https://doi.org/10.1007/s00701-012-1463-5 19. Kamp MA, Dibué M, Santacroce A et al (2013) The tumour is not enough or is it? Problems and new concepts in the surgery of cerebral metastases. Ecancermedicalscience 7:306. https://doi.org/10.3332/ecancer.2013.306 20. Gaspar L, Scott C, Rotman M et al (1997) Recursive partitioning analysis (RPA) of prognostic factors in three Radiation Therapy Oncology Group (RTOG) brain metastases trials. Int J Radiat Oncol Biol Phys 37:745–751. References 1. Shen CJ, Lim M, Kleinberg LR (2016) Controversies in the Therapy of Brain Metastases: Shifting Paradigms in an Era of Effective Systemic Therapy and Longer-Term Survivorship. Curr Treat Options Oncol 17:. https://doi.org/10.1007/s11864-016-0423-3 2. Steeg PS, Camphausen KA, Smith QR (2011) Brain metastases as preventive and therapeutic targets. Nature Reviews Cancer 2. Steeg PS, Camphausen KA, Smith QR (2011) Brain metastases as preventive and therapeutic targets Nature Reviews Cancer 3. Chamberlain MC, Baik CS, Gadi VK et al (2017) Systemic therapy of brain metastases: Non-small cell lung cancer, breast cancer, and melanoma. Neuro-Oncology 19:i1–i24. https://doi.org/10.1093/neuonc/now197 4. 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Neurosurg Rev 28:115–119. https://doi.org/10.1007/s10143- 004-0364-3 Page 9/12 Tables Table 1: Baseline demographic characteristics and parameters Table 1: Baseline demographic characteristics and parameters Parameter No. % Median Range Age at operation ≤ 65 years > 65 years 75 32 70.1 29.9 61 26–83 Gender Male female 43 64 40.2 59.8 Primary tumor non-small cell lung cancer Breast cancer Melanoma Gastro-intestinal tumorOther 40 21 14 11 21 37.4 19.6 13.1 10.3 19.6 Extracranial disease Stable Non-stable 46 61 43.0 57.0 Symptoms (multiple references possible) Cerebellar Cognitive Hemiparesis Seizures Headache Impaired vision Aphasia Others 19 11 25 14 22 15 13 18 17.8 10.3 23.4 13.1 20.6 14.0 12.1 16.8 Table 2: Pre- and postsurgical treatment, surgery, and complications Table 2: Pre- and postsurgical treatment, surgery, and complications Table 2: Pre- and postsurgical treatment, surgery, and complications Page 10/12 Parameter No. % Previous treatment Resection Radiotherapy Whole brain radiotherapy Partial brain radiotherapy Stereotactic radiosurgery Brachytherapy 44 30 24 53 8 41.1 28.0 22.4 49.5 7.5 Number of recurrent BM 1 BM 2–3 BM ≥ 4 BM 80 19 8 74.8 17.8 7.5 Extent of resection Gross total Subtotal 78 29 72.9 27.1 Adjuvant local treatment None Radiotherapy Whole brain radiotherapy Partial brain radiotherapy Stereotactic radiosurgery Combination Postsurgical systemic therapy 40 67 5 49 11 2 37 37.4 62.6 4.7 45.8 10.3 1.9 34.6 Cause of death (n = 73) Neurological Systemic Others unknown 37 12 2 22 34.6 11.2 1.9 20.6 Table 3: Complications stratified according to CTCAE (Common Terminology Criteria of Adverse Events) Parameter No. Tables % Previous treatment Resection Radiotherapy Whole brain radiotherapy Partial brain radiotherapy Stereotactic radiosurgery Brachytherapy 44 30 24 53 8 41.1 28.0 22.4 49.5 7.5 Number of recurrent BM 1 BM 2–3 BM ≥ 4 BM 80 19 8 74.8 17.8 7.5 Extent of resection Gross total Subtotal 78 29 72.9 27.1 Adjuvant local treatment None Radiotherapy Whole brain radiotherapy Partial brain radiotherapy Stereotactic radiosurgery Combination Postsurgical systemic therapy 40 67 5 49 11 2 37 37.4 62.6 4.7 45.8 10.3 1.9 34.6 Cause of death (n = 73) Neurological Systemic Others unknown 37 12 2 22 34.6 11.2 1.9 20.6 Table 3: Complications stratified according to CTCAE (Common Terminology Criteria of Adverse Events) Table 3: Complications stratified according to CTCAE (Common Terminology Criteria of Adverse Events) Page 11/12 Complication CTCAE grade n New neurological deficit 2 6 Wound healing disorder 2 5 Wound healing disorder requiring surgery (revision, external drain) 3 4 CSF disorder requiring surgery 3 3 Postoperative haemorrhage requiring intervention 4 1 Cerebral ischemia 4 1 Cerebral edema 4 1 Pulmonary embolism 3 2 Carotic artery dissection 4 1 Pneumonia, sepsis 3 2 Postoperative death 5 2 Carotic artery dissection 4 1 Pneumonia, sepsis 3 2 Postoperative death 5 2 Figures Figure 1 Overall survival (OS), depicting the impact of the clinical status after surgery. Kaplan-Meier plot. Figures Figures Figure 1 Overall survival (OS), depicting the impact of the clinical status after surgery. Kaplan-Meier plot. Page 12/12 Page 12/12
https://openalex.org/W3095503612	https://arpi.unipi.it/bitstream/11568/1058862/1/Onchocerca%20in%20donkeys.pdf	English	null	Incidental Detection of Onchocerca Microfilariae in Donkeys (Equus asinus) in Italy: Report of Four Cases	Frontiers in veterinary science	2,020	cc-by	8,177	ORIGINAL RESEARCH published: 06 November 2020 doi: 10.3389/fvets.2020.569916 Edited by: Edited by: Donato Traversa, University of Teramo, Italy Reviewed by: Charles D. Mackenzie, Task Force for Global Health, United States Susan Catherine Cork, University of Calgary, Canada Reviewed by: Charles D. Mackenzie, Task Force for Global Health, United States Susan Catherine Cork, University of Calgary, Canada Correspondence: Roberto Amerigo Papini roberto.amerigo.papini@unipi.it orcid.org/0000-0002-5928-0663 Reviewed by: Charles D. Mackenzie, Task Force for Global Health, United States Susan Catherine Cork, University of Calgary, Canada Correspondence: Roberto Amerigo Papini roberto.amerigo.papini@unipi.it orcid.org/0000-0002-5928-0663 Incidental Detection of Onchocerca Microﬁlariae in Donkeys (Equus asinus) in Italy: Report of Four Cases Roberto Amerigo Papini, George Lubas and Micaela Sgorbini Department of Veterinary Sciences, Veterinary Teaching Hospital “Mario Modenato”, University of Pisa, Pisa, Italy This paper reports the occurrence for the ﬁrst time in Italy of autochthonous Onchocerca infection in donkeys. Four jennies, bred on the same farm, were referred to the Veterinary Teaching Hospital of Pisa for a check-up on ovarian activity (n = 3) or for veterinary support during the delivery (n = 1). Microﬁlariae were incidentally detected during the blood smear examination of one jenny. Peripheral blood samples were then collected from the other three jennies and the presence of microﬁlariae was investigated by Knott’s test. Circulating unsheathed microﬁlariae were identiﬁed in all the animals. The level of microﬁlaraemia was between 1 and 31 microﬁlariae in 2 mL of blood. Hematological changes showed moderate eosinophilia in one case or both remarkable eosinophilia and basophilia in another case. Based on molecular ﬁndings by PCR and sequencing, the microﬁlariae showed 98% sequence similarity with Onchocerca sp. in the NCBI GenBank database (Accession No.: MK541848.1). The present report provides evidence that Onchocerca is an etiological agent of parasitic infection in donkeys in Italy. Our ﬁndings highlight the importance of screening in donkeys for Onchocerca even in the absence of clinical indications. Keywords: microﬁlariae, Onchocerca, onchocerciasis, donkey, Equus asinus, Italy INTRODUCTION Onchocerca cervicalis, Onchocerca reticulata, and Onchocerca raillieti (Onchocercidae, Spirurida) are ﬁlarial thread-like nematodes. O. cervicalis and O. reticulata infect equines worldwide (1). O. cervicalis adult females (50–70 cm long) and males (7–10 cm long) live in the ligamentum nuchae. O. reticulata adult worms can measure up to 75 cm (females) or 15–20 cm (males) and inhabit the ﬂexor tendons and suspensory ligament of the fetlock (2). O. raillietti has only been reported in donkeys in Africa (1) and adult worms live in the ligamentum nuchae (3). They are all viviparous. Adult females release microﬁlariae (L1) measuring 190–310 µm for O. cervicalis, 330–370 µm for O. reticulata (4), and 180–217 µm for O. raillieti (3). When microﬁlariae are released by the females, they migrate through connective tissues and accumulate in the lymphatics of the upper dermis (1, 2). The highest concentrations can be found along the linea alba in the ventral midline, especially near the umbilicus (1, 3, 4). Microﬁlariae are also commonly found in the dermis of the face, neck, withers, thorax, and eyes (1). O. raillietti microﬁlariae have also occasionally been found in the dermis of the head, back, hips, forelimbs, hind limbs, and perineum, though to a limited extent (3). Information is only available for the life cycle of O. cervicalis (4), however the transmission pathways of O. cervicalis, O. reticulata, and O. raillieti are likely to be similar. These parasites are all transmitted by blood-sucking midges of Culicoides Specialty section: This article was submitted to Comparative and Clinical Medicine, a section of the journal Frontiers in Veterinary Science Specialty section: This article was submitted to Comparative and Clinical Medicine, a section of the journal Frontiers in Veterinary Science Received: 05 June 2020 Accepted: 13 October 2020 Published: 06 November 2020 ase Presentation and Blood Collection Case Presentation and Blood Collection In June 2019, four adult, pluriparous jennies of the Amiatina breed with ages ranging from 6 to 14 years (median age 13 years) were presented to the Veterinary Teaching Hospital (VTH) of the University of Pisa (geographical coordinates: 43◦40 ′48 ′′N 10◦20 ′55 ′′E). The jennies were from a donkey farm located in central Italy, where animals are used for milk production and reared outdoor in a free animal housing system. On the farm, water ad libitum and food were available in shaded resting spots. The serum biochemical assays were performed on a combined spectrophotometric-immunoturbidimetric device (Analyzer SAT 450 R⃝, Medical System, Guidonia, Rome, Italy) including: total plasma protein, albumin, gamma glutamyl transferase, aspartate aminotransferase, creatine phosphokinase, blood urea nitrogen, creatinine, calcium, phosphate, total bilirubin, and direct bilirubin. In addition, few electrolytes such as sodium, potassium and chloride were assessed with an ion-selective electrodes device (Electrolyte Analyzer GE200 R⃝, Medical System, Guidonia, Rome, Italy). Three out of the four were barren jennies admitted to the VTH for an examination of ovarian activity. The other jenny was pregnant and was diagnosed with rupture of the prepubic tendon before pregnancy, thus this jenny needed veterinary care at the time of delivery. The blood cell counter was set up to evaluate the parameters as equine species. All the reference intervals (RIs) used were derived from an internal assessment carried out by the veterinary clinical pathology laboratory with multi-year experience in examining donkey blood. When admitted to the VTH, jennies were housed in collective paddocks 24 h a day, fed with meadow hay ad libitum along with commercial equine feed (Equiﬁoc R⃝, Molitoria Val di Serchio, Italy) according to the NCR energy recommendations (National Research Council, NRC, 2007), as well as having free access to clean and fresh water. Detection of Circulating Microﬁlariae Since microﬁlariae were detected through the blood smear examination of jenny II (Figure 1), the occurrence of peripherally-circulating microﬁlariae was also evaluated in the other three jennies. Microﬁlariae detection was performed using a modiﬁed Knott’s test on peripheral EDTA blood samples collected, as described above. Brieﬂy, 8 mL of distilled water were added to 2 mL of whole EDTA blood and mixed thoroughly. Following centrifugation at 3,000 rpm for 5 min, the supernatant was discarded. Blood Analysis Blood Analysis Complete blood counts were performed using a combined laser-impedance cell counter (ProCyte R⃝, Idexx Laboratories, Milan, Italy) including red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell distribution width, total leukocyte count, absolute values of neutrophils, lymphocytes, monocytes, eosinophils, and basophils, platelet count, and mean platelet volume. In order to complete the information on the morphological aspects of red blood cells, white blood cells, and platelets, and to assess the leukocyte diﬀerential counts in comparison to the results provided by the cell counter, fresh blood smears were prepared and stained with May-Grundwald-Giemsa (MGG) using an automatic stainer (Wescor Aerospray 7150 R⃝, Delcon, Milan, Italy). Using bright-ﬁeld light microscopy, blood smears were screened at 20× and then examined at 100× by a trained clinical pathologist. q Many studies have been conducted on epidemiological, clinical, diagnostic, pathological, and therapeutic aspects of Onchocerca infection in horses as reviewed by Dagnaw et al. (1). However, with the exception of a relatively recent case report in Italy (7), all these studies are dated. Moreover, only a limited number of studies have investigated the parasitosis in donkeys, almost all of the published studies in donkeys were from African countries, and most focus on O. raillieti (3, 5, 8–10). Currently, little or nothing is known about the current spread of Onchocerca infections in donkeys in Italy. In order to ﬁll this gap, we report a case of autochthonous infection in jennies. We discuss various epidemiological and clinical aspects of the infection in this host species. Frontiers in Veterinary Science \| www.frontiersin.org Citation: Papini RA, Lubas G and Sgorbini M (2020) Incidental Detection of Onchocerca Microﬁlariae in Donkeys (Equus asinus) in Italy: Report of Four Cases. Front. Vet. Sci. 7:569916. doi: 10.3389/fvets.2020.569916 November 2020 \| Volume 7 \| Article 569916 Frontiers in Veterinary Science \| www.frontiersin.org Papini et al. Onchocerca in Donkeys species (1, 2, 4, 5). Mosquitoes (Aedes aegypti) also act as intermediate hosts for O. cervicalis (6). Microﬁlariae are ingested by insect vectors while feeding on an infected animal. After development within the intermediate host to reach the infective third-stage, L3 larvae are transmitted to other susceptible deﬁnitive hosts at the subsequent blood meal (4). same donkey farm. Blood collection was performed in the same way as previously described. The samples were labeled for animal identiﬁcation (jennies II–IV). RESULTS All four jennies were apparently healthy on the basis of history and physical examination at admission to the VTH. Three of them (II, III, and IV) remained in this condition throughout hospitalization. However, one jenny (I) was apparently healthy at admission to the VTH but presented signs of colic 2 days after delivering her foal. The results of the complete blood count for all four jennies are reported in Table 1. In jenny I, the values evidenced an anemic condition with normocytic hyperchromic red blood cells. Hyperchromia was due the lipemic sample collected, which interferes with the correct spectrophotometric reading of hemoglobin and induces a high value of mean corpuscular hemoglobin concentration. In the leukogram, neutrophilia, and monocytosis were found along with lymphopenia, all three conditions could be related to a long-lasting inﬂammatory process or simply related to stress. In jennies II and III, the values did not reveal anything particular in the red blood cell compartment. In the leukogram, eosinophilia was found in both animals and in jenny III it was substantial as was the occurrence of basophilia. In jenny IV, the results of the complete blood count were all within the RIs. All four jennies were apparently healthy on the basis of history and physical examination at admission to the VTH. Three of them (II, III, and IV) remained in this condition throughout hospitalization. However, one jenny (I) was apparently healthy at admission to the VTH but presented signs of colic 2 days after delivering her foal. The results of the complete blood count for all four jennies are reported in Table 1. In jenny I, the values evidenced an anemic condition with normocytic hyperchromic red blood cells. Hyperchromia was due the lipemic sample collected, which interferes with the correct spectrophotometric reading of hemoglobin and induces a high value of mean corpuscular hemoglobin concentration. In the leukogram, neutrophilia, and monocytosis were found along with lymphopenia, all three conditions could be related to a long-lasting inﬂammatory process or simply related to stress. In jennies II and III, the values did not reveal anything particular in the red blood cell compartment. In the leukogram, eosinophilia was found in both animals and in jenny III it was substantial as was the occurrence of basophilia. In jenny IV, the results of the complete blood count were all within the RIs. ase Presentation and Blood Collection The sediment was re-suspended in two drops of distilled water, removed with a 1 mL Pasteur pipette, and examined with a light microscope at 100× magniﬁcation. The number of microﬁlariae was counted in the Knott’s test sediment. See the Results section for details on morphometric features. After microscopic examination, another aliquot (2 mL) of EDTA blood sample from each positive jenny was stored at −20◦C prior to shipment to a sequencing service provider. Upon admission to the VTH, all jennies underwent a complete clinical assessment and coprological examinations by means of a ﬂotation technique with saturated solution of NaCl (speciﬁc gravity = 1.20). The pregnant jenny showed signs of colic 2 days after delivery and thus underwent further clinical investigations. A complete blood count and biochemistry analyses were also performed. For this purpose, a venous blood sample was drawn via jugular venipuncture and collected in two sterile 10 mL vacutainer tubes, one with K3-EDTA and one plain tube for serum harvesting. As soon as the blood collection was completed, the sample was labeled (jenny I), immediately transferred to the laboratory of veterinary clinical pathology, and examined. Likewise, a blood work was also carried out in the three barren jennies from the November 2020 \| Volume 7 \| Article 569916 2 Papini et al. Onchocerca in Donkeys FIGURE 1 \| Giemsa stained blood smear showing a single microﬁlaria of Onchoerca sp. detected in a jenny in Italy. In the microscopic ﬁeld, one eosinophil with characteristic feature of the asinine species, three lymphocytes, and one neutrophil can also be identiﬁed (×400 magniﬁcation). FIGURE 1 \| Giemsa stained blood smear showing a single microﬁlaria of Onchoerca sp. detected in a jenny in Italy. In the microscopic ﬁeld, one eosinophil with characteristic feature of the asinine species, three lymphocytes, and one neutrophil can also be identiﬁed (×400 magniﬁcation). Molecular Procedures from the owners for the participation of their animals in this study. Molecular analyses were performed by BMR Genomics (Padua, Italy). After thawing at room temperature, genomic DNA was extracted from positive blood samples using the commercial DNA IQTM System R⃝(Promega) based on paramagnetic beads, following the manufacturer’s instructions. A fragment (about 700 base pairs in length) of the mitochondrial gene for cytochrome c oxidase subunit 1 (COX1) was used as a DNA barcoding system and ampliﬁed. The PCR ampliﬁcation was carried out in a ﬁnal mixture containing 12.5 µL of Master Mix 2X (GoTaq R⃝Green Master Mix, Promega), 2 µL of genomic DNA extracted from blood, 1 µL (10 µM) of forward primer (5 ′- TGATTGGTGGTTTTGGTAA-3 ′), 1 µL (10 µM) of reverse primer (5 ′-ATAAGTACGAGTATCAATATC-3 ′), and deionized water, reaching a total reaction volume of 25 µL. The PCR reactions were subjected to the following conditions in thermal cycler (Mastercycler R⃝, Eppendorf): 95◦C × 2 min, then 5 cycles (95◦C × 40 s, 45◦C for 1.5 min, and 72◦C for 1.5 min), followed by 35 cycles (95◦C for 40 s, 50◦C for 1.5 min, and 72◦C for 1 min), and ﬁnally 72◦C for 10 min. The ampliﬁcation products were visualized after electrophoresis on 1.5% agarose gel. PCR products were puriﬁed by AMPure XP Beads (Beckman Coulter), sequenced, and aligned via BLAST analysis to detect their identity by retrieving similar sequences deposited in NCBI’s GenBank database (11, 12). RESULTS The biochemistry analysis in jenny I revealed renal insuﬃciency highlighted by an increase in both urea (48 mg/dL, RI = 9–31) and creatinine (2.2 mg/dL, RI = 0.59–1.3) as well as a decrease in total proteins (5.6 g/dL, RI = 5.8–7.6) due to a mild reduction in albumin (2.2 g/dL, RI = 2.5–3.2). Both the cholesterol (195 mg/dl, RI = 55–115) and triglycerides Ethics Approval for the Study The mild increase in urea could be related both to anorexia, or along with high levels of creatinine, to an acute pre-renal insuﬃciency secondary to low ingestion of water. In the other three jennies all parameters on serum biochemistry and electrolytes investigated were within the RIs (values not reported). Since circulating microﬁlariae were incidentally detected by microscopic examination of a blood smear from one of the jennies, we thus examined the peripheral blood samples from each jenny by a modiﬁed Knott’s test, as described above. The Knott’s test results showed that all the four jennies presented microﬁlariae in their peripheral blood samples. Circulating microﬁlariae appeared to be morphologically identical to each other and from one jenny to another. They were actively motile with serpentine movements, showing the lack of a sac-like hyaline sheath at both ends and a short tail, which matched the characteristics exhibited by the microﬁlariae in MGG stained blood smears. The characteristic features of the microﬁlariae appeared to be consistent with Onchocerca microﬁlariae and no further morphometric investigations were carried out, as microﬁlariae found in Knott’s test sediments and in MGG stained smears appeared to be morphologically identical. However, the unusual site of the ﬁnding (i.e., peripheral blood stream) precluded any deﬁnitive conclusion and only a presumptive diagnosis of asymptomatic onchocerciasis could be made at that time. The levels of microﬁlaraemia were 31, 4, 3, and 1 microﬁlariae in 2 mL of blood samples from jennies II, I, IV, and III, respectively. The highest level of microﬁlaraemia was detected in jenny II which had been found positive from the blood smear. This suggests that positivity at the blood smear was probably related to the higher burden of circulating microﬁlariae. Table 1 shows the Knott’s test results. In jenny II, microﬁlaraemia was detected when the blood smear examination by conventional staining and optical microscopy reading was performed by an expert clinical pathologist (Figure 1). On the other hand, no microﬁlariae were detected through the blood smear evaluation in the other three jennies. MGG stained microﬁlariae had a wider and rounded anterior end, while the posterior end showed a short, thin, and sharply pointed tail. The microﬁlariae measured ∼214–229 µm in length and 4–5 µm in width. The most prominent feature was the lack of a faintly stained anterior and posterior sheath. Ethics Approval for the Study Ethical review was not required as per institutional guidelines/local legislation due to natural infections that occurred spontaneously. Written informed consent was obtained November 2020 \| Volume 7 \| Article 569916 Frontiers in Veterinary Science \| www.frontiersin.org 3 Onchocerca in Donkeys Papini et al. TABLE 1 \| Alterations detected in the complete blood count of four jennies found incidentally infected with Onchocerca microﬁlariae. TABLE 1 \| Alterations detected in the complete blood count of four jennies found incidentally infected with Onchocerca microﬁlariae. Hematological values Reference range I II III IV Red blood cells M/µL 3.40↓ 6.47 5.42 5.25 4.4–7.1 Hematocrit % 18.5 35.2 30.9 29.8 27–42 Hemoglobin g/dL 7.1↓ 12.1 10.9 10.5 9.1–14.7 Mean corpuscular volume fL 54.4 54.4 57.0 56.8 53–67 Mean corpuscular hemoglobin pg 20.9 18.7 20.1 20.0 17.6–23.1 Mean corpuscular hemoglobin concentration g/dL 38.4↑ 34.4 35.3 35.2 32–37.5 Red blood cell distribution width % 21.4 22.9↑ 20.5 22.4 16.1–22.5 Total leukocytes K/µL 14.78 10.95 15.69 6.40 6.2–16 Neutrophils K/µL 11.82↑ 5.04 5.49 3.07 2.4–6.3 Lymphocytes K/µL 1.18↓ 4.27 6.12 2.28 1.6–8.5 Monocytes K/µL 1.77↑ 0.33 0.31 0.32 0.0–0.8 Eosinophils K/µL 0.00 1.31↑ 3.45↑ 0.73 0.1–1.0 Basophils K/µL 0.00 0.00 0.31↑ 0.00 0.0–0.2 Platelets K/µL 296 190 295 190 95–360 Mean platelet volume fL 10.4 10.0 10.4 10.1 8.8–12.0 Appearance of plasma Lipemia ++ Clear Clear Clear NA Microﬁlaraemia at the blood smear 0 2 0 0 NA Microﬁlaraemia by Knott’s technique 4 31 1 3 NA *Reference range used internally at the Clinical Pathology Laboratory; the values out-the-range are in italics; ↓, decreased values; ↑, increased values; NA, not applicable. microﬁlariae between the diﬀerent species parasitizing donkeys falls within the range of 180–370 µm (3, 4). (663 mg/dl, RI = 53–248) showed high values. All the other parameters tested, namely calcium, phosphate, gamma glutamyl transferase, aspartate aminotransferase, creatine phosphokinase, total bilirubin, and direct bilirubin were within the RIs. In the electrolytes, sodium (125 mEq/L, RI = 128–138) and potassium (1.6 mEq/L, RI = 3.2–5.1) were low probably due to the interference of the lipemic sample, while chloride was in the normal range. Overall, the trend of these parameters along with the complete blood count results highlighted lipidosis due to a negative energy intake balance related to obstructive colic in a lactating jenny. The clinical symptoms were depression, anorexia, and oliguria (the jenny refused to drink). Frontiers in Veterinary Science \| www.frontiersin.org Ethics Approval for the Study Unfortunately, no follow-up was possible as the jenny with the ruptured prepubic tendon died after delivery, while the others returned to the farm a few days after check-up on ovarian activity. There have been few epidemiological studies on Onchocerca infection in donkeys. Reported prevalence values are 65.38% (21) and 82.98% (13) for O. cervicalis in Egypt, 34% for O. raillieti in Sudan (10), and 4.26% for O. reticulata in again Egypt (13). In another study, only one donkey was examined and found to be infected with O. cervicalis (22). No inﬂuence of sex on the infection rate has been observed (5, 10, 13). However, a statistically (p < 0.05) higher prevalence has been reported for O. raillieti (64.3%) in donkeys aged between 7 and 10 years (10) and for O. cervicalis (100%) in donkeys older than 15 years (10). Similarly, Onchocerca infection has been reported to be more common in older horses rather younger horses (23–25), particularly over 15 years of age (25, 26), as the prevalence of infection increases with age (25). Our detection of Onchocerca infection in jennies aged 6–14 years thus appears to be in agreement with the results of previous prevalence studies on donkeys and horses. Frontiers in Veterinary Science \| www.frontiersin.org Ethics Approval for the Study This suggested that on the basis of morphological features, they could be diﬀerentiated from the sheathed microﬁlariae of Setaria spp and belonged to the genus Onchocerca, in which the length of The coprological results were positive for gastrointestinal strongyle eggs. November 2020 \| Volume 7 \| Article 569916 4 Onchocerca in Donkeys Papini et al. TABLE 2 \| Accession numbers and description of sequences retrieved from GenBank producing signiﬁcant alignments with COX1 mitochondrial gene of microﬁlariae incidentally found in peripheral blood samples of four jennies in Italy. Accession no. Parasitic organism identiﬁcation Bit score E-value Max identity MK541848.1 Onchocerca sp. JJP-2019 1,138 0.0 98% KX898458.1 Onchocerca boehmi Supperer, 1934 835 0.0 90% LC318284.1 Onchocerca ﬂexuosa 833 0.0 90% AJ271616.1 Onchocerca gibsoni 833 0.0 90% AM749269.1 Onchocerca skrjabini 824 0.0 91% AP017692.1 Onchocerca ﬂexuosa 817 0.0 89% KX265050.1 Diroﬁlaria sp. “hongkongensis” 817 0.0 89% AM749270.1 Onchocerca skrjabini 813 0.0 91% AB518693.1 Onchocerca sp. wild boar 808 0.0 89% KX853323.1 Onchocerca boehmi Supperer, 1934 806 0.0 90% TABLE 2 \| Accession numbers and description of sequences retrieved from GenBank producing signiﬁcant alignments with COX1 mitochondrial gene of microﬁlariae incidentally found in peripheral blood samples of four jennies in Italy. However, since our four jennies were milk-producing animals, no treatment for ectoparasite or endoparasite control had never been performed at the donkey farm in order to prevent the risk of contamination with drug residues in milk intended for human consumption. Moreover, the Culicoides species, which may act as intermediate hosts for Onchocerca, are spread throughout Italy, including central regions (20) where the farm of the infected jennies is located. Therefore, the examined jennies likely acquired the infection locally on the farm and the source of the infection was the bite of infected midges. The BLAST search results showed that COX1 of microﬁlariae from the four examined jennies had the closest sequence similarity (i.e., 98%) with that of Onchocerca sp. available from GenBank (Accession Number MK541848.1). Sequences producing signiﬁcant alignments are shown in Table 2. Unfortunately, no follow-up was possible as the jenny with the ruptured prepubic tendon died after delivery, while the others returned to the farm a few days after check-up on ovarian activity. The owner was unwilling either to take them back to the VHT for further examination or to allow us to visit his farm at a later time. DISCUSSION cervicalis infection in horses with a variety of ocular lesions of the conjunctiva, cornea, uveal tract, lens and retina (35), ﬁstulous withers (36), and dermatitis of the ventral midline and/or limbs, shoulders, thorax, and withers (17). It is thought that microﬁlariae migrate through the bloodstream reaching the eyelids, cornea, conjunctiva, and uvea where they concentrate and that a marked inﬂammatory reaction is triggered by immune responses to antigens released by dead and dying microﬁlariae (1). However, the pathogenic role of O. cervicalis still remains uncertain and controversial (2, 37) as the infection can be seen in large numbers of horses with or without clinical signs (38). Since we observed the absence of dermatological and ocular signs, we concluded that neither adult worms nor microﬁlariae appear to cause any clinical evidence in donkeys. It is possible that donkeys may have a higher tolerance than horses to Onchocerca infection. Detection of Onchocerca microﬁlariae in the peripheral blood smear of donkeys or other equids is unusual and thus molecular analysis for conﬁrmatory purposes was needed. In our study, donkeys did not show any dermal or ocular sign that might suggest Onchocerca infection at the time of admittance to the VTH. Therefore, skin biopsies (see the Section Discussion below) were not included in the initial diagnostic workup. On the other hand, onchocerciasis diagnosis with skin biopsy is laborious and time consuming for equine practitioners and laboratory technicians, as well as being expensive for owners. Even when Onchocerca infection was suspected because of microﬁlariae morphological features, performing skin biopsies of the ventral midline on at least one of the jennies would have required the speciﬁc owner’s informed consent. Unfortunately, for this veterinary procedure, the jennies need to be sedated to avoid any risk for the operators and also an extended hospitalization time at the VTH, thus leading to economic losses because of the additional costs and lack of income for the owner of a milk production farm. This means that skin biopsies would never have been permitted by the owner given the lack of any clinical evidence and above all with no apparent economic beneﬁt for him. Consequently, we opted to use molecular analysis rather than skin biopsy to conﬁrm a case of microﬁlaraemia compatible with presumptive laboratory diagnosis of asymptomatic Onchocerca infection. This was despite the fact that a skin biopsy is the standard technique for diagnosing equine onchocerciasis. DISCUSSION Onchocerca in Donkeys climate, thus aﬀecting the seasonal distribution of the insect vectors (27), and that environmental conditions may arrest the development of microﬁlariae or inﬂuence their distribution in the host’s tissues (28). Similar seasonal variations likely occur in the donkey too in terms of the concentration, distribution, and occurrence of microﬁlariae in tissues, including the bloodstream. Therefore, our ﬁnding of Onchocerca microﬁlariae circulating in the peripheral blood of jennies in late spring (June) is in agreement with the results of other studies (27, 28). and 8 to 55,600, respectively (31). In other surveys on horses, microﬁlarial counts ranged from 18 to 42,446 microﬁlariae/skin snip (16) and a concentration of 19,770 microﬁlariae/mg was detected (26). All four Onchocerca infected jennies appeared to be healthy and clinically normal at the initial clinical examination. This is in agreement with Hussein and El Sammani (5) and Matov et al. (33) in O. cervicalis and O. raillieti infected donkeys, respectively. Matov et al. (33) reported that 12 out of 41 healthy donkeys harbored adults of O. cervicalis and eight had microﬁlariae in their eyes. Hussein and El Sammani (5) found that at post- mortem examination, ﬁbrous nodules ranging from 5–10 mm to 2–5 cm in diameter, containing degenerated parasites or ﬁlled with caseous and calciﬁed necrotic material, were scattered in the ligamentum nuchae. At histological examination, the authors found that live adult worms induced mild inﬂammation whereas dead worms elicited an intense inﬂammatory response with the inﬁltration of large numbers of lymphocytes, macrophages, and ﬁbroblasts intermingled with eosinophils. Moreover, heavy microﬁlarial skin densities were detected, as mentioned above, and 5 to 15 microﬁlariae per eye were found, mostly in the anterior chamber. However, despite these ﬁndings, none of the 373 infected donkeys had shown clinical signs that could be attributed to the infection, such as poll-evil and ﬁstulous withers, pruritus, blindness or periodic ophthalmia. Likewise, after surveying over 120 horses infected with O. cervicalis, Mellor (34) reported that pathological eﬀects of adult worms on the ligamentum nuchae were of little importance and that no pathological conditions were seen, in either the skin or the eyes, that could be deﬁnitely attributed to microﬁlariae. Matov et al. (33) reported that microﬁlariae of O. cervicalis cannot be considered as the primary cause of periodic ophthalmia in horses. However, other authors have associated O. DISCUSSION To the best of our knowledge, this is the ﬁrst report of Onchocerca infection in donkeys in Italy. The presence of microﬁlariae in peripheral blood samples was ascertained by laboratory and molecular ﬁndings. Unfortunately, the species of microﬁlariae was not identiﬁed. The two Onchocerca species infecting donkeys in Europe are O. cervicalis and O. reticulata, which are both found worldwide (1). The geographical range of O. raillieti infection in donkeys known to date is restricted to some countries in Africa (3, 5, 8). In previous studies, when the occurrence of O. cervicalis and O. reticulata was investigated concurrently, O. cervicalis was more prevalent than O. reticulata. In fact, prevalence values of 82.98 and 4.26% have been reported for O. cervicalis and O. reticulata in donkeys, respectively (13). Similarly, prevalence values of 25.42, 5.93, and 2.54% have been reported for O. cervicalis, O. reticulata, or both infections in horses, respectively (14). Therefore, based on the results of previous surveys, we believe that O. cervicalis was most likely the species involved in our study. The occurrence, distribution, and population density of microﬁlariae in tissues of Onchocerca infected hosts may vary by season. The peak of distribution of O. cervicalis microﬁlariae in ventral-midline skin of 15 naturally infected pony mares was investigated over a 13-month period and was shown to be highest during the spring and lowest in the winter, disappearing in the surface layers of the dermis during the winter months (27). A distinct pattern of distribution was also reported in blood samples from 284 camels (Camelus dromedarius) where the highest monthly prevalence of Onchocerca microﬁlariae throughout a period of 14 months was detected in June, disappearing in July to September and February (28). The authors concluded that this is correlated with an adaptation of the parasite to the All the four Onchocerca infected jennies were bred on the same farm and were kept under the same management conditions. They had always lived on the same farm since their birth and there were no movements throughout the country except for their transportation to the VTH. Onchocerca infection in horses is considered rare when macrocyclic lactones are used regularly (15), since treatments with ivermectin (16, 17), moxidectin (18, 19) or doramectin (9) are eﬀective in killing microﬁlariae (but not against adult worms located in the nuchal ligament). November 2020 \| Volume 7 \| Article 569916 5 Papini et al. DISCUSSION The concentration of microﬁlariae that we found in the peripheral blood samples (range 1–31 microﬁlariae in 2 mL) is likely to be much lower than the actual microﬁlariae concentration in the tissues of the examined jennies because Knott’s test is not the most suitable method to detect microﬁlariae in Onchocerca infected equids. Onchocerca microﬁlariae have a very uneven distribution in the dermis of their hosts (29). The most eﬀective method to detect Onchocerca microﬁlariae and accurately estimate their concentration is based on skin biopsies of the midline of the abdomen, preferably near the umbilical region, both in infected donkeys (5, 10) and horses (15, 30–32). For instance, Hussein and El Sammani (5) reported that the density of O. raillieti microﬁlariae in donkeys ranged from 1,200 to 26,200 per gram of skin in the linea alba along the mid-ventral line and from 1,100 to 16,900 per gram of skin in the region of the wither. In donkeys within the age groups of 1–3, 4–6, and 7–10 years, increasing counts per gram of skin were detected with mean values of 1,083 (range 100–2,400), 1,444 (1,000– 2,000), and 2,040 (1,330–9,000) microﬁlariae, respectively (10). The number of O. cervicalis microﬁlariae per 8 or 6 mm of biopsied skin in ponies and horses ranged from 1 to 21,570 Detection of Onchocerca microﬁlariae in the peripheral blood smear of donkeys or other equids is unusual and thus molecular analysis for conﬁrmatory purposes was needed. In our study, donkeys did not show any dermal or ocular sign that might suggest Onchocerca infection at the time of admittance to the VTH. Therefore, skin biopsies (see the Section Discussion below) were not included in the initial diagnostic workup. On the other hand, onchocerciasis diagnosis with skin biopsy is laborious and time consuming for equine practitioners and laboratory technicians, as well as being expensive for owners. Even when Onchocerca infection was suspected because of microﬁlariae morphological features, performing skin biopsies of the ventral midline on at least one of the jennies would have required the speciﬁc owner’s informed consent. Unfortunately, for this veterinary procedure, the jennies need to be sedated to avoid any risk for the operators and also an extended hospitalization time at the VTH, thus leading to economic losses because of the additional costs and lack of income for the owner of a milk production farm. DISCUSSION This means that skin biopsies would never have been permitted by the owner given the lack of any clinical evidence and above all with no apparent economic beneﬁt for him. Consequently, we opted to use molecular analysis rather than skin biopsy to conﬁrm a case of microﬁlaraemia compatible with presumptive laboratory diagnosis of asymptomatic Onchocerca infection. This was despite the fact that a skin biopsy is the standard technique for diagnosing equine onchocerciasis. The concentration of microﬁlariae that we found in the peripheral blood samples (range 1–31 microﬁlariae in 2 mL) is likely to be much lower than the actual microﬁlariae concentration in the tissues of the examined jennies because Knott’s test is not the most suitable method to detect microﬁlariae in Onchocerca infected equids. Onchocerca microﬁlariae have a very uneven distribution in the dermis of their hosts (29). The most eﬀective method to detect Onchocerca microﬁlariae and accurately estimate their concentration is based on skin biopsies of the midline of the abdomen, preferably near the umbilical region, both in infected donkeys (5, 10) and horses (15, 30–32). For instance, Hussein and El Sammani (5) reported that the density of O. raillieti microﬁlariae in donkeys ranged from 1,200 to 26,200 per gram of skin in the linea alba along the mid-ventral line and from 1,100 to 16,900 per gram of skin in the region of the wither. In donkeys within the age groups of 1–3, 4–6, and 7–10 years, increasing counts per gram of skin were detected with mean values of 1,083 (range 100–2,400), 1,444 (1,000– 2,000), and 2,040 (1,330–9,000) microﬁlariae, respectively (10). The number of O. cervicalis microﬁlariae per 8 or 6 mm of biopsied skin in ponies and horses ranged from 1 to 21,570 To the best of our knowledge, only two studies have previously investigated hematological values in donkeys naturally infected with Onchocerca. The results of one of these studies showed that levels of serum total proteins, albumin, and globulins were signiﬁcantly (p < 0.05) higher compared to uninfected donkeys. Other variations included an increase in the level of glucose and a lower concentration of serum cholesterol (10). By contrast, in jenny I we found low values of serum total proteins and a mild reduction in albumin along with increased cholesterol. November 2020 \| Volume 7 \| Article 569916 Frontiers in Veterinary Science \| www.frontiersin.org 6 Papini et al. CONCLUSIONS Onchocerca microﬁlariae were incidentally detected in four jennies in Italy. Microﬁlariae were identiﬁed by blood smear, Knott’s test, and ﬁnally DNA analysis. All the cases were of autochthonous origin because the jennies were born and reared on the same farm located in central Italy and they had never moved outside the farm. The transmission of Onchocerca infection to these animals is linked to their exposure to bites of infected Culicoides, suggesting that vectors in turn could have acquired the infection locally. The reproductive lifespan of Onchocerca volvulus, the etiological agent of river blindness in humans, ranges from 9 to 11 years (45) and adult females can release 700 to 900 microﬁlariae daily (46). At present, the lifespan of Onchocerca species in donkeys is unknown but we can assume that adult females live for years and likewise release hundreds of microﬁlariae daily during their lifespan. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. DISCUSSION The relationship between basophilia and helminth infections has also been well established in humans (43) and several animal models (44). However, it cannot be ruled out that these hematological alterations were caused by concurrent infections with other pathogens. Therefore, further investigations are needed to determine whether most donkeys naturally aﬀected by subclinical infection with Onchocerca may develop eosinophilia and basophilia. Insect vectors that are suitable for transmitting the infection are widespread in Italy (20). Since the infection can be easily overlooked, a high proportion of donkeys may be infected with minimal or no clinical or hematological evidence, and may act as clinically asymptomatic carriers. Our report demonstrates the occurrence of Onchocerca in donkeys in Italy. We thus believe that equine practitioners should be aware of our ﬁndings and of their implications for equine health. Our ﬁndings highlight: (i) the risk of spreading the infection to other susceptible hosts (donkeys, mules, horses, ponies), (ii) the risk of progression in parasitized equines toward clinical disease, and (iii) the need to control the occurrence of Onchocerca infections in the breeding systems of donkeys and horses reared in Italy. The possibility of accidental transmission of O. cervicalis to humans has also been reported (47, 48). Our report also provides a basis for further studies to determine infection rates in donkeys and horses, to identify the insect vectors that may act as intermediate hosts, and to implement possible prevention and control measures. ETHICS STATEMENT Ethical review was not required as per institutional guidelines/local legislation due to natural infections that occurred spontaneously. Written informed consent was obtained from the owners for the participation of their animals in this study. FUNDING This study was supported by the University of Pisa (Fondi di Ateneo 2018). AUTHOR CONTRIBUTIONS RP performed parasitological examinations and wrote the ﬁrst draft of the manuscript. GL performed hematological examinations. MS performed all clinical examinations. All the authors critically edited and reviewed the manuscript. All authors contributed to the article and approved the submitted version. DISCUSSION Onchocerca in Donkeys However, comparing our ﬁndings with those previously reported is not reliable because of the large diﬀerences in study design between our report and previous studies. In fact, one study investigated blood biochemical changes following treatment with doramectin against O. raillietti microﬁlariae (9), while another study compared the eﬀects of O. raillieti infection on serum total proteins, albumin, glucose, and cholesterol in naturally infected and uninfected donkeys (10). We detected a variety of hematological alterations in one of the jennies (I) with subclinical Onchocerca infection. However, all these alterations were probably due to the blood lipemic status secondary to obstructive colic in a lactating jenny and it is very unlikely that Onchocerca infection plays a key role in this context. Diﬀerential white blood cell counts revealed moderate eosinophilia in jenny II while remarkable eosinophilia and basophilia were present in jenny III. The role of eosinophils in parasitic helminth infections in mammals has been widely demonstrated (39, 40), including in cases of Onchocerca infection in humans (41, 42). The relationship between basophilia and helminth infections has also been well established in humans (43) and several animal models (44). However, it cannot be ruled out that these hematological alterations were caused by concurrent infections with other pathogens. Therefore, further investigations are needed to determine whether most donkeys naturally aﬀected by subclinical infection with Onchocerca may develop eosinophilia and basophilia. However, comparing our ﬁndings with those previously reported is not reliable because of the large diﬀerences in study design between our report and previous studies. In fact, one study investigated blood biochemical changes following treatment with doramectin against O. raillietti microﬁlariae (9), while another study compared the eﬀects of O. raillieti infection on serum total proteins, albumin, glucose, and cholesterol in naturally infected and uninfected donkeys (10). We detected a variety of hematological alterations in one of the jennies (I) with subclinical Onchocerca infection. However, all these alterations were probably due to the blood lipemic status secondary to obstructive colic in a lactating jenny and it is very unlikely that Onchocerca infection plays a key role in this context. Diﬀerential white blood cell counts revealed moderate eosinophilia in jenny II while remarkable eosinophilia and basophilia were present in jenny III. The role of eosinophils in parasitic helminth infections in mammals has been widely demonstrated (39, 40), including in cases of Onchocerca infection in humans (41, 42). REFERENCES 5. Hussein HS, El Sammani SE. Onchocerca raillieti: prevalence and pathology in Sudanese donkeys. J Comp Pathol. (1985) 95:345–51. doi: 10.1016/0021-9975(85)90038-6 1. Dagnaw M, Zemene M, Getaneh G, Tibenu S. A review on equine onchocerchiasis. Afr J Basic Appl Sci. (2016) 8:27–33. doi: 10.5829/idosi.ajbas.2016.8.1.22966 6. Lok JB, Cupp EW, Braide EI, Bernardo MJ. Aedes aegypti as a surrogate host for Onchocerca spp. Am J Trop Med Hyg. (1980) 29:382–8. doi: 10.4269/ajtmh.1980.29.382 2. Melhorn H. Encyclopedia of Parasitology. Berlin; Heidelberg; New York, NY: Springer-Verlag (2008). 2. Melhorn H. Encyclopedia of Parasitology. Berlin; Heidelberg; New York, NY: Springer-Verlag (2008). 7. Lia RP, Mutafchiev Y, Veneziano V, Giannelli A, Abramo F, Santoro M, et al. Filarial infection caused by Onchocerca boehmi (Supperer, 1953) in a horse from Italy. Parasitol Res. (2017) 116:191–8. doi: 10.1007/s00436-016-5 277-x 7. Lia RP, Mutafchiev Y, Veneziano V, Giannelli A, Abramo F, Santoro M, et al. 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Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 27. Foil LD, Klei TR, Miller RI, Church GE, Foil CS, French DD, et al. Seasonal changes in density and tissue distribution of Onchocerca cervicalis microﬁlariae in ponies and related changes in Culicoides variipennis populations in Louisiana. J Parasitol. (1987) 73:320–6. doi: 10.2307/328 2085 Copyright © 2020 Papini, Lubas and Sgorbini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2020 Papini, Lubas and Sgorbini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Copyright © 2020 Papini, Lubas and Sgorbini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 28. Abdul-Salam J, al-Taqui M. Seasonal prevalence of Onchocerca-like microﬁlaria in camels in Kuwait. J Egypt Soc Parasitol. (1995) 25:19–24. f The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 29. Mellor PS. Studies on Onchocerca cervicalis Railliet and Henry 1910: I. Onchocerca cervicalis in British horses. J Helminthol. (1973) 47:97–110. doi: 10.1017/S0022149X00023774 November 2020 \| Volume 7 \| Article 569916 Frontiers in Veterinary Science \| www.frontiersin.org
https://openalex.org/W3022372991	https://europepmc.org/articles/pmc7247825?pdf=render	English	null	Cognitive Intervention Targeting Autobiographical Memory Impairment in Patients With Schizophrenia Using a Wearable Camera: A Proof-of-Concept Study	Frontiers in psychiatry	2,020	cc-by	12,158	ORIGINAL RESEARCH published: 08 May 2020 doi: 10.3389/fpsyt.2020.00397 Cognitive Intervention Targeting Autobiographical Memory Impairment in Patients With Schizophrenia Using a Wearable Camera: A Proof-of-Concept Study Romane Dassing 1,2†, Me´lissa C. Alle´ 1,3†, Mathieu Cerbai 1,2, Alexandre Obrecht 4, Nicolas Meyer 2,5,6, Pierre Vidailhet 1,2,4, Jean-Marie Danion 1,2,4, Amaury C. Mengin 2,4 and Fabrice Berna 1,2,4 Romane Dassing 1,2†, Me´lissa C. Alle´ 1,3†, Mathieu Cerbai 1,2, Alexandre Obrecht 4, Nicolas Meyer 2,5,6, Pierre Vidailhet 1,2,4, Jean-Marie Danion 1,2,4, Amaury C. Mengin 2,4 and Fabrice Berna 1,2,4 Keywords: schizophrenia, autobiographical memory, cognitive remediation, wearable camera, rehabilitation Correspondence: Romane Dassing romane.dassing@hotmail.fr †These authors have contributed equally to this work Specialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry Received: 21 February 2020 Accepted: 20 April 2020 Published: 08 May 2020 Citation: Dassing R, Alle´ MC, Cerbai M, Obrecht A, Meyer N, Vidailhet P, Danion J-M, Mengin AC and Berna F (2020) Cognitive Intervention Targeting Autobiographical Memory Impairment in Patients With Schizophrenia Using a Wearable Camera: A Proof-of-Concept Study. Specialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry Specialty section: This article was submitted to Schizophrenia, a section of the journal Frontiers in Psychiatry Received: 21 February 2020 Accepted: 20 April 2020 Published: 08 May 2020 Received: 21 February 2020 Accepted: 20 April 2020 Published: 08 May 2020 Edited by: Judith M. Ford, University of California, San Francisco, United States 1 INSERM U1114 Neuropsychologie Cognitive et Physiopathologie de la Schizophre´ nie, Strasbourg, France, 2 Faculte´ de Me´ decine, Universite´ de Strasbourg, Strasbourg, France, 3 Center on Autobiographical Memory Research, Department of Psychology and Behavioural Sciences, Aarhus University, Aarhus, Denmark, 4 Hô pitaux Universitaires de Strasbourg, Service de Psychiatrie, Santé Mentale et Addictologie, Strasbourg, France, 5 Hô pitaux Universitaires de Strasbourg, Département de Santé Publique, Strasbourg, France, 6 Université de Strasbourg, Laboratoire de Biostatistique et Informatique Mé dicale, iCUBE UMR 7357, Illkirch, France Reviewed by: Ian Spicer Ramsay, University of Minnesota Twin Cities, United States Martin A. Conway, City University of London, United Kingdom Correspondence: Romane Dassing romane.dassing@hotmail.fr †These authors have contributed equally to this work Reviewed by: Ian Spicer Ramsay, University of Minnesota Twin Cities, United States Martin A. Conway, City University of London, United Kingdom Autobiographical memory (AM) impairment in schizophrenia affects the richness of detail in personal memories and is one of the major predictors of patients’ social functioning. Despite the empirical evidence attributing these difﬁculties to a defective encoding process, cognitive remediation interventions targeting AM in schizophrenia often focus on the remote past, making it difﬁcult to address the consequences of poor encoding. Our study evaluated the efﬁcacy of an innovative approach using a wearable camera (NarrativeClip®) in reinforcing the encoding of recent daily life events in patients with schizophrenia. Seventeen patients with schizophrenia and 15 control participants wore the camera during four consecutive days. Then, memories of events experienced during these days were reinforced using different types of retrospective, i.e. interventions designed to promote a re-encoding of the event. We evaluated two types of retrospective using the camera pictures: a simple visual retrospective and a visual retrospective associated with a speciﬁc event-cueing (VisR+EC). These two techniques were compared to a verbal retrospective and to the absence of retrospective. Our results showed that the VisR+EC allowed patients to retrieve as many details as the control group at a two-week interval. However, patients’ memories remained impaired when a simple visual or a verbal retrospective was used. Our study provides encouraging results to foster the use of a wearable camera in individualized cognitive remediation programs for AM impairment in schizophrenia. Dassing R, Alle´ MC, Cerbai M, Obrecht A, Meyer N, Vidailhet P, Danion J-M, Mengin AC and Berna F (2020) Cognitive Intervention Targeting Autobiographical Memory Impairment in Patients With Schizophrenia Using a Wearable Camera: A Proof-of-Concept Study. Front. Psychiatry 11:397. doi: 10.3389/fpsyt.2020.00397 INTRODUCTION Autobiographical memory allows individuals to capture spatio-temporal, emotional, sensory, and cognitive representations of speciﬁc past personal events over long time intervals, which can extend up to several decades for particularly signiﬁcant events (9–11). The retrieval of these episodic details is generally accompanied by a conscious recollection which confers the capacity to mentally travel back in time and re-experience a speciﬁc event. Autobiographical memory is a crucial function of human cognition, as it contributes to the emergence and construction of a coherent sense of identity (9, 12). It plays an important role in social relationships (13) and promotes a relevant anticipation of the future (14). Several studies investigating autobiographical memory in schizophrenia have shown that patients have difﬁculty accessing unique past experiences in comparison to control subjects (15–19). Indeed, patients’ autobiographical memories are described as being “over-general”, more similar to summaries of repeated life experiences than to unique detailed events (20). Moreover, conscious recollection at the time of memory retrieval appears reduced in schizophrenia in comparison to controls (16, 21–23). A meta-analysis by Berna et al. (5) highlighted moderate to large effect sizes associated with these decreased speciﬁcity (g = -0.97), level of detail (g = -1.40), and conscious recollection (g = -0.62), all of which are key features of the episodicity level of autobiographical memory. g p p Two studies have evaluated the effect of supporting memories of recent (less than a week old) daily life events on the encoding process in patients with schizophrenia using the diary method in a group setting (38, 39). This method asks patients to write down one or more recent events in a diary on a daily basis, using this tool as an external memory aid. Subsequently, patients are invited to share their memories with the other members of the group. The results of these two studies were conclusive, since they showed an improvement in memory speciﬁcity following the diary intervention that was maintained for up to three months (38). However, this method has several disadvantages: it is very time-consuming and it requires a high level of motivation, which can be difﬁcult to mobilize in patients with schizophrenia (40). Furthermore, the material used (diary content) is limited by the accuracy of the patient’s memory, yet this is precisely the therapeutic target of the intervention, since the memories of patients with schizophrenia tend to be overly general. INTRODUCTION Reminiscence or “life-review” therapies (30) were among the ﬁrst therapeutic programs aimed at stimulating autobiographical memory capabilities. Designed to promote the retrieval of vivid and detailed autobiographical past events, they usually target speciﬁc life periods, ranging from the past year to early childhood. Two studies (31, 32) have evaluated the efﬁcacy of such programs, respectively the Autobiographical Reminiscence Therapy (REMau) (33) and the Life Review Therapy focusing on positive events (LRTspev) (34) in patients with schizophrenia. An improvement in the capacity to retrieve speciﬁc autobiographical memories was highlighted with both programs, and a greater number of details in patients’ recalled memories was observed with the LRTspev (32). A large number of studies have documented the presence of cognitive impairments in patients with schizophrenia (1–3), and such impairments have come to be considered a core feature of this disorder. Although the entire set of cognitive functions seems to be affected in schizophrenia, these alterations are heterogeneous (3). Memory impairment is among the most pronounced cognitive deﬁcits in schizophrenia (4) and goes beyond memory processes evaluated in laboratory, affecting complex cognitive functions such as autobiographical memory (5). Although the pure neurocognitive nature of these deﬁcits is at some point discussed (6, 7), they have a concrete impact on patients’ daily functioning, often hindering patients’ access to a traditional educational or professional trajectory (8). Another autobiographical memory intervention consists of applying a speciﬁc event-cueing at the time of memory retrieval (35). This speciﬁc event-cueing follows a series of questions inspired by the Memory Characteristics Questionnaire (36) and asks for different categories of episodic details (contextual, perceptual/sensory, temporal, cognitive, and emotional) not mentioned by participants during their free recall. Potheegadoo et al. (37) have shown its efﬁcacy in patients with schizophrenia. Indeed, the use of speciﬁc event-cueing at retrieval allowed patients to reach a level of detail in memory recall similar to that of healthy subjects. Unfortunately, this was not true for the richness of perceptual/sensory, temporal, and contextual details, which remained reduced in patients despite the assistance of the event-cueing. The authors proposed that the event-cueing helped to overcome patients’ retrieval difﬁculties but did not alleviate the consequences of poor encoding of the event. It therefore seemed necessary to develop cognitive interventions to compensate this defective encoding process in schizophrenia. Citation: Dassing R, Alle´ MC, Cerbai M, Obrecht A, Meyer N, Vidailhet P, Danion J-M, Mengin AC and Berna F (2020) Cognitive Intervention Targeting Autobiographical Memory Impairment in Patients With Schizophrenia Using a Wearable Camera: A Proof-of-Concept Study. Front. Psychiatry 11:397. doi: 10.3389/fpsyt.2020.00397 May 2020 \| Volume 11 \| Article 397 1 Frontiers in Psychiatry \| www.frontiersin.org Recent Memories Remediation in Schizophrenia Dassing et al. Frontiers in Psychiatry \| www.frontiersin.org Participants Thirty-two participants took part in the study. These included a group of 17 outpatients (six women) diagnosed with schizophrenia according to the DSM-5 criteria (49), who were recruited from the Strasbourg University Hospital Psychiatric Department, and a comparison group of 15 control participants (seven women). Patients and controls did not differ in either age or level of schooling. All the patients had been clinically stabilized under antipsychotic treatment for at least two months. We did not include patients treated with benzodiazepines due to their adverse effects on memory (50). None of the study’s participants had a history of neurological or major somatic illness, a current alcohol or substance abuse problem, or a current severe depressive episode, as deﬁned by a score higher than 6 on the Calgary Depression Scale for Schizophrenia (CDSS) (51) for patients and by a score higher than 9 on the Beck Depression Inventory (BDI) (52) for controls. Furthermore, control participants had no history of psychiatric disorder. In addition, we measured pre-morbid IQ in each group using a validated French version of the National Adult Reading Test (f-NART) (53). Current IQ was measured using a validated abbreviated version of the Wechsler Adult Intelligence Scale – third edition (WAIS-III) (54), which included three subtests: vocabulary, matrix reasoning, and arithmetic (55). Participants with a current IQ score below 70 were excluded from the study. Considering that deﬁcient encoding processes may contribute to autobiographical memory impairment in patients with schizophrenia, we were interested in evaluating the relevance of material obtained with a wearable camera in fostering patients’ memories of recent daily life events. In order to collect the most ecological daily life events possible, we did not intervene at the time of the initial encoding of the events. For this reason, the interventions described in this study cannot be considered as direct interventions on encoding processes, but have rather been used to 1) reinforce the consolidation of the initially encoded experience by structuring its content and 2) promote a new and richer encoding of the lived event. This study was approved by the Committee for the Protection of Persons of Paris X (reference 2013-A00402-43), and all participants gave their written informed consent in accordance with the Declaration of Helsinki. More precisely, the central aim of this proof-of-concept study was to test the efﬁcacy of the NarrativeClip® camera (a more recent version of the Sensecam® camera) in improving the richness (i.e. INTRODUCTION The number of cues patients provide may thus be insufﬁcient to reﬂect their initial experiences. Finally, this diary method does not allow the experimenter to verify the accuracy of the content used as remediation material, and so altered or even false memories can be retrieved and trained during these interventions. The functional mechanisms responsible for patients’ memory impairment have primarily been studied using standard laboratory episodic memory tasks. Although memory impairment was ﬁrst attributed to a retrieval deﬁcit (24, 25), empirical evidence has since emphasized the major implication of a defective encoding process in patients’ memory difﬁculties (26). Finally, autobiographical memory deﬁcit in schizophrenia has been described as one of the major predictors of social functioning, surpassing the inﬂuence of both clinical symptoms and basic cognitive deﬁcits (27). As such, it is one of the major therapeutic targets in the cognitive landscape of schizophrenia. The antipsychotics used in schizophrenia to overcome clinical symptoms either have no particular effect or a deleterious effect on patients’ memory disorders (28). Psychological interventions have therefore begun to be developed in the past few decades, including cognitive intervention programs (29). Considering the limitations of the diary method, several studies have begun to investigate memory using a wearable May 2020 \| Volume 11 \| Article 397 2 Recent Memories Remediation in Schizophrenia Dassing et al. camera as a cognitive remediation tool for autobiographical memory disorders. The ﬁrst device used for such purposes was the Sensecam® camera, which can be worn around the neck and is typically programmed to take pictures automatically every 30 s. This tool does not require any particular intervention from the participant, which facilitates its use in patients with initiation or motivation difﬁculties. As using a wearable camera makes it possible to create dynamic slideshows with a very large number of pictures taken from a ﬁrst-person perspective, the participant beneﬁts from a cueing method very close to the initial lived experience (41, 42). Over the past decade, several studies have investigated the effect of viewing such event-related slideshows on autobiographical memory performance across multiple pathological situations (41–47). Results showed that the use of the Sensecam® camera triggered the recollection of more speciﬁc and detailed memories, even in the case of severe amnesia. In addition, several studies have compared the use of a wearable camera to the diary method previously described (41, 43, 45, 46). INTRODUCTION Results showed that the improvement in autobiographical memory retrieval is more pronounced in the Sensecam® condition. This inter-condition difference seems to expand over time, as shown by Woodberry et al. (46), who found memories to be up to three times more detailed in the Sensecam® condition than in the diary condition at a three- month follow-up. Interestingly, Loveday and Conway (41) also highlighted an effect of the type of cueing method on the nature of the reported details, with an advantage for episodic details for events trained with the Sensecam® pictures, and an advantage for general knowledge in the diary condition. In addition, several studies have suggested that viewing Sensecam® pictures can trigger a “Proustian moment”, i.e. the intense retrieval of a speciﬁc memory including also non-sensory details (emotions, thoughts) experienced at the time of the initial experience, although not perceivable on the pictures (48). These previous studies have primarily assessed the effect of wearable cameras on autobiographical memory in neurological populations (e.g. post- encephalitic amnesia, traumatic brain injury, mild cognitive impairment, and Alzheimer’s disease). experiences. We compared the effect of two interventions using the NarrativeClip® camera: 1) a simple visual retrospective involving only the viewing of a slideshow of pictures collected during the day and 2) a visual retrospective associating this viewing with the speciﬁc event-cueing used by Potheegadoo et al. (37). These two methods were then compared to a verbal retrospective without viewing the pictures of the day and to the absence of retrospective. In light of the literature, we expected that the visual retrospectives using the NarrativeClip® pictures would promote a greater improvement in the number of details recalled in comparison to the verbal retrospective and, even more so, to the absence of retrospective. Moreover, we predicted that patients with schizophrenia would beneﬁt more from the visual retrospective with event-cueing than from the simple visual retrospective, considering the dual modality (visual and verbal) of this condition and the active involvement required of the participant. Participants number of details) and episodicity level with which patients with schizophrenia recall recent personal Frontiers in Psychiatry \| www.frontiersin.org Events Collection and Interventions At the end of each day, we collected the day’s pictures and each time the memories of the day were subjected to a different type of intervention (the order of which being randomly assigned; for a schematic of the events collection and interventions, see Figure 1). g Here is one of the possible combinations of interventions: Materials and Procedure Control participants Patients with schizophrenia Statistics n = 15 n = 17 M (SD) M (SD) Pr (P > C) Results are presented as the probability that patients’ scores to be higher than the controls’ scores is above 0: Pr(P > C). A large Pr(P > C) value (e.g., > .95, > .975, or > .99) must be interpreted as indicating higher values for patients compared with controls. A small value of Pr(P > C) (e.g., < .05, < .025, or < .01) reﬂects lower values for patients compared with controls. It is worth noting that the probability Pr(P > C) can be interpreted as 1 – Pr(P < C). Probability values near 1, and near 0, both indicate a meaningful effect of the group. aPositive And Negative Syndrome Scale for Schizophrenia. bBeck Depression Inventory. cCalgary Depression Scale for Schizophrenia. dIntelligence Quotient. eFrench National Adult Reading Test. fWechsler Memory Scale – third edition. gTrail Making Test. Clinical Assessments experimenter. The purpose of this was ﬁrstly to familiarize the participant with the tool and estimate any possible associated discomfort or anxiety, and secondly to train the participant to position the device correctly on his/her clothing. To assess the severity of clinical symptoms of schizophrenia, we used the Positive and Negative Syndrome Scale (PANSS) (56) in patients. Subsequently, if the person agreed to participate in the study, he/she was invited to carry the camera for four consecutive days, for a minimum of seven hours a day. The events experienced during each day were collected and allocated to a different type of retrospective (i.e. intervention). Then, two weeks later, a memory test was conducted to evaluate the robustness of these events’ memories and thus the respective effect of each type of retrospective. Cognitive Assessments To investigate long-term memory performance, we used the Family Scenes subtest of the Wechsler Memory Scale-III (WMS- III) (57). We selected this standardized task because its material is more similar to what our study evaluated (recall of autobiographical events) than the lists of words usually employed in conventional verbal memory tests. In addition, knowing that long-term memory encoding and retrieval processes depend on executive processes, we measured working memory performance using the WMS-III’s Spatial Memory subtest. Participants’ mental ﬂexibility was also measured using a verbal ﬂuency task (58), which assessed both the phonological and semantic aspects of ﬂuency, and the non- verbal Trail Making Test (TMT) (59). Results are presented as the probability that patients’ scores to be higher than the controls’ scores is above 0: Pr(P > C). A large Pr(P > C) value (e.g., > .95, > .975, or > .99) must be interpreted as indicating higher values for patients compared with controls. A small value of Pr(P > C) (e.g., < .05, < .025, or < .01) reﬂects lower values for patients compared with controls. It is worth noting that the probability Pr(P > C) can be interpreted as 1 – Pr(P < C). Probability values near 1, and near 0, both indicate a meaningful effect of the group. aPositive And Negative Syndrome Scale for Schizophrenia. bBeck Depression Inventory. cCalgary Depression Scale for Schizophrenia. dIntelligence Quotient. eFrench National Adult Reading Test. fWechsler Memory Scale – third edition. gTrail Making Test. Materials and Procedure Table 1 presents the clinical and cognitive measures administered to participants. May 2020 \| Volume 11 \| Article 397 3 Recent Memories Remediation in Schizophrenia Dassing et al. TABLE 1 \| Means (M) and standard deviations (SD) of sociodemographic, clinical, and cognitive measures for patients with schizophrenia and controls. Control participants Patients with schizophrenia Statistics n = 15 n = 17 M (SD) M (SD) Pr (P > C) Sociodemographic characteristics Gender (number of women, %) 7 (46.7) 6 (35.3) 0.262 Age (years) 40.6 (10.58) 40.24 (10.29) 0.739 Level of schooling (years) 13.67 (2.02) 12.29 (2.62) 0.063 Clinical variables PANSSa Total Scale – 57.63 (19.3) Positive Scale – 12.94 (5.63) Negative Scale – 18 (9.44) Depression BDIb 3.07 (2.31) – CDSSc – 2.38 (1.61) Duration of illness (years) – 13.29 (8.78) Chlorpromazine equivalents (mg) – 322.25 (191.61) General intelligence Total IQd scores f-NARTe (premorbid IQd) 106.87 (7.04) 106.73 (8.75) 0.938 Short version of WAIS-III (current IQd) 96.48 (11.87) 90.05 (11.27) 0.793 Cognitive assessments Standard scores WMS-IIIf Family Scenes I 9.67 (2.72) 6.65 (4.06) 0.013 Family Scenes II 9.87 (2.59) 6.71 (3.79) 0.007 Family Scenes (percentage of retention) 10.00 (2.88) 8.88 (3.64) 0.192 Spatial Memory 9.57 (1.99) 8.75 (3.15) 0.221 Z-scores TMTg B - A (time) -0.48 (0.97) -0.70 (1.34) 0.305 B - A (number of errors) -0.64 (1.80) -0.85 (2.36) 0.397 Fluency Phonological -0.41 (0.52) -0.16 (0.69) 0.849 Semantic -0.27 (0.76) -0.89 (0.90) 0.031 Results are presented as the probability that patients’ scores to be higher than the controls’ scores is above 0: Pr(P > C). A large Pr(P > C) value (e.g., > .95, > .975, or > .99) must be interpreted as indicating higher values for patients compared with controls. A small value of Pr(P > C) (e.g., < .05, < .025, or < .01) reﬂects lower values for patients compared with controls. It is worth noting that the probability Pr(P > C) can be interpreted as 1 – Pr(P < C). Probability values near 1, and near 0, both indicate a meaningful effect of the group. aPositive And Negative Syndrome Scale for Schizophrenia. bBeck Depression Inventory. cCalgary Depression Scale for Schizophrenia. dIntelligence Quotient. eFrench National Adult Reading Test. fWechsler Memory Scale – third edition. gTrail Making Test. TABLE 1 \| Means (M) and standard deviations (SD) of sociodemographic, clinical, and cognitive measures for patients with schizophrenia and controls. Frontiers in Psychiatry \| www.frontiersin.org Experimental Protocol This questionnaire consisted of two central questions, measuring 1) the level of constraint associated with the camera and 2) the usability of the camera, using Likert scales ranging from 1 (constraining/difﬁcult to use) to 7 (pleasant/easy to use). After each event recall, the participant was asked to complete a subjective questionnaire to evaluate various characteristics related to his/her memory on a seven-point Likert-type scale, among which were its episodicity level, its associated valence and emotional intensity, and its importance for the participant (see the detailed questionnaire in Supplementary Material S2). Memory Test The memory test was conducted two weeks after the last day of wearing the camera and included two different tasks (for a schematic of the memory test, see Figure 1): Recognition Task. In the recognition phase, the purpose was to investigate in which extent participants subjectively estimated the robustness of their memories when we gave them the visual content of each experience. Cued-Recall Task. The aim of the cued-recall task was to measure the level of detail remembered by the participants two weeks after the initial events. Sixteen events were randomly selected from the verbal, simple visual, and visual retrospective associated with event-cueing conditions, as well as from the control condition (four events per condition). For the conditions with retrospective, the 43 selected events were those that had not been discussed during the For this task, 23 events titles were randomly selected from the 63 events identiﬁed by the participant during the verbal, simple visual, and visual retrospectives associated with event- cueing (two events per condition). Since the condition with no retrospective was not subjected to event selection by the TABLE 2 \| Examples of details for each of the categories described in the scoring method of the Autobiographical Interview (Levine et al., 2002). Experimental Protocol With the picture of the event displayed before him/her, the participant is then questioned in depth about the event, following the pro- cedure of Levine et al. (35). The questions follow a predeﬁned order and aim to encourage the participant to recall visual and non-visual details of the scene (see the detailed procedure in Supplementary Material S1). participant, it was not included in this task. The experimenter gave the written title of each of these events as a cue to the participant, who was directly invited to recall aloud all the information he/she remembered concerning the event. All these autobiographical memories were recorded and then transcribed. All the verbatim transcripts were analyzed according to the scoring method of Levine et al. (35). This objective method translates qualitative content into quantitative content, adding and classifying the various details present in the narratives. Indeed, each piece of information provided by the participant was counted and classiﬁed in a speciﬁc category, starting with a distinction between internal and external details, that is, details related or unrelated to the selected event (see descriptions and examples presented in Table 2). The participants of both groups were able to select six events experienced during their day, without any major difﬁculty for the three conditions with retrospective (VerR, VisR, and VisR+EC). The 63 event titles collected in these conditions were then used for the memory test two weeks later. Two out of the six event titles were randomly selected in each condition to be used as cues in the cued-recall task. The remaining event titles were used to create the slideshows of the recognition task (for a schematic of the event selection, see Figure 1). All narratives were analyzed by the ﬁrst author (RD), and a trained second rater (fourth author, AO) analyzed a random selection of 60% of the transcripts. At the time of the analysis of the memories, the raters were naive to the group to which the participants belonged and the experimental condition. An intra- class correlation coefﬁcient was calculated to assess the consistency of the two contributors’ ratings. This analysis showed excellent inter-rater reliability, with Cronbach’s alpha of 0.97 and 0.91 for internal and external detail ratings, respectively. At the end of these four days of collecting events, a questionnaire was presented to each participant, which evaluated his/her subjective impressions of the NarrativeClip® tool. Experimental Protocol Day 1: Absence of retrospective – control condition (AbsR). The participant is invited to carry out the vocabulary and matrix During the ﬁrst visit, we asked each participant to wear the camera (NarrativeClip®) during a short trip with the May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org Dassing et al. Recent Memories Remediation in Schizophrenia matic of the experimental protocol’s design, including the events collection phase and the memory test. he experimental protocol’s design, including the events collection phase and the memory test. FIGURE 1 \| Schematic of the experimental protocol’s design, including the events collection phase and the memory test. used as a cue in the memory test, we therefore opted for an oral task. reasoning subtests of the WAIS-III (54), without making any reference to the course of his/her day. used as a cue in the memory test, we therefore opted for an oral task. Day 2: Verbal retrospective (VerR). The experimenter asks the participant to give a title to six of the most signiﬁcant events experienced during the day. The participant is invited to orally recount the events experienced during the day in chronological order, making sure that his/her story includes the six pre-selected events. As described above, the production of a written content requires a high level of motivation. In order to avoid disadvan- taging the group of patients, and since this narrative will not be Day 3: Simple visual retrospective (VisR). Similarly to the verbal condition, the participant is asked to give a title to six of the most signiﬁcant events experienced during the day. Subsequently, a slideshow comprising the pictures collected during his/her day is presented to the participant, without any verbal intervention from the experimenter. The participant is invited to silently pay attention to the pictures of his/her day. The pictures scroll at a rate of one picture per second. May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org 5 Recent Memories Remediation in Schizophrenia Dassing et al. Day 4: Visual retrospective associated with event-cueing (VisR +EC). The participant is asked to give a title to six of the most signiﬁcant events experienced during the day. Subsequently, a slideshow comprising the pictures collected during the day is presented to him/her. The participant is asked to stop the slideshow at the time of each of the six pre-selected events. May 2020 \| Volume 11 \| Article 397 Clinical and Cognitive Measures Each of these events was presented in random order to the participant in a short slideshow including approximately 20 pictures. If the participant felt that he/she had not lived the event presented, he/she was not given a questionnaire, and we proceeded to the following event. If the participant recognized the event as personally experienced, he/she was invited to complete the same subjective questionnaire as in the cued- recall task, evaluating several characteristics related to his/ her memory. Concerning sociodemographic characteristics, our two groups did not differ in terms of age, gender, or level of education (see Table 1). Similarly, the two groups did not differ in their premorbid and current IQ levels. With regard to cognitive measures, the patient group presented poorer performances than the control participants in information storage (both short- and long-term) and executive functioning, in the context of semantic ﬂuency. Correlation analyses showed no relevant association between cognitive or clinical evaluations and measures of autobiographical memory detail in either group. Measures of Autobiographical Memory Detail In all analyses, we computed the mean difference of each parameter tested and its 95% credible interval (indicated as CI95%). We then calculated the probability that the score of each measure was higher in the patient group than in the control group [indicated as Pr(P > C)]. Correlation coefﬁcients are provided with their means and 95% posterior credible intervals and the probability that this coefﬁcient is larger than 0 [indicated as Pr(r > 0)]. Probabilities higher than 95% or lower than 5% were considered meaningful. Experimental Protocol Event Recollection Internal details External details directly related to the selected speciﬁc event not related to the selected speciﬁc event Event details Place details Time details Perceptual details Emotion/ Thought details Other event details Semantic details Repetitions Other details Actions, people and weather From a country to a part of a room From a period of life to a precise schedule Related to the ﬁve senses or perceptions of a position/duration Mental state at the time of the event Belonging to another speciﬁc event General knowledge about the world and oneself Second occurrence of a detail Comments or metacognitive reﬂections I went to withdraw money. It was in a mall. It was around 10:30 a.m. The cash dispenser screen was blue. I thought of the balance of my account. The last time I came to this mall, I bought a sweater. This shopping center contains ﬁfty or so stores. — I do not remember that well… May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org 6 Recent Memories Remediation in Schizophrenia Dassing et al. cued recall. For the control condition (without retrospective), the experimenter himself selected four events from the picture bank of the day. distribution. Convergence of the MCMC sample chains was checked graphically and was observed in each case. All computations were done in the R computing environment with the required additional packages (in particular r2jags). These 16 events were mixed with eight “lure events” created by the experimenter using the wearable camera and therefore not personally experienced by the participant. These events allowed us to verify that each participant was able to recognize whether he/she had experienced an event personally or not, both in the control group and the group of patients with schizophrenia. Internal Details A smaller number of internal details was observed in the patients’ memories compared to those of the controls [OR=0.67, CI95%: 0.43–1.03, Pr(patients > controls) = 0.03], and a larger number of internal details was found in the VisR+EC condition than in the VerR condition [OR=1.21, CI95%: 1.03–1.41, Pr(VisR+EC > VerR) > 0.99]. Other pairwise comparisons between conditions showed that the number of internal details was higher in VisR +EC vs. VisR vs. VerR, but the probabilities that these differences differed from 0 were only between 0.87 and 0.88. Finally, a meaningful group by type of retrospective interaction was found [OR=1.56, CI95%: 1.22–2.00, Pr(OR > 1) > 0.99], showing that the number of internal details was lower in the patient group than in the control group in the VerR condition, but not in the VisR+EC condition (see Figure 2). We used informative priors to perform each of these regressions; priors were estimated according to previous studies that 1) demonstrated autobiographical memory impairments in schizophrenia (5, 15, 16, 21, 61) and 2) have recently successfully used a wearable camera for autobiographical memory remediation in amnesic, brain-damaged, or mildly cognitively impaired patients (41, 43–46). Subsequently, to evaluate the robustness of our ﬁndings concerning the transcript ratings, non-informative priors and pessimistic priors (where the values of the informative priors were entered in the regression analyses, but in the opposite direction to what was expected) were used (see details in Supplementary Material S3). Statistical Analyses Bayesian methods were used to analyze the study’s data (60). Between-group comparisons of sociodemographic (age, level of education), IQ and cognitive measures were performed using univariate linear regressions. The number of details recalled in the cued-recall task was analyzed using a mixed Poisson regression model with group (patients vs. controls) and type of retrospective (verbal vs. simple visual vs. visual associated with event-cueing) as predictors. Because the ratings of the subjective memory characteristics presented in both the cued recall and the recognition tasks were bounded variables and not normally distributed, mixed Beta regression models were calculated using the same predictors (group and type of retrospective) to analyze these data. Frontiers in Psychiatry \| www.frontiersin.org Experimental Protocol No participants expressed dissatisfaction regarding the NarrativeClip® camera during the study. Indeed, they estimated it to be relatively simple (M = 6.28, SD = 1.28) and pleasant (M = 5.08, SD = 1.51) (range: 1–7) to use. Scores did not differ between the groups (all Prs(patients > controls) between 0.14 and 0.23). Measures of Autobiographical Memory Detail In total, 141 autobiographical event transcripts were included in the analysis, of which 63 were from the patient group (22 for the verbal retrospective condition, 21 for the simple visual retrospective condition, and 20 for the visual retrospective with event-cueing condition) and 78 from the control group (26 for each type of retrospective). Cued-Recall Task We computed a composite episodicity score by averaging the scores for two ratings: the ability to relive the event mentally and the level of detail (see Figure 4). The results indicated lower scores in the patient group compared to the control group [OR=0.36, CI95%: 0.18-0.69, Pr(patients > controls) = 0.001], but scores did not differ across the various types of retrospective [all Prs(OR > 1) between 0.41 and 0.82]. Similarly, there was no meaningful interaction between group and type of retrospective [all Prs(OR > 1) between 0.62 and 0.69]. ] Regarding the remaining single items (see Table 4), patients displayed lower scores in the ability to relive the event mentally [OR=0.39, CI95%: 0.21–0.70, Pr(patients > controls) = 0.001] and in the level of detail [OR=0.35, CI95%: 0.20–0.64, Pr(patients > controls) = 0.001] than control participants. Similarly, ratings of emotional valence were lower in patients than in controls [OR = 0.51, CI95%: 0.27– 0.95, Pr(patients > controls) = 0.02], showing patients’ tendency to judge event memories more negatively than controls did. The pairwise comparisons between the experimental conditions demonstrated higher estimations of the ability to relive the event mentally [OR=1.66, CI95%: 1.12–2.43, Pr(VisR+EC > AbsR) = 0.99] and of the level of detail [OR=1.65, CI95%: 1.12–2.45, Pr(VisR+EC > AbsR) = 0.99] in the visual with event-cueing condition compared to the absence of retrospective condition. Scores in the ability to mentally relive the event were also higher in the VisR+EC condition compared to the VerR condition [OR=1.37, CI95%: 0.93–2.07, Pr(VisR+EC > VerR) = 0.95]. No other relevant differences were observed across the remaining types of retrospective [all Prs(OR > 1) between 0.08 and 0.93]. Subsequently, each remaining item was analyzed separately (see Table 3). Patients displayed a reduced ability to relive the event mentally [OR=0.30, CI95%: 0.16–0.58, Pr(patients > controls) < 0.001] and lower levels of memory detail [OR=0.46, CI95%: 0.23–0.91, Pr(patients > controls) = 0.01] and emotional intensity [OR=0.49, CI95%: 0.23–0.99, Pr(patients > controls) = 0.02] compared to controls. The results also showed that the memory detail scores were higher in the VisR condition than the VerR condition [OR=1.58, CI95%: 0.92–2.73, Pr(VisR > VerR) = 0.95]. The scores in the other conditions did not differ [all Prs (OR > 1) between 0.40 and 0.70]. Finally, no relevant interaction between group and type of retrospective was observed [all Prs (OR > 1) between 0.09 and 0.87]. Recognition Task External Details A smaller number of external details was found in the patients’ memories compared to those of the controls [OR=0.6, CI95%: 0.18–0.71, Pr(patients > controls) = 0.002]. No meaningful differences in score were observed across the different types of A burn-in of 5,000 iterations followed by 100,000 iterations was used for each of the three chains, yielding a ﬁnal 300,000- iteration sample for retrieving the characteristics of the posterior May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org 7 Recent Memories Remediation in Schizophrenia Dassing et al. retrospective [all Prs(OR > 1) between 0.20 and 0.38]. The interaction between group and type of retrospective was also not meaningful [all Prs(OR > 1) were between 0.82 and 0.89] (see Figure 3). non-personal events (eight in the patient group, three in the control group). This result conﬁrms the ability of all participants to discriminate personally experienced events from lure events. Lower episodicity scores were observed in the patient compared to the control group [OR=0.36, CI95%: 0.2–0.66, Pr(patients > controls) < 0.001] (see Figure 5). Episodicity scores were higher in the visual retrospective with event-cueing condition than in the absence of retrospective condition [OR=1.66, CI95%: 1.14–2.42, Pr (VisR+EC > AbsR) > 0.99]. The scores in the other conditions did not differ [all Prs(OR > 1) between 0.51 and 0.94], and no group by condition interaction was observed [all Prs(OR > 1) between 0.46 and 0.76]. Recognition Task A meaningful group effect was observed showing a smaller level of external details in the patient group, compared to the control group. No relevant differences were observed across the different types of retrospective. FIGURE 4 \| Mean episodicity level (and standard deviations) evaluated by the participants during the cued recall task. These results highlighted lower scores of episodicity in the patient group compared to controls, but no relevant differences across the different types of retrospective. FIGURE 4 \| Mean episodicity level (and standard deviations) evaluated by the participants during the cued recall task. These results highlighted lower scores of episodicity in the patient group compared to controls, but no relevant differences across the different types of retrospective. Finally, there was no interaction between group and type of retrospective [all Prs(OR > 1) between 0.17 and 0.94]. primarily driven by the data collected and not by our expected results. Recognition Task Only one lure event was recognized as having been personally experienced, and 11 of the 768 events assessed were classiﬁed as FIGURE 2 \| Mean numbers of internal details in participants’ recalls in the cued recall task, analyzed according to the scoring method of the Autobiographical Interview (35). A larger number of internal details was found in the Visual + Event cueing condition, compared to the Verbal condition. A meaningful group by type of retrospective interaction was found, with an overall smaller number of internal details in the group of patients compared to controls, except for the Visual + Event- cueing condition. FIGURE 2 \| Mean numbers of internal details in participants’ recalls in the cued recall task, analyzed according to the scoring method of the Autobiographical Interview (35). A larger number of internal details was found in the Visual + Event cueing condition, compared to the Verbal condition. A meaningful group by type of retrospective interaction was found, with an overall smaller number of internal details in the group of patients compared to controls, except for the Visual + Event- cueing condition. May 2020 \| Volume 11 \| Article 397 8 Frontiers in Psychiatry \| www.frontiersin.org Recent Memories Remediation in Schizophrenia Dassing et al. FIGURE 3 \| Mean numbers of external details in participants’ recalls in the cued recall task, analyzed according to the scoring method of the Autobiographical Interview (35). A meaningful group effect was observed showing a smaller level of external details in the patient group, compared to the control group. No relevant differences were observed across the different types of retrospective. FIGURE 4 \| Mean episodicity level (and standard deviations) evaluated by the participants during the cued recall task. These results highlighted lower scores of episodicity in the patient group compared to controls, but no relevant differences across the different types of retrospective. FIGURE 3 \| Mean numbers of external details in participants’ recalls in the cued recall task, analyzed according to the scoring method of the Autobiographical Interview (35). A meaningful group effect was observed showing a smaller level of external details in the patient group, compared to the control group. No relevant differences were observed across the different types of retrospective. FIGURE 3 \| Mean numbers of external details in participants’ recalls in the cued recall task, analyzed according to the scoring method of the Autobiographical Interview (35). DISCUSSION No other relevant differences were observed across the conditions. FIGURE 5 \| Mean episodicity level (and standard deviations) evaluated by the participants during the recognition task. Episodicity levels were estimated as lower in the patient group compared to the control group. No other relevant differences were observed across the conditions. memory in schizophrenia (15–19), which report difﬁculties for patients in accessing episodic details related to a past personal event. A meta-analysis by Berna et al. (5) has also highlighted a large effect size of -1.40 associated with this lower level of detail in patients’ memories. Our results extend existing knowledge by showing that these deﬁcits also affect the memory of recent personal events, as studies on autobiographical memory in schizophrenia have typically investigated remote personal events that happened months or years before. schizophrenia’s memories of recent personally experienced events. Our results showed that strengthening these memories with a visual retrospective method combined with event-cueing allowed patients to retrieve as many internal details as the control group at a two- week interval. This improvement was however not found when patients were asked to subjectively assess the vividness of their memories. Furthermore, the level of detail of patients’ memories remained impaired when a simple visual retrospective or a verbal retrospective was used. To our knowledge, this is the ﬁrst study to investigate the efﬁcacy of a wearable camera as a tool for the remediation of memories of recent personal events in patients with schizophrenia. What is however surprising and new in the present study is the lack of difference between the verbal and the simple visual retrospectives in terms of internal details recalled. Our initial hypothesis predicted a larger number of details to be recalled for memories trained with the simple visual method, based on the literature on wearable cameras used in a cognitive remediation context. Indeed, the effectiveness of this type of tool in comparison to verbal interventions in improving retrieval of past personal experiences has previously been demonstrated (41, 43, 45, 46). Nevertheless, our study suggests that the simple use of pictures as DISCUSSION Control participants n=15 Patients with schizophrenia n=17 Verbal Simple visual Visual + Event-cueing Verbal Simple visual Visual + Event-cueing n=30 n=30 n=30 n=34 n=34 n=34 M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) Level of details 4.93 (1.95) 5.60 (1.33) 5.50 (1.46) 3.79 (1.78) 4.15 (1.81) 4.18 (1.77) Ability to mentally relive 5.50 (1.25) 5.63 (1.27) 5.63 (1.38) 3.70 (2.07) 4.38 (1.79) 4.12 (1.79) Emotional valence 1.43 (1.28) 1.43 (1.30) 1.67 (1.15) 0.79 (1.41) 0.79 (1.47) 0.29 (1.71) Emotional intensity 4.27 (1.41) 4.43 (1.38) 4.53 (1.68) 3.30 (1.19) 3.68 (1.61) 3.53 (1.46) Importance 4.17 (1.64) 4.57 (1.59) 4.30 (1.84) 3.33 (1.53) 3.76 (1.79) 3.79 (1.75) Each of these characteristics was assessed by the participants on Likert scales ranging from 1 to 7, except emotional valence which was estimated on a Likert scale ranging from -3 to 3. FIGURE 5 \| Mean episodicity level (and standard deviations) evaluated by the participants during the recognition task. Episodicity levels were estimated as lower in the patient group compared to the control group. No other relevant differences were observed across the conditions. TABLE 3 \| Means (M) and standard deviations (SD) of memories characteristics subjectively evaluated by participants during the cued recall task. Control participants n=15 Patients with schizophrenia n=17 Verbal Simple visual Visual + Event-cueing Verbal Simple visual Visual + Event-cueing n=30 n=30 n=30 n=34 n=34 n=34 M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) Level of details 4.93 (1.95) 5.60 (1.33) 5.50 (1.46) 3.79 (1.78) 4.15 (1.81) 4.18 (1.77) Ability to mentally relive 5.50 (1.25) 5.63 (1.27) 5.63 (1.38) 3.70 (2.07) 4.38 (1.79) 4.12 (1.79) Emotional valence 1.43 (1.28) 1.43 (1.30) 1.67 (1.15) 0.79 (1.41) 0.79 (1.47) 0.29 (1.71) Emotional intensity 4.27 (1.41) 4.43 (1.38) 4.53 (1.68) 3.30 (1.19) 3.68 (1.61) 3.53 (1.46) Importance 4.17 (1.64) 4.57 (1.59) 4.30 (1.84) 3.33 (1.53) 3.76 (1.79) 3.79 (1.75) Each of these characteristics was assessed by the participants on Likert scales ranging from 1 to 7, except emotional valence which was estimated on a Likert scale ranging from -3 to 3. TABLE 3 \| Means (M) and standard deviations (SD) of memories characteristics subjectively evaluated by participants during the cued recall task. FIGURE 5 \| Mean episodicity level (and standard deviations) evaluated by the participants during the recognition task. Episodicity levels were estimated as lower in the patient group compared to the control group. DISCUSSION To test the robustness of the statistical analyses, we performed sensitivity analyses using non-informative and pessimistic priors concerning the two types of detail (internal and external) in the objective evaluations. The estimated coefﬁcients remained unchanged, suggesting that the coefﬁcient estimations were The aim of the present proof-of-concept study was to explore whether and how an external memory aid, such as pictures taken by a wearable camera, can enhance the richness of patients with May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org 9 Recent Memories Remediation in Schizophrenia Dassing et al. TABLE 3 \| Means (M) and standard deviations (SD) of memories characteristics subjectively evaluated by participants during the cued recall task. Control participants n=15 Patients with schizophrenia n=17 Verbal Simple visual Visual + Event-cueing Verbal Simple visual Visual + Event-cueing n=30 n=30 n=30 n=34 n=34 n=34 M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) Level of details 4.93 (1.95) 5.60 (1.33) 5.50 (1.46) 3.79 (1.78) 4.15 (1.81) 4.18 (1.77) Ability to mentally relive 5.50 (1.25) 5.63 (1.27) 5.63 (1.38) 3.70 (2.07) 4.38 (1.79) 4.12 (1.79) Emotional valence 1.43 (1.28) 1.43 (1.30) 1.67 (1.15) 0.79 (1.41) 0.79 (1.47) 0.29 (1.71) Emotional intensity 4.27 (1.41) 4.43 (1.38) 4.53 (1.68) 3.30 (1.19) 3.68 (1.61) 3.53 (1.46) Importance 4.17 (1.64) 4.57 (1.59) 4.30 (1.84) 3.33 (1.53) 3.76 (1.79) 3.79 (1.75) Each of these characteristics was assessed by the participants on Likert scales ranging from 1 to 7, except emotional valence which was estimated on a Likert scale ranging from -3 to 3. FIGURE 5 \| Mean episodicity level (and standard deviations) evaluated by the participants during the recognition task. Episodicity levels were estimated as lower in the patient group compared to the control group. No other relevant differences were observed across the conditions. TABLE 3 \| Means (M) and standard deviations (SD) of memories characteristics subjectively evaluated by participants during the cued recall task. Measures of Autobiographical Memory Detail First of all, reduced richness of internal and external detail was observed in the patients’ compared to the controls’ memories in the simple verbal or visual retrospectives conditions. This observation corroborates existing ﬁndings on autobiographical May 2020 \| Volume 11 \| Article 397 Frontiers in Psychiatry \| www.frontiersin.org Recent Memories Remediation in Schizophrenia Dassing et al. TABLE 4 \| Means (M) and standard deviations (SD) of memories characteristics subjectively evaluated by participants during the recognition task. Control participants Patients with schizophrenia n=15 n=17 Absence Verbal Simple visual Visual + Event-cueing Absence Verbal Simple visual Visual + Event-cueing n=60 n=60 n=60 n=60 n=68 n=68 n=68 n=68 M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) M (SD) Level of details 5.00 (2.00) 5.29 (1.72) 5.18 (1.49) 5.67 (1.40) 3.27 (1.81) 3.59 (1.76) 3.91 (1.80) 4.10 (1.93) Ability to mentally relive 5.04 (2.05) 5.16 (1.71) 5.20 (1.49) 5.70 (1.44) 3.58 (1.87) 3.82 (1.82) 3.97 (1.80) 4.06 (1.86) Emotional valence 1.09 (1.40) 1.16 (1.41) 0.88 (1.46) 1.17 (1.32) 0.24 (1.45) 0.34 (1.53) 0.51 (1.48) 0.33 (1.50) Emotional intensity 4.28 (1.47) 4.15 (1.22) 4.20 (1.70) 4.10 (1.68) 3.63 (1.52) 3.30 (1.41) 3.66 (1.70) 3.63 (1.54) Importance 3.67 (1.88) 4.00 (1.63) 3.72 (1.75) 3.73 (1.87) 3.68 (1.89) 3.61 (1.73) 3.57 (1.79) 3.67 (1.66) Each of these characteristics was assessed by the participants on Likert scales ranging from 1 to 7, except emotional valence which was estimated on a Likert scale ranging from -3 to 3. andard deviations (SD) of memories characteristics subjectively evaluated by participants during the recognition task. testing effect has been established in the healthy population (63) as well as in several clinical populations (64–69), including schizophrenia (70). Indeed, patients, similarly to controls, showed better recall performances when the material to be remembered (a list of word pairs) was subjected to a test in comparison to a restudying condition. Although to our knowledge the literature on the testing effect has not yet been evaluated in autobiographical memory tasks, it seems appropriate to question its potential implications for our results. Indeed, the low cognitive effort involved in the passive viewing of the slideshow certainly did not make the simple visual retrospective effective enough to distinguish itself from the verbal retrospective, which can be viewed as an active memory test of the events experienced during the day. Measures of Autobiographical Memory Detail In addition, previous literature has shown that a retention interval of more than one day favours the occurrence of a testing effect (63). These ﬁndings are consistent with our results, since our protocol employed a long retention interval (two weeks). To bypass this possible bias, it would have been necessary to include a “passive” verbal retrospective. However, this is unfortunately impossible to conceive without the involvement of a third person, who would be required to narrate to the participant the events he/ she had experienced during the day. re-encoding material without speciﬁc event-cueing does not improve the level of detail more than a verbal retrospective at a two-week interval. Firstly, this result could be explained by the use of different methodological parameters in our study compared to previous studies. In the existing literature, the majority of studies (43–46) have operationalized memory enhancement using a set of approximately 15 details pre-established by a third person (typically the patient’s spouse). Indeed, patients’ recalls were represented as a percentage relative to these pre-deﬁned details, which could reach 100% if the participant recalled all the details initially chosen by the third person. In our study, the evaluation of the retrospectives’ effect was based directly on the number of details recalled by the patient, according to the rating method of Levine et al. (35). Loveday and Conway (41) used a similar rating method by counting the number of details in the patient’s memories. However, in their study, the cued recall was carried out directly following the viewing of the slideshow (which constituted the cueing), while our participants were only given the title of the event during the cued-recall task. Indeed, we only showed the slideshow during the event collection phase, as soon as possible after the event encoding (two weeks before the cued- recall task), and only for the events related to these types of retrospective. A ﬁrst explanatory hypothesis would be that in the previous studies, the beneﬁt of the simple viewing of the pictures without any additional cueing was dependent on the rating method used (percentage of recall related to a pre-established list of details in previous studies vs. number of recalled details in ours) and on the interval between the slideshow viewing and the participant’s recall (immediate recall vs. two week delay). Frontiers in Psychiatry \| www.frontiersin.org Ratings of Subjective Autobiographical Memory Characteristics Inspired by previous experimental protocols (43, 46), it would also be crucial to train selected events repeatedly with a verbal retrospective based on the diary method (in which the re-encoding material would be a written summary of the event produced by the patient himself/ herself) and other selected events with a visual retrospective based on the pictures taken by the wearable camera and comparing these two approaches to a non-interventional control condition. Furthermore, to deepen the evaluation of the respective effects of both verbal and visual retrospectives, it would be wise to associate both with the event- cueing used in the current study. To ensure the therapeutic value of these cognitive remediation programs, it will be necessary to carry out measures of generalization and transfer of the interventions to untrained cognitive and functional areas. All in all, since remembering personally experienced events allows us to clarify our sense of identity (71) and helps us to mentally anticipate future events (14), one can reasonably expect positive outcomes of these interventions, such as a reduction in disability and an increased quality of life in patients. However, these points need to be further investigated in future. Lower overall memory episodicity was found in the patient group compared to the control group for both the cued-recall and recognition tasks. This result aligns with patients’ difﬁculty in accessing rich details related to daily life events and in mentally reliving these events (5, 23). Regarding inter-condition differences, episodicity was evaluated as higher for the events trained with the visual retrospective associated with event-cueing compared to the control condition (absence of retrospective) in the recognition task. No other differences were found between the different types of retrospective, neither in the cued-recall task nor in the recognition task. We could therefore interpret this higher episodicity rating as the result of the patients having developed familiarity with the slideshows, which were presented again in the recognition task. In other words, the participants may have remembered the corresponding slideshow that they had seen previously in the events collection phase two weeks earlier rather than accurately re-experiencing the original event. In line with this, Loveday and Conway (41) reported that although the post-cue self-rating of memory vivacity was higher than the initial rating, no difference was found between the different cueing methods (diary vs. wearable camera). Measures of Autobiographical Memory Detail It is worth stressing that memory performance did not differ across the patient and control groups when the memories were trained with the visual retrospective coupled to event-cueing, suggesting that patients may have normalized their memory performances. This result aligns with that of Potheegadoo et al. (37) which also showed a normalization of patients’ performance following a speciﬁc cueing procedure. However, one major distinction from Potheegadoo’s study (37) is that the event- cueing procedure was used during a “re-encoding” phase and not at the time of event retrieval. Thus, our result tentatively suggests that our intervention made it possible to compensate the deﬁcient encoding of real-world events in patients when those events were retrieved a few days later. It however remains difﬁcult to explain the weak effect of the simple visual retrospective, in view of its efﬁcacy when coupled with the event-cueing. In order to more ﬁnely discriminate the contribution of the verbal and visual retrospective and better grasp the cueing procedure’s contribution to memory improvement, further studies would beneﬁt from adding a similar event-cueing procedure to the verbal retrospective condition. A complementary explanation of our results can be provided by work done in the ﬁeld of retrieval practice. In fact, one may consider that our verbal and simple visual retrospectives did not require the same degree of involvement from the participant. While in the simple visual retrospective task the participant was only asked to passively watch the slideshow (and thus to restudy the material to be learned), the verbal retrospective task required additional engagement insofar as the participant tested his/her own memory by organizing the events of his/her day in as complete a narrative as possible. Roediger et al. (62) demonstrated that the retention of material is strengthened when the individual is invited to test his/her learning compared to when the material is “simply” re-studied. This May 2020 \| Volume 11 \| Article 397 11 Recent Memories Remediation in Schizophrenia Dassing et al. Ratings of Subjective Autobiographical Memory Characteristics Loveday and Conway’s study (41) and the present work, taken together, indicate the difﬁculty of highlighting an inter-condition difference in subjective ratings, even if the participant has just watched the slideshow. This observation raises the question of the sensitivity to change of subjective measures of autobiographical memory characteristics. Conclusion and Future Perspectives This work was supported by INSERM (Institut National de la Santé et de la Recherche Médicale), France and by the University Hospital of Strasbourg under a Grant for Young researcher (API 2012 – HUS N°5539). p This ﬁrst proof-of-concept study in schizophrenia has substantial clinical implications, since it provides encouraging results concerning both the usefulness and acceptability of wearable cameras for improving autobiographical memory in these patients. The next step is to investigate whether patients with schizophrenia reporting autobiographical memory difﬁculties are likely to use this type of camera in a therapeutic setting. In line with previous studies conducted in a variety of clinical conditions (41–47), future studies should envision personalized cognitive remediation programs reinforcing autobiographical memories over longer periods of time. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. AUTHOR CONTRIBUTIONS MA, J-MD, and FB designed the study. RD, MA, and MC collected the data. RD, AO, and FB conducted the analyses. NM supervised the method and statistical analyses. RD wrote the ﬁrst draft of the manuscript. MA, PV, J-MD, AM, and FB provided substantial contributions to the manuscript. All authors read and approved the ﬁnal version of the manuscript. RD and MA contributed equally to the study. MA, J-MD, and FB designed the study. RD, MA, and MC collected the data. RD, AO, and FB conducted the analyses. NM supervised the method and statistical analyses. RD wrote the Limitations Several limitations of our work should be acknowledged. Keeping in mind that our study was conceived as a proof-of-concept, it is premature to conclude the utility of wearable cameras in improving autobiographical memory of daily life events in patients with schizophrenia. In fact, a limited number of events was considered here over a brief period of time, and these events mostly consisted of trivial daily life events with limited self-relevance. Furthermore, we didnotinvestigate the presence orabsence of amemory complaintin our patients. One may assume that patients with memory complaints would beneﬁt to a greater extent from these memory interventions than patients without such complaints. In addition, since we did not target patients in demand for cognitive intervention, we did not investigate to which extent they were likely to use a camera to improve their memory capacities. These points should be addressed in future studies. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Committee for the Protection of Persons of Paris X (reference 2013-A00402-43). The patients/participants provided their written informed consent to participate in this study. 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W4313154688.txt	http://acta.bibl.u-szeged.hu/76515/1/belvedere_2022_002_173-189.pdf	en		A fakultatív hitoktatás a Rákosi-korszakban az adatok tükrében	Belvedere meridionale	2,022	cc-by-sa	6,084	Sz. Nagy Gábor PhD sz.nagy.gabor@gmail.com tudományos munkatárs, Nemzeti Közszolgálati Egyetem Hadtudományi és Honvédtisztképző Kar A fakultatív hitoktatás a Rákosi-korszakban az adatok tükrében The optional religious education in the Rakosi-era in the light of the national datas. Abstract In September 1949, the religious education in public education was abolished, and optional religious education was replaced. The Hungarian Working People’s Party (MDP) called on the Party, the ministerial and central government bodies (in co-operation with the trade unions and the mass movement) to make every effort to reduce the number of students enrolling in religious education. Between 1949 and 1953, administrative measures were mostly used, while between 1953 and 1956, the emphasis was more on persuasion by parents. The plans and instructions made by the various bodies had to be implemented by the regional Party and state administration bodies (city, county, district councils) and data had to be provided. These data were forwarded to the Party’s bodies (especially the MDP Agitation and Propaganda Department) and to the State Office for Church Affairs (established in 1951), which also submitted it to the MDP’s bodies. Keywords religious education, communism, national datas, Churches, enrollment DOI 10.14232/belv.2022.2.12 https://doi.org/10.14232/belv.2022.2.12 174 Tanulmányok 2022. 2. Cikkre való hivatkozás / How to cite this article: Sz. Nagy Gábor (2022): A fakultatív hitoktatás a Rákosi-korszakban az adatok tükrében. Belvedere Meridionale vol. 34. no. 2. pp 173–189. ISSN 1419-0222 (print) ISSN 2064-5929 (online, pdf) (Creative Commons) Nevezd meg! – Így add tovább! 4.0 (CC BY-SA 4.0) (Creative Commons) Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) www.belvedere-meridionale.hu Magyarországon 1949. szeptemberében a Népköztársaság Elnöki Tanácsának 1949. évi 5. számú törvényerejű rendelete értelmében megszűnt a közoktatásban a kötelező vallásoktatás, és helyére a választható hitoktatás került. A mindössze két paragrafusból álló törvényerejű rendelet értelmében „A Magyar Népköztársaság Alkotmányának 54. §-ban foglalt, az állam és az egyház különválasztására vonatkozó rendelkezésnek megfelelően a vallásoktatás az iskolákban nem kötelező”.1 Még aznap a Vallás- és Közoktatásügyi Minisztérium kiadta másnapi hatálybalépéssel a rendelethez tartozó végrehajtási utasítást is, amely duplája volt a rendeletnek, ugyanis négy paragrafusból állt. Az utasítás mindössze annyival mondott többet a rendeletnél, hogy „Azok a szülők, akik gyermekeik vallásoktatását kívánják, legkésőbb szeptember 15-ig szóban vagy írásban az iskolában ezt a szándékukat bejelentik”, valamint azt, hogy a vallásoktatás felmerülő költségét a Vallás- és Közoktatásügyi Minisztérium állja.2 A törvényerejű rendelet kiadását nem előzte meg sem politikai, sem szakmai egyeztetés. Az egyházak a rendelet kiadásának a tervéről semmit nem tudtak, a politikai döntéshozók nem vonták be őket sem az egyeztetésbe. Az akkor már csak időszakosan működő, minden funkciójától megfosztott országgyűlésben sem tartottak vitát a törvényerejű rendelet kiadásáról, hanem csak Olt Károly, a Népköztársaság Elnöki Tanácsának titkára pár mondatos tájékoztatóját hallgathatták meg erről, az Országgyűlés 1949. december 5-i ülésnapján.3 Amikor 1947 tavaszán először felmerült a hitoktatás választhatóvá tétele, komoly politikai és társadalmi nyomás nehezedett az akkor még, névlegesen legalább is, koalíciós alapon működő kormányra.4 Egy évvel később, 1948-ban az iskolák államosítása során már egy jóval inkább kommunista befolyás alatt álló politikai mezőben került erre sor. Ráadásul, az 1947-es fakultatív hitoktatás bevezetésére irányuló lépéssel szemben, 1948-ban a katolikus egyház hivatalosan semmit nem sejtett arról, hogy az iskolák államosítása napirendben van.5 Ugyanakkor a nyilvánosság értesült az államosítás ellenzőinek és támogatóinak a véleményéről, 1 Az 1949. évi 5. számú törvényerejű rendelet a vallásoktatásról. Balogh – Gergely 2005. 894. 2 Az 1101/1-1949. (IX. 6.) VKM rendelet a vallásoktatásról szóló 1949. évi 5. számú törvényerejű rendelet végrehajtása tárgyában. Balogh – Gergely 2005. 895. 3 Az 1949. évi június hó 8-ára összehívott Országgyűlés Naplója. Hiteles Kiad. Első kötet. 1–25. ülés (1949. VI. 8. – 1949. XII. 22.) Budapest, 1950. 248 col. 4 Balogh 2012. 109–114., Nagy P. 2000a. 38–40., Sz. Nagy 2018. 202–203. 5 Balogh 2008. 76. 2022. 2. Tanulmányok 175 nem egy esetben tüntetések is voltak. Tehát 1947 és 1948-ban, bár a politikai helyzet egyre inkább a sztálinista berendezkedésű állam irányába mozdult el, a szólás szabadsága még, ha egyre korlátozottabban is, de létező valóság volt. Ugyanakkor, amit 1947-ben még el lehetett érni, az 1948-ban már nem volt realitás. A hatalom ekkor már nem engedett sem a politikai, sem az egyházi nyomásnak. A Magyar Dolgozók Pártja (MDP) a párt-, a miniszteriális- és a központi kormányzati szervek feladatául szabta (együttműködve a szakszervezetekkel és a tömegmozgalmi szervekkel), hogy minden eszközzel igyekezzenek csökkenteni a hitoktatásra beiratkozó tanulók számát. 1949–1953 között leginkább adminisztratív eszközöket vetettek be, míg 1953–1956 között inkább a szülők meggyőzésre helyezték a hangsúlyt. A különböző szervek által meghozott terveket, utasításokat pedig a területi párt és államigazgatási szerveknek (városi, megyei, járási tanácsok) kellett végrehajtaniuk, és adatot szolgáltatni. Ezeket az adatokat a párt központjába (az MDP Központi Vezetőség Agitációs és Propaganda Osztályához) és az 1951ben felállított Állami Egyházügyi Hivatalba (ÁEH) továbbították, akik azt felterjesztették az MDP vezető szerveihez. A területi közigazgatási szervek települések szerinti bontásban szolgáltattak adatokat az alap- és középfokú iskolákba beiratkozott tanulók számáról, valamint az ebből hitoktatásra jelentkezőkét, ezekből azonban a vezető szervekhez az adatok már csak a megyei és megyei jogú városi bontásban kerültek. Az 1954/1955. tanévtől megyei tanács oktatási osztályai az Oktatásügyi Minisztérium statisztikai osztályához is megküldték az intézmény működéséről szóló adatokat (beiratkozott tanulók, pedagógusok száma, hitoktatásra beiratkozott tanulók, stb.). Tanulmányomban ezeknek az adatoknak a mentén vizsgálom a fakultatív hitoktatás helyzetét a Rákosi-korszakban. Az országos adatok elemzése Az első, 1949 szeptemberében kelt adatok még nem tükrözték a szeptember elején kiadott törvényerejű rendelet hatását, hiszen országosan 92,15% volt a beiratkozottak aránya az általános iskolai hitoktatásra. Három helyről nem rendelkezünk adatokkal: Pest-Pilis-Solt-Kiskun vármegye északi részéről, valamint Zemplén és Szatmár vármegyéből. A 23 érintett vármegyéből (Pest-Pilis-Solt-Kiskun vármegye déli részéről rendelkezünk adatokkal), 13 megyében haladta meg a beiratkozottak száma az országos átlagot, míg 10 megye ez alatt szerepelt. (1. ábra) Az első beiratkozási időszakban az idő rövidsége miatt (ne felejtsük el, hogy a törvényerejű rendelet kiadása és a beiratkozás között néhány nap telt csak el) az állami és a pártgépezet nem készülhetett fel, így se a párt-, se a tömegmozgalmi szervek nem tudtak a rendelkezésre álló rövid idő alatt számottevő agitációt végezni. Ráadásul a hitoktatás az előző tanév végéig az alapfokú oktatásban kivétel nélkül kötelező volt, amely azt vonta maga után, hogy a szülők egyáltalán nem mérlegelték annak a lehetőségét, hogy a gyerek részt vegyen-e egyáltalán a hittanórákon. A bevezetésről elmondható, hogy elhamarkodottan történt, amely felkészületlenül érte a párt-, az állami- és a tömegszerveket, míg az egyházak helyzeti előnyben voltak az eddigi kötelező vallásoktatás miatt. 176 Tanulmányok 2022. 2. 1. ábra Az 1949/1950. tanévben, az általános iskolai hitoktatásra beiratkozottak aránya százalékosan6 A következő, 1950. júliusi beiratkozási adatok azonban már teljesen más számokat mutattak. A párt-, az állami- és a tömegszervezeteknek egy évük volt előkészíteni a hitoktatás beiratkozása elleni agitációs- és propagandamunkát. Ez elsősorban az egyházak ellen irányult, ugyanakkor olyan adminisztrációs intézkedéseket is végeztek, amelyek döntő mértékben befolyásolták a beiratkozást: amit a rendes iskolai beiratkozástól függetlenül kellett megtenni, amely plusz időt igényelt a szülőktől; a hitoktatásra való jelentkezést írásban vagy személyesen kellett bejelenteni, valamint a beiratkozás időpontját olyan későn hozták nyilvánosságra,7 hogy ezzel csak minimális időt biztosítottak az egyházak részére, hogy a szülőket a beiratkozás fontosságáról meggyőzzék. Ugyanakkor nem volt egy konkrét kijelölt nap, hanem a beíratást július 1-jéig lehetett megtenni. Ugyan szeptember elején pótbeiratkozást is biztosítottak, de ekkor már csak nagyon alacsony számban írattak be tanulókat. Az MDP Központi Vezetőségének (KV) Agitációs és Propaganda Osztálya (APO) ezeket a lépéseket hatékonynak találta, hiszen ezáltal „a politikailag legérettebb szülőkön kívül az ingadozók egy része sem íratta be hittanra gyerekét”. Ugyanakkor a jelentés arra is rámutatott, hogy a beiratkozás időpontjának eltitkolása és a beíratás adminisztratív akadályozása helytelen volt, mert ezzel lehetőséget biztosított az egyházak számára a pótbeiratkozások követelésére és a párt egyházpolitikájának bírálatára a hívek körében.8 Az országos átlag ebben a tanévben az általános és középiskolák együttes adataival 42,99% volt, amely számszerűleg azt jelentette, hogy az 1 272 402 beiratkozott tanuló közül 546 968an jelentkeztek vallásoktatásra. 9 Az általános iskolákra vonatkozó adat 44,45% volt, amely 6 MNL OL XIX-A-1-v-2-b-20-1949. Jelentés a szabad vallásoktatás bevezetéséről, 1949. szeptember 21. 152. 7 Szabad Nép, 1950. június 25. 7. 8 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Jelentés a hitoktatásról, 1951. május 21., 22. 9 MNL OL XIX-I-5-e-6. tétel, Vallásoktatásra jelentkezett tanulók száma megyénként az 1950/1951. iskolai évben (általános iskolai és általános gimnáziumi tanulók együtt), 1950.október 13. A statisztikából hiányzik 29 általános iskola adata, valamint a gimnáziumok adatai között nem szerepel a 8 katolikus gimnázium adata. 2022. 2. Tanulmányok 177 azt jelentette, hogy 1 223 657 tanulóból 543 941 tanuló jelentkezett hitoktatásra.10 Ebből a két adatból jól látszik, hogy a középfokú intézményekben csak minimális volt a hitoktatásra jelentkezők száma. Ugyanakkor félévkor a hitoktatásra járó általános iskolás tanulók száma országosan 54 664 fővel csökkent (ez 10%-os lemorzsolódás). Mindezek ellenére 13 megye adatai meghaladták az országos átlagot, amelyből kiemelkedtek Győr-Sopron, Vas és Veszprém megye adatai. Az országos átlag alatt 6 megye található. (2. ábra) Az év közbeni lemorzsolódást egy jelentéskészítő azzal magyarázta, hogy a szülők a beiratkozást csak próbaként használták arra, hogy megbizonyosodjanak arról, hogy mennyire gátolja vagy engedi a rendszer a beiratkozást.11 A már idézett MDP KV APO jelentés pedig arra mutatott rá ezzel kapcsolatban, hogy a lemorzsolódás nem tudatos agitációs munka vagy szülői döntés eredménye, hanem egyszerűen a gyerekek az utolsó tanóra után más elfoglaltság után néztek, és így lassan kimaradtak a hitoktatásból.12 2. ábra Az 1950/1951. tanévben, az általános iskolai hitoktatásra beiratkozottak aránya százalékosan13 Az előző évi adatokkal összehasonlítva jól látható, hogy a hitoktatásra beiratkozottak száma a felére csökkent egy év alatt, de ennek oka nem a különböző szervezetek hatékony munkája volt, hanem az, hogy a szülők egyrészt az adminisztratív intézkedések miatt elálltak a beíratástól, másrészt pedig azt is mérlegelhették, hogy a hitoktatásra való jelentkezést milyen hátrányos 10 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Jelentés a hitoktatásról, 1951. május 21., 22. 11 Nagy P. 2000b. 70. A szerző ugyan szószerinti idézetet használt, azonban az idézet pontos lelőhelyét nem adta meg. 12 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Jelentés a hitoktatásról, 1951. május 21., 23. 13 MNL OL XIX-I-5-e-6.t.-1950. Vallásoktatásra jelentkezett tanulók száma megyénként az 1950/1951. iskolai évben (általános iskolai és általános gimnáziumi tanulók együtt), 1950.október 13. 178 Tanulmányok 2022. 2. helyzetbe hozhatja őket a mindennapi életükben.14 Az állam erős egyházellenes tevékenysége, a média által folyamatosan sugallt egyházellenes propaganda is megtehette a hatását azok között, akikre az MDP KV APO fentebb már ismertetett jelentésében is utaltak. Ugyanakkor az MDP KV 1950. június 1-jén hozott határozata, valamint a Vallás- és Közoktatásügyi Minisztérium 1950. szeptember 15-i rendelete is szerepet játszott a beiratkozási adatok csökkenésében. Az 1951/1952. tanévre a hittanbeiratkozás időpontját a Közoktatásügyi Minisztérium június 27–28-ára tűzte ki, amelyet június 22-én hoztak nyilvánosságra.15 Ez az előző évvel ellentétben már pontosan szabályozta azt a két napot, amikor ezt meg lehetett tenni. A beiratkozási adatok kis növekedést mutatnak, de ez még jóval a hibahatáron belül maradt. Országos szinten a beiratkozottak aránya 44,55%-os volt (az előző évi 44,45%-hoz képest)16. Ebben a tanévben összesen 1 177 395 általános iskolás iratkozott be a tanintézményekbe, ebből 524 578 gyereket írattak be vallásoktatásra.17 Ahogy az előző évben, ebben az évben is 13 megye adatai haladták meg az országos átlagot, kiugró, 70% feletti beiratkozási adatot csak két megye produkált: Győr-Sopron és Vas megye. (3. ábra) 3. ábra Az 1951/1952. tanévben hittanra beiratkozottak számaránya százalékban18 14 Ahogy azt majd a későbbiekben látni fogjuk Pest megye példáján, járási és települési szinten erős szóródás volt a szülői aktivitás között 15 Szabad Nép, 1951. június 22. 6. 16 Ebben az évben már csak az általános iskolák adatait tartalmazza a jelentés 17 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 20.; Érdemes megjegyezni, hogy az MDP KV APO jelentésében 1 213 251 tanulóból iratkozott be 524 578 diák hitoktatásra, amely 43,2%-os arány. MNL OL M-KS 276. f. 89. cs. 276. ő.e. Feljegyzés a hitoktatás helyzetéről, [1952. nyara] 69. 18 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 20. 2022. 2. Tanulmányok 179 A lemorzsolódás továbbra is magas volt: fél év alatt a beiratkozott tanulók 16%-a maradt ki a hitoktatásból (ez kicsivel több, mint 85 ezer gyereket jelentett). A hitoktatás helyzetéről készült MDP KV APO jelentés kiemelte, hogy bár Győr-Sopron és Vas megyében továbbra is magas a beiratkozottak aránya, Budapesten (2,79%) az ipari kerületekben (pl. Újpesten, Angyalföldön és Pesterzsébeten), az ipari (Diósgyőr és Ózd 14,6%), és a szocialista városokban (Karcag 16%, Túrkeve 6,79%, Kisújszállás 12,7%) ezek a számok nagyon alacsonyak. Ugyanakkor a jelentés azt is hangsúlyozta, hogy egyelőre ezek az adatok még nem jellemzőek minden szocialista városra. A jelentés szerint jónak értékelték a párt- és tömegszervezetek munkáját, valamint a természettudományos előadások tartását, azonban véleményük szerint az ifjúsági szervezetek hatékonyságát fokozni kell. Ahogy az előző évben, úgy ebben az évben is problémaként jelezték, hogy a lemorzsolódás nem a szülők között végzett hatékony politikai agitáció eredménye, hanem az, hogy a gyerekek „a szülők tudta és beleegyezése nélkül nem jár[nak] hittanra”, noha a szülők beíratták őket. Az APO szerint a hatékonyságot csak tudatos agitációs- és propagandamunkával lehet növelni, amelyben a párt-, a megyei és járási közigazgatási- és tömegszervezetek mellett a sajtó is tevékenyen kiveszi a részét.19 Ebben a tanévben már kivették a részüket a munkából a megyei és a járási tanácsok is, valamint megindult a természettudományos előadássorozat is. Azonban továbbra is főleg az adminisztrációs lépések voltak azok, amelyek igyekeztek eltántorítani a szülőket és a diákokat a hitoktatástól. Azonban az adatokat nézve a hitoktásra jelentkezők arányának a csökkentése, noha a párt a munka egyes elemeit jónak értékelte, továbbra sem volt hatékony. Ezért is merült fel az MDP APO KV javaslataiban, hogy nagyobb gondot kell fordítani az anyák meggyőzésére, főleg a falusi környezetben. A következő, 1952/1953-as tanévre a hitoktásra való beiratkozás időpontjának hivatalos kihirdetése az előző évek hagyományait követte: minél később nyilvánosságra hozni, így próbálva megakadályozni azt, hogy mind az egyházak, mind a szülők részéről kellő idő legyen a felkészülésre. A hittanbeiratkozás időpontját az MDP KV Titkársága határozatában, akár csak az előző évben, június 27–28-ára tűzte ki,20 amelyet a határozat értelmében június 24-én hoztak nyilvánosságra,21 míg pótbeiratkozás időpontját, amely augusztus 24-e volt, augusztus 21-én.22 A vizsgált tanévre vonatkozóan már négy forrásból is van adatunk, ezek azonban lényegesen nem tértek el egymástól. Az MDP KV APO jelentésében az általános iskolai tanulók 26,1%-a jelentkezett vallásoktatásra, azonban teljes megyei adatokat nem közölnek, csak a legmagasabb és a legalacsonyabb százalékot „produkáló” megyéket tüntetik fel.23 A legátfogóbb adatokat az ÁEH közölte. A közölt adatok szerint az 1 176 859 beiratkozott általános iskolai tanulóból 305 845 diákot írattak be hittanra, amely 25,99%-os arány.24 Az Oktatásügyi Minisztérium 19 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Feljegyzés a hitoktatás helyzetéről, [1952. nyara] 69–82. 20 MNL OL M-KS 276. f. 54. cs. 196. ő.e. A Titkárság 1954. június 4-i határozata a hittanbeiratkozásokkal kapcsolatos tennivalókról, 1952. június 4. 24–25. 21 Szabad Nép, 1952. június 24. 8. 22 Szabad Nép, 1952. augusztus 21. 4. 23 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Javaslat az 1953/54. tanévi hittanbeíratásra, 1953. május 13. 109. 24 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 20. 180 Tanulmányok 2022. 2. adatai szerint az általános iskolások 29,63%,25 míg a Pedagógusok Szakszervezete adatai szerint 30,23% iratkozott be vallásoktatásra.26 A számok közötti eltérést az okozhatta, hogy elképzelhető, hogy az adatszolgáltatás során az adott intézmények vagy járási, megyei hivatalok rossz, vagy nem teljeskörű adatot adtak meg a kérelmező felé. Ez azonban sejtés, a forrásokban erre semmiféle utalás nincs. Mivel azonban a százalékok alig térnek el egymástól, elmondhatjuk, hogy átlagban az általános iskolai tanulók nagyjából 29%-a iratkozott be hittanra. Ebben az évben már csak kilenc megye átlaga haladta meg az országos átlagot, és ezek közül mindegyik megye 40%, vagy afeletti beiratkozási adatot ért el. Ami leginkább meglepő, hogy Zala megyében 30%-kal csökkent a beiratkozottak aránya az előző évhez képest.27 (4. ábra) 4. ábra Az 1952/1953. tanévben hittanra beiratkozottak számaránya százalékban (átlag)28 A tanév közbeni lemorzsolódásról hivatalos statisztikai adat nem található, de a már idézett MDP KV APO jelentés szerint az általános iskolákban országosan 18,6% volt. Az okok azonban 25 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Oktatásügyi Minisztérium jelentése a hittanbeíratás tapasztalatairól, 1954. július 6. 26. 26 MNL OL XIX-A-21-a-300/1953. A Pedagógusok Szakszervezete Kultúr-nevelési Osztályának jelentése az 1953/1954.-ik tanévi hittanbeiratások tapasztalatai, további feladatai a klerikális reakció, a vallásos ideológia elleni harcban, 1953. augusztus 17. 27 Ez különösen annak a fényében meglepő, hogy a következő évben ismét a korábbi számokat „hozta”. Feltételezhető, hogy adatfelvételi probléma állhat a háttérben 28 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Javaslat az 1953/54. tanévi hittanbeíratásra, 1953. május 13. 109., MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 20., MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Oktatásügyi Minisztérium jelentése a hittanbeíratás tapasztalatairól, 1954. július 6. 26., MNL OL XIX-A-21-a-300/1953. A Pedagógusok Szakszervezete Kultúr-nevelési Osztályának jelentése az 1953/1954.-ik tanévi hittanbeiratások tapasztalatai, további feladatai a klerikális reakció, a vallásos ideológia elleni harcban, 1953. augusztus 17. 2022. 2. Tanulmányok 181 nem változtak: továbbra is ritkább volt a szülő által kiíratott gyerek, mint az „órák következetes nem látogatása”. A jelentés kiemelte az egyre aktívabb felvilágosító munkát, a párt- és tanácstagok, funkcionáriusok gyerekeinek távoltartását a hitoktatástól, a hittanórák megfelelő ellenőrzését a közoktatási szervek részéről. A pedagógusok is egyre javuló mértékben végezték el az ezzel kapcsolatos feladataikat: vallási kérdések cáfolata a természettudomány eszközeivel, szakkörök szervezése, ifjúsági szervezetek támogatása, valamint a pedagógusgyerekek egyre kisebb számú részvétele a vallásoktatásban. Az ifjúsági szervezetek is egyre hatékonyabban vették ki a részüket a politikai munkából, főleg a hétvégi foglalkozások megszervezéséből. Ugyanakkor a jelentés arra is kitért, hogy a politikai feladatokat továbbra is az adminisztratív intézkedésekkel (beiratkozás, az órák megtartásának megnehezítése, hitoktatói megbízólevelek késve kiadása, stb.) próbálták meg kiváltani. Ugyanakkor a pedagógusok között is akadnak még nagy számban olyanok, akik továbbra is templomba járnak, és emiatt passzívan állnak az egyházi agitációval szemben.29 Az 1953/1954-es tanév már Nagy Imre kormányalakítása után történt, a beiratkozási számokon azonban még nem érződött a kormányprogramban megfogalmazott nagyobb fokú türelmesség a vallással kapcsolatban. A beiratkozás továbbra is előre kijelölt két napon történt (június 26–27.), és abban sem volt változás, hogy a lehető legkésőbb hozták ezt nyilvánosságra.30 Ugyanakkor ebben az évben lehetőség volt pótbeiratkozásra31 (augusztus 24.), de ebben az esetben is megtartották a „lehető legkésőbb közreadni”-elvét.32 Erről a tanévről is több forrásból vannak adataink. Az előző évekkel ellentétben ebben az évben az Oktatásügyi Minisztérium közölte az MDP KV APO-val a rendes és a pótbeiratkozások adatait. Noha fentebb már említettem, hogy az összesített adatok továbbra is csökkentek, azonban a júniusi és az augusztusi adatok között látványos emelkedés volt. A júniusi beiratkozás után az országos átlag 14,46% volt, míg a pótbeiratkozás után ez az adat több mint a duplájára, 31,35%-ra emelkedett. (5. ábra) A egyes megyékben is számottevő emelkedés volt. (6. ábra) A két adatszolgáltatás egyik legfőbb oka az lehetett, hogy a júniusi és az augusztusi beiratkozás között komoly politikai változás történt az országban: az 1953. évi választások után július 3-án alakult meg a Nagy Imre vezette kormány,33 amely az egyházzal és a vallással kapcsolatban is új irányt szabott a kormánypolitikában. Nagy Imre az Országgyűlés előtt kihirdetett kormányprogramjában a következőképpen jelölte meg az egyházzal kapcsolatos feladatokat: „Nagyobb türelmességet kell tanúsítani vallási kérdésekben. Megengedhetetlen e téren adminisztratív eszközök alkalmazása, ami eddig bizony néha előfordult. A kormány ebben a kérdésben a türelmesség alapján áll, melynek a felvilágosítás és a meggyőzés az eszköze. Adminisztratív vagy más kényszerítő eszközök alkalmazását a kormány elítéli és nem fogja tűrni.”34 A pótbeiratkozások után, ahogy a korábbi években is, Győr-Sopron és Vas megyében volt a legnagyobb a beiratkozók aránya. 29 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Javaslat az 1953/54. tanévi hittanbeíratásra, 1953. május 13. 109–110. 30 Szabad Nép, 1953. június 23. 4. 31 MNL OL M-KS 276. f. 53. cs. 131. ő.e. Határozat hittan pótbeiratás engedélyezésére, 1953. augusztus 15. 81. 32 Szabad Nép, 1953. augusztus 21. 4. 33 Gyarmati 2011. 329–342. 34 Az 1953. évi július hó 3-ára összehívott Országgyűlés naplója. Hiteles Kiadás. I. kötet. 1–27. ülés (1953. VII. 3–1956. II. 11.). Budapest, 1967. 21. col., idézi még: A kormány programmja az országgyűlés előtt. Szabad Nép, 1953. július 5. 2. 182 Tanulmányok 2022. 2. 5. ábra Az 1953. júniusi és augusztusi beiratkozási adatok35 6. ábra Az 1953. júniusi és augusztusi beiratkozási adatok közötti különbség36 35 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Kimutatás a hittanra beiratkozott tanulókról, 1953. augusztus 27. 134. 36 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Kimutatás a hittanra beiratkozott tanulókról, 1953. augusztus 27. 134. alapján 2022. 2. Tanulmányok 183 A júniusi beiratkozással kapcsolatban MDP KV APO jelentés kiemelte, hogy a korábbi évekkel ellentétben az adminisztratív intézkedések jelentős mértékben visszaszorultak, ugyanakkor „párt és állami szerveink az adminisztratív módszerekkel együtt magáról a világnézeti és felvilágosító munkáról is lemondtak”. A tapasztalat az volt, hogy a júniusi párthatározatban foglaltak elbizonytalanították a különböző szerveket és a pedagógusokat is azzal kapcsolatban, hogy milyen módszereket lehet alkalmazni. Ez pedig lehetőséget adott az egyházak számára, hogy fokozott tevékenységet folytassanak a hittanbeiratkozások mellett. A pótbeiratkozásról az ÁEH készített feljegyzést az MDP KV APO részére. Ebben külön értékelték az egyházak, valamint a párt- és az állami szervek tevékenységét is. Az ÁEH kiemelte, hogy a pótbeiratkozáskor az egyházak igen komoly munkát végeztek a hívek körében: a templomkapukra függesztették ki a beiratkozás időpontját, elmagyarázták a szülőknek a beiratkozás feltételeit, előre elkészített nyomtatványokat helyeztek ki, a távolabb élőknek segítettek eljutni az iskolákhoz, valamint a beiratkozás során tapaszt visszaéléseket a püspökök azonnal jelezték az ÁEH felé. Ezzel szemben a párt- és állami szervek esetében bizonyos fokú bénultságot tapasztaltak. Ez annak köszönhető, hogy az információáramlás a megyei szervektől nem valósult meg a járási szervek, majd innen a tantestületek felé, ez pedig azt eredményezte, hogy – akárcsak az előző években – ismételten nem a politikai felvilágosító munkára, hanem az adminisztratív eszközök használatára helyezték a hangsúlyt. Ezt pedig a szülők úgy értelmezték, hogy a helyi szervek és az iskola képtelen végrehajtani a minisztériumok által előírt feladatukat: „a tömegek hangulatából kiérződik az, hogy amikor sérelmeiket nem orvosolják, vagy követeléseiket nem teljesítik, arra hivatkoznak, hogy nem ez áll a kormányprogrammban [!sic]”.37 Mind az MDP KV APO, mind az ebből készült, az 1954/1955. tanévre szóló MDP KV határozat megállapította, hogy a pótbeiratkozás lehetőségének biztosításával a beiratkozottak számának nagyarányú növekedése „reális volt”.38 Az 1954/1955. tanévre való beiratkozás rendjét a fentebb már említett MDP KV határozat tartalmazta. Eszerint a beiratkozáson legalább az egyik szülőnek jelen kell lennie és a beiratkozási lapon mind a két szülőnek az aláírása szükséges. Ettől indokolt esetben lehet eltérni („katonai szolgálat, betegség, távoli munkahely, stb.”), ugyanakkor a tanév megkezdéséig az aláírást pótolni kell. A határozat a gyerek személyes jelenlétét ebben az évben nem írta elő. A beiratkozást 1954. június 28–29-én kellett megtartani, az egyházak erről két héttel korábban kaphatnak értesítést. A határozat külön kiemelte, hogy a pártfunkcionáriusok a gyerekeiket ne írassák be, valamint arra is felhívta a figyelmet, hogy a „tekintéllyel bíró párttag és a párton kívüli szülők” se írassák be a gyerekeiket. A határozat arra is felhívta az iskolaigazgatók és a pedagógusok figyelmét, hogy „messzemenően kerüljék a helytelen adminisztratív eszközök igénybevételét”.39 A időpontot a Szabad Nép három nappal a beiratkozás előtt hozta nyilvánosságra.40 A tanévről több forrásból rendelkezünk adatokkal. Egyrészt az MDP KV APO részére megküldött adatokkal az Oktatásügyi Minisztérium (OM), az ÁEH és az MDP KV Tudományos 37 MNL OL M-KS 276. f. 89. cs. 276. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos feljegyzése az augusztus 24-i hittanpótbeiratásról, 1953. augusztus 25. 127–128. 38 MNL OL M-KS 276. f. 53. cs. 180. ő.e. Határozat az 1955–1955. évi hittanbeiratkozásokra, 1954. június 9. 94. 39 MNL OL M-KS 276. f. 53. cs. 180. ő.e. Határozat az 1955–1955. évi hittanbeiratkozásokra, 1954. június 9. 95–96. 40 Szabad Nép, 1954. június 25. 4. 184 Tanulmányok 2022. 2. és Kulturális Osztálya (TKO) részéről. Ebben az évben egy új statisztikai szolgáltatás is megjelent, amelyben az alap- és középfokú intézmények közöltek részletes statisztikákat az adott intézmény teljes működéséről a megyei tanácsok oktatási osztálya részére, amelyet ők továbbítottak az OM statisztikai osztálya felé, megyei összesítések formájában. Az ÁEH adatai szerint a hitoktatásra beiratkozott tanulók a teljes létszám 35,62%-át,41 az MDP KV TKO szerint 35,49%-át,42 míg az OM adatai alapján 40,53%-át tette ki.43 Az oktatási intézmények adatai alapján ez az érték 32,70% volt.44 Az országos átlag ezen adatok szerint 36,09% volt, amely 5%-os emelkedést mutatott az előző év adataihoz képest. A beiratkozott tanulók számának vizsgálatából jól látszik, hogy az ÁEH és az MDP KV TKO adatai előzetes adatok lehettek, hiszen jóval kevesebb beiratkozóval számoltak, mint az oktatási intézmények által leadott adatok.45 (1 123 016 és 1 115 754 szemben az 1 207 455 tanulóval). Mivel az OM statisztikai osztálya felé leadott adatokat tekinthetjük hivatalos adatközlésnek, így ezeket az adatokat fogadhatjuk el hivatalosnak, ezért csak ennek az adatait közlöm. Az 1954/1955. tanévben összesen 1 207 455 tanuló iratkozott be az általános iskolákba, ebből 394 880 gyerek járt hittanra. Az adatok alapján látható, hogy ebben a tanévben 15 megye került az országos átlag fölé. Négy megye beiratkozási aránya 50%, két megye pedig 45% felett volt. A legmagasabb értékeket, ahogy a korábbi években is, Vas és Győr-Sopron megye produkálta. Ugyanakkor tovább folytatódott Nógrád megye messze az átlagon felüli aránya. (7. ábra) A jelentés egyik érdekessége, hogy külön megadta az alsó és a felső tagozatra beiratkozottak arányát is. Alsó tagozaton a beiratkozottak 34,48%-a, míg felső tagozaton 30,74%-a járt hittanra. Az ÁEH által készített összefoglaló jelentés kiemelte, hogy az előző évhez képest az egyházak kiemelt figyelmet fordítottak a beiratkozásra. Ennek okát abban látták, hogy a kormány döntése értelmében az adminisztratív eszközöket a párt- és azállami szervek igyekeztek visszaszorítani. Az aktivitást főként a katolikus egyház részéről érzékelték: ők leginkább a „régebben is jól bevált egyéni meggyőzés módszerét választotta”, de ugyanakkor használta a tavalyi pótbeiratkozáskor használt közösségi tevékenységét is (plakátok, körlevelek, prédikációk). A pártés az állami szervek részéről a jelentés rámutatott, hogy „az idei hittanbeíratást a párt- és az állami szervek többsége elég nagyfokú bizonytalansággal és bizonyosfokú szorongással várta”. Ez főként az adminisztratív módszerek visszaszorítására tett kormányintézkedések miatt történt. Ugyanakkor nagy segítséget kaptak az MDP KV által kiadott határozat által, amely egyértelműen megfogalmazta a különböző szintű párt- és állami szervek feladatát. A jelentés ugyanakkor továbbra is gyengének minősítette a politikai felvilágosító munka hatékonyságát. Ez pedig, 41 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 20. 42 MNL OL M-KS 276. f. 89. cs. 277. ő.e. MDP KV TKO kimutatása az 1953/54. és 1954/55. évi hittanbeiratkozásokról, d.n. [1954]. 11. 43 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Oktatásügyi Minisztérium jelentése a hittanbeíratás tapasztalatairól, 1954. július 6. 26. 44 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1954–1955 45 Az OM csak százalékokat adott meg az MDP KV APO felé. MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Oktatásügyi Minisztérium jelentése a hittanbeíratás tapasztalatairól, 1954. július 6. 26. Az OM felé a megyei tanácsok szeptember 15-i határidővel kellett, hogy adatot szolgáltassanak: MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1954–1955 Tanulmányok 2022. 2. 185 az adminisztratív eszközök visszaszorítása mellett nagyban csökkentette a beiratkozás elleni lépések hatékonyságát.46 Ugyan a jelentések nem foglalkoztak a lemorzsolódások számával, de az OM statisztikai osztálya részére a tanév végén megküldött jelentésekből látható, hogy országos szinten a lemorzsolódás aránya mindösszesen 1% volt (összesen 19 883 fő), ugyanakkor ezt csak tájékoztató adatként lehet kezelni, mert az általános iskolai oktatásból kiesettek száma nem szerepelt az adatok között.47 7. ábra Az 1954/1955. tanévben hittanra beiratkozottak számaránya százalékban48 Az 1955/1956. tanévre a beiratkozás ismételten egy megváltozott politikai környezetben történt. 1955. áprilisában az Országgyűlés felmentette Nagy Imre miniszterelnököt és helyette Hegedűs Andrást nevezte ki. Ez pedig azt eredményezte, hogy az eddig háttérbe szorított Rákosi Mátyás ismét visszatért a politikai hatalomba.49 A tanévre a hittanbeiratkozás rendjét nem szabályozta párthatározat. Az MDP KV APO tájékoztatója szerint a beiratkozást 1955. június 24–25-én kell megtartani. Az első napon 14–18 óra, míg a második napon 8–13 között. A pontos időpontot mind a sajtó (Szabad Nép, Magyar Nemzet), mind az iskolaigazgatók két nappal a beiratkozás előtt tehették nyilvánossá. A beiratkozás menete nem változott az előző évihez képest.50 Az előírtakkal szemben azonban 46 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1954. évben történt hittanbeiratkozásokról, 1954. július 2. 14–16. 47 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1954–1955 48 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1954–1955 49 Gyarmati 2011. 358–373. 50 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az MDP KV APO szigorúan bizalmas tájékoztatója az 1955. évi hittanbeiratkozásokkal kapcsolatban, 1955. május 19. 145–146. 186 Tanulmányok 2022. 2. a Szabad Nép és a Magyar Nemzet is csak a beiratkozás elő napján hozta nyilvánosságra az időpontokról szóló MTI-hírt.51 A beiratkozási adatokról három forrásból tájékozódhatunk. Az ÁEH ebben az évben is közölt adatokat az MDP KV APO-val,52 viszont újdonság, hogy a Belügyminisztérium is hasonlóan járt el.53 Ahogy az előző évben, úgy 1955-ben az OM statisztikai osztálya is bekérte a hitoktatásra vonatkozó adatokat a megyei tanácsok oktatási osztályaitól.54 A három adat között lényegi eltérés nem található, ami a beiratkozottak arányát illeti (ÁEH: 30,40%, BM: 29,86%). A továbbiakban csak az OM statisztikai osztálya által összeállított adatsorral foglalkozom. A statisztika szerint országosan 1 226 200 tanuló iratkozott be az általános iskolákba, ebből 331 856 tanuló járt hittanra. Ez a beiratkozottak 27,06%-a, amely 5%-os csökkenést mutat az előző év adataihoz képest. A vizsgált évben 13 megye adatai haladták meg az országos átlagot. Ahogy a korábbi években, ebben az évben is Győr-Sopron és Vas megyében volt a legmagasabb a beiratkozók aránya. További hat megye ért el 40% feletti arányt. (8. ábra) 8. ábra Az 1955/1956. tanévben hittanra beiratkozottak számaránya százalékban55 51 Szabad Nép, 1955. június 24. 4., Magyar Nemzet, 1955. június 24. 6. 52 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1951–1955. évben történt hittanbeiratkozásokról, 1955. június 29. 357. 53 MNL OL M-KS 276. f. 89. cs. 278. ő.e. A Belügyminisztérium szigorúan titkos tájékoztató jelentése a hittanra való beiratkozásokról, 1955. augusztus 4. 3. 54 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1955–1956 55 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1955–1956 2022. 2. Tanulmányok 187 Az MDP KV APO jelentése a beiratkozás tapasztalatairól nem fedte le az egész ország területét: mindössze nyolc megyei és a budapesti pártbizottság, valamint a Vasútpolitikai Osztály jelentései alapján állították össze. A beiratkozottak szülei legtöbb esetben nem a politikailag preferált osztályok tagjai közül került ki (értelmiségiek, kisiparosok), ugyanakkor a jelentés szerint továbbra is magas a párttagok és állami funkcionárius szülők gyerekeinek a száma. Az egyházak és a pártszervezetek munkája az előző évekhez hasonlóan alakult: míg az egyházak továbbra is kiemelt fordítottak a szülők meggyőzésére és a minél szélesebb körű felvilágosító munkára, addig a pártszervezetek továbbra sem voltak képesek érdemi agitációs és propagandamunkát kifejteni „a vallásos ideológia, a klerikális reakció leleplezésére”, mégpedig azért mert a Nagy Imre miniszterelnöksége alatt „a jobboldali nézetek elterjedésének következtében – párt és állami szerveinknél a liberalizmus, elnézés ütötte fel a fejét”. Ennek ellenére a pártszervek munkáját a jelentés jónak értékelte, hiszen a hittanbeiratkozás aránya csökkent az előző évekhez képest. Mindezt pedig úgy, hogy az adminisztratív eszközök igénybevétele nem volt jelentős, ami a felvilágosító munka térnyerését mutatta – legalább is a jelentések szerint.56 A Rákosi-korszak utolsó hittanbeiratkozása ismét egy megváltozott politikai térben folyt le. A beiratkozást az ÁEH előterjesztése nyomán 1956. június 27–28-án tartották meg. Az első napon 14–18 óra, míg a második napon 8–13 között. A beiratkozás módja az előző évekhez képest nem változott (szülői aláírások stb.).57 A tájékoztatót hivatalosan az MDP KV küldte meg a pártszervek részére május végén. A tájékoztatója külön kiemelte, hogy az előző beiratkozáskor felülprezentált párt- és állami funkcionáriusok ne írassák be a gyerekeiket a hitoktatásra. 58 A beiratkozás időpontját az Szabad Nép az MTI-hír alapján június 25-én hozta nyilvánosságra.59 A tárgyalt tanévről csak két szerv szolgáltatott adatokat: az ÁEH és az OM statisztikai osztálya. Az ÁEH adatai szerint az országos átlag 30,18%,60 míg OM szerint ez az adat 28,30%. volt.61 A két adatból látszik, hogy az 1956/57. tanévben enyhe emelkedés volt a beiratkozottak arányában. Ebben a tanévben tizenhárom megye adatai meghaladták, míg két megye adatai közel azonosak voltak az országos átlaggal. A két legmagasabb százalékot elérő megye ebben a tanévben is Győr-Sopron és Vas megye volt. (9. ábra) Erről a tanévről csak egy előzetes jelentés áll a rendelkezésünkre, amelyet az ÁEH készített az MDP KV APO részére. A jelentés megállapította, hogy az egyház részéről az előző években tapasztalt tevékenységet végezték ebben a ciklusban is, ugyanakkor jóval több helyen is „a kiadott rendelkezések megsértését, ellenségeskedést, meg nem engedett módszereket tapasztaltunk a papok részéről”. Ilyen volt a papok részvétele az oktatási intézményben a beiratkozáskor, pedagógusok, ingadózó szülők egyéni meggyőzésére tett kísérletek, stb. A pártszervek 56 MNL OL M-KS 276. f. 89. cs. 277. ő.e. Az MDP KV APO bizalmas információja a hittanbeíratás tapasztalatairól, 1955. július 19. 292–296. 57 MNL OL M-KS 276. f. 89. cs. 278. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos javaslata az 1956/47. évi hittanbeiratkozásokkal kapcsolatban, 1956. április 16. 307–310. 58 MNL OL M-KS 276. f. 89. cs. 278. ő.e. Az MDP KV szigorúan bizalmas tájékoztatója az 1956/47. évi hittanbeiratkozásokkal kapcsolatban, 1956. május 23. 334–336. 59 Szabad Nép,1956. június 25. 4. 60 MNL OL M-KS 276. f. 89. cs. 278. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos összesítő kimutatása az 1953–1956. évben történt hittanbeiratkozásokról, 1956. július 6. 372. 61 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1956–1957 188 Tanulmányok 2022. 2. részéről végzett munkát a jelentés jónak értékelte (különösen a politikai felvilágosító munka terén), ugyanakkor az adminisztratív eszközök alkalmazása továbbra sem szűnt meg, „amelyek elsősorban az esetleges százalék-növekedés megakadályozásából eredtek”.62 9. ábra Az 1956/1957. tanévben hittanra beiratkozottak számaránya százalékban63 Összefoglalás A hitoktatás adatainak vizsgálatából jól látható, hogy az adatok folyamatosan változtak, amelyek nem voltak függetlenek a korszakban lejátszódott politikai változásokból. Az első évben még nem beszélhetünk csökkenésről, hiszen a párt- és állami szervek részére nem állt elegendő idő arra, hogy a párthatározatban és a törvényerejű rendeletben foglaltakat elvégezzék. Az igazi hatást 1952-re sikerült elérni, amikor drasztikus módon lecsökkent a beiratkozások száma. Azonban ez nem a politikai munka eredménye volt, hanem az adminisztratív eszközök túlzott használata, amellyel a szülőket minden módon megpróbálták távol tartani a beiratkozásoktól. A Nagy Imre-kormány időszakában egy kicsit ugyan emelkedtek a számok (ez főleg az 1953-as pótbeiratkozásoknál látszódott), de a kormány megbuktatása után 1955-ben már ismét a korábbi szintre esett vissza, és ezen az 1956-os tavaszi újabb fordulat sem tudott változtatni. 62 MNL OL M-KS 276. f. 89. cs. 278. ő.e. Az Állami Egyházügyi Hivatal szigorúan titkos tájékoztató gyors-jelentése az 1956/57. évi hittanbeiratkozásokról, 1956. június 30. 368–370. 63 MNL OL XXXII-20-XXXII. Általános iskolák megyei összesítői (kötet), 1956–1957 2022. 2. Tanulmányok 189 Az is jól látható, hogy az alacsony számok mellett minden évben volt legalább tíz olyan megye, ahol a számok mindig meghaladták az országos átlagot, így kijelenthető, hogy az MDP hitoktatásellenes politikája kudarcos volt. Ennek oka a már említett politikai, felvilágosító munka hiánya, valamit az, hogy (a kutatás további időszakára vonatkozó hipotézist felvázolva) a kisebb lélekszámú településeken jóval magasabb volt (akár duplája is) a beiratkozások száma, mint a nagyobb lélekszámú településeken. Ez pedig azt is jelenti, hogy a párt-, az állami- és a tömegmozgalmi szervek egyházellenes propagandája a kisebb településeken nem volt hatékony. Ugyanakkor nem sikerült a pedagógusok, párttagok, párt-, állami- és tömegszervezeti funkcionáriusok, tagok között sem az elvárt eredményt elérni az agitáció során. A további kutatások során járási, települési szintre kell koncentrálni (adatokra, egyházi és egyházellenes tevékenységre, stb.), amely pontosabb képet fog adni egyrészt arról, hogy miért nem sikerült a hitoktatást a párt legfelsőbb vezetősége által elvárt szintre csökkenteni, másrészt pedig arról is, hogy milyen egyéb adatok jelennek meg (származási, felekezeti) a statisztikában. Irodalomjegyzék Az 1949. évi június hó 8-ára összehívott Országgyűlés Naplója. Hiteles Kiad. Első kötet. 1–25. ülés (1949. VI. 8. – 1949. XII. 22.) Budapest, Athenaeum, 1950. Balogh Margit – Gergely Jenő (2005): Állam, egyházak, vallásgyakorlás Magyarországon, 1790–2005. Dokumentumok. II. 1944–2005. Budapest, MTA (História Könyvtár. Okmánytárak, 2.) Balogh Margit (2008): Kötélhúzás a kulisszák mögött. Czapik Gyula egri érsek tárgyalásai 1948-ban. In Szabó Csaba – Szigeti László (szerk.): Az egyházi iskolák államosítása Magyarországon 1948. A Lénárd Ödön Közhasznú Alapítvány évkönyve 2008. Budapest, Lénárd Ödön Közhasznú Alapítvány – Új Ember. 65–102 Balogh Margit (2012): Fakultatív hitoktatás és konkordátum-fiaskó. Magyar Egyháztörténeti Vázlatok. 24. évf. 3–4. sz. 109–119. Nagy Péter Tibor (2000a): Hittanoktatás az ötvenes években. Iskolakutúra. 10. évf. 2. az. 38–46. Nagy Péter Tibor (2000b): Fakultativitás és lajstrom. Hittan és politika, 1949–1957. Világosság. 41. évf. 1. sz. 59–77. Sz. Nagy Gábor (2018): A katolikus egyház a baloldali sajtó tükrében. In Klestenitz Tibor – Paál Vince (szerk.): Médiatörténeti tanulmányok 2018. Budapest, MTA BTK. 185–208. (Médiatudományi Könyvek) Gyarmati György (2011): A Rákosi-korszak. Rendszerváltó fordulatok évtizede Magyarországon, 1945–1956. Budapest, ÁBTL–Rubicon.
https://openalex.org/W4225115356	https://www.researchsquare.com/article/rs-1591133/latest.pdf	English	null	Preoperative and Postoperative Factors Affecting Functional Success in Anatomically Successful Retinal Detachment Surgery	Research Square (Research Square)	2,022	cc-by	5,317	Preoperative and Postoperative Factors Affecting Functional Success in Anatomically Successful Retinal Detachment Surgery Mehmet Eren Guner (  mehmeterenmugla@gmail.com ) University of Cincinnati Methods 75 eyes of 75 patients with rhegmatogenous retinal detachment (RRD) who underwent anatomically successful surgery, from 2014 to 2019 with more than one year follow up were included in the study. Demographic characteristics, ocular examination findings, preoperative and postoperative Spectral Domain Optic Coherence Tomography (SD-OCT) images were retrospectively evaluated. Conclusion Early intervention, presence of preoperative ELM integrity and restoration of postoperative outer retinal layers positively affected functional success. The presence of preoperative VH, PVR and the extent of retinal detachment had a negative effect on prognosis at 12 months. Purpose To investigate preoperative and postoperative factors affecting functional success in anatomically successful retinal detachment surgery. Results The mean age was 53.9 ± 17 (range: 11–85). The mean follow-up period was 36.7 ± 16 (range: 14–72) months. The mean best corrected visual acuity (BCVA) of the patients before surgery was 1.35 ± 1.24, and at postoperative 12 months was 0.66 ± 0.5 logMAR. Patients who were operated in 7 days of visual symptoms onset were found to have better visual acuity at the first and subsequent postoperative exams. Preoperative proliferative vitreoretinopathy (PVR), vitreous hemorrhage (VH) and extent of retinal detachment were found to have a negative effect on functional success at 12 months. Regeneration of the outer retinal layers had a positive effect on visual acuity at 12 months but did not significantly increase visual acuity after 12 months. Multivariate analysis showed that preoperative external limiting membrane (ELM) integrity was significantly associated with better functional outcomes. Research Article Keywords: Retinal Detachment, Vitrectomy, Optical Coherence Tomography, Retinal Regenereation Posted Date: April 27th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1591133/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/10 Page 1/10 Introduction Rhegmatogenous retinal detachment (RRD) can cause vision impairment in certain cases even after anatomically successful surgery. The incidence of RRD ranges from 5.3 to 12.6 cases per 100,000[1]. In uncomplicated cases, primary anatomical success rates are generally high[2–4]. Patients who were treated for macula off RRD might have broad range of visual acuity. It is important to evaluate the preoperative and postoperative parameters that could affect the final functional outcome. Spectral domain optical coherence tomography (SD-OCT) allows visualizing retinal morphology in detail. In previous studies, the integrity of the ellipsoid zone (EZ) and the external limiting membrane (ELM) and the presence of foveal bulge were associated with the final best corrected visual acuity (BCVA) [5–10]. The other factors that were associated with postoperative visual outcome were preoperative visual acuity, age, proliferative vitreoretinopathy (PVR), and detachment duration [4, 11–13]. However, these prognostic factors demonstrated some differences between studies. The aim of this study is to evaluate the preoperative clinical and OCT findings that affect postoperative functional and visual improvement at different time points after RRD surgery in a large patient group. Materials And Method Surgical Procedure Twenty-three gauge pars plana vitrectomy with gas or silicone oil was performed in all patients. Pars plana vitrectomy with additional scleral buckle was indicated in patients with PVR. Patients with visually significant cataracts underwent combined cataract and detachment repair surgery. Materials And Method Page 2/10 Page 2/10 Charts of the patients who underwent surgery in Istanbul University Medical School Ophthalmology Department between January 2014 and May 2019 due to macula-off RRD were reviewed retrospectively. Study was approved by Medical, Surgical and Drug Research Ethics Committee of Istanbul University Istanbul Medical Faculty. Patients who had successful RRD repair, who showed up to regular follow ups and had SD-OCT (Heidelberg Engineering, Germany) images at every follow-up were included in the study. Exclusion criteria included patients with any other ophthalmologic pathology that affects vision, degenerative myopia, non rhegmatogenous etiology, recurrent retinal detachment, ambient opacities hindering imaging, macula-on RRD, history of ocular surgeries except cataract surgery. Statistical analysis Statistical analyses were performed using SPSS 23.0 (IBM corp. Armonk, NY, USA) statistics program. Kolmogorov-Smirnov test is used to determine distribution of variables in each group. Independent t-test and one-way analysis of variance (ANOVA) tests used to evaluate normally distributed data. For non-normally distributed data, Mann-Whitney U and chi-square tests were used. A linear regression analysis was used to estimate the relationship between the potential influencing preoperative factors and BCVA at 12 months. In addition, a linear mixed effects regression model was performed to estimate the relationship between the changes of postoperative factors and the changes of BCVA after surgery. For the results, in the 95% confidence interval, p < 0.05 was considered statistically significant. Protocol The demographic characteristics of the patients, BCVA, biomicroscopic examination findings, extent of detachment, detachment location, presence of accompanying PVR, vitreous hemorrhage (VH), and SD-OCT characteristics were recorded preoperatively. Surgical methods, duration of symptoms, central macular thickness, presence of subretinal fluid, ELM integrity, EZ integrity, cone interdigitation zone integrity (CIZ) and foveal bulge formation on SD-OCT at one, three, six, twelve months and at last examination were recorded. BCVA was evaluated with Snellen chart and was converted to Logarithm of Minimal Angle of Resolution (LogMAR) equivalent for statistical analysis. Macula-off duration was defined as the time between surgery and onset of subjective complaints (deterioration of visual acuity, visual field defects, photopsia, and floaters). Preoperative ELM integrity was defined as absent, in cases which the integrity could not be evaluated because of folding, undulation, edema, and/or the cases in which ELM continuity was disrupted[6]. To evaluate retinal morphology, horizontal cross-sectional images by SD-OCT at each visit were analyzed. Retinal layers were defined as intact, disrupted (attenuation, discontinuity or disruption) and absent (total absence of layer) at central fovea. The foveal bulge was defined as an outer segment (OS) length at the central fovea being 10 µm longer than the average OS length at 250 µm temporal and nasal from the central fovea. OCT images were evaluated by two retina specialists who were blinded to patient’s examinations. Findings that were scored differently by two clinicians were discussed, re-evaluated and the final decision was recorded. Results Seventy-five eyes of 75 RRD patients were included in the study. The mean follow-up period was 36.7 ± 16 months (Range: 14– 72 months). Demographics and clinical features were shown in Table 1. Age, gender, lens status, surgery type, endotamponade Page 3/10 Page 3/10 type and silicone oil retention time were not significantly associated with BCVA at 12 months. Table 1 Demographics and Clinical Characteristics Total n = 75 (%) Age 53.9 ± 17 years (Range: 11–85) Gender (M/F) (%) 41 (54.7)/34 (45.3) Duration of Symptoms 12.8 ± 13.5 days (Range: 1–90) Lens Status (phakic/pseudophakic) (%) 48 (64.0)/27 (36.0) Extent of retinal detachment 1 quadrant 2 quadrants 3 quadrants 4 quadrants 19 (25.3) 34 (45.3) 8 (10.7) 14 (18.7) Surgery type (%) PPV PPV + scleral buckle PPV + CEIOL 47 (67.2) 22 (29.3) 6 (8.0) Endotamponde type (%) Silicone oil SF6 C3F8 52 (69.3) 1 (1.3) 22 (29.3) Silicone oil retention time 8.3 ± 4.73 months CEIOL: cataract extraction and intraocular lens implantation; C3F8: perfluoropropane; F: female; M: male; PPV: pars plana vitrectomy; SF6: Sulfur hexafluoride Preoperative Factors Affecting Visual Acuity and silicone oil retention time were not significantly associated with BCVA at 12 months. type and silicone oil retention time were not significantly associated with BCVA at 12 months. Table 1 Demographics and Clinical Characteristics Total n = 75 (%) Age 53.9 ± 17 years (Range: 11–85) Gender (M/F) (%) 41 (54.7)/34 (45.3) Duration of Symptoms 12.8 ± 13.5 days (Range: 1–90) Lens Status (phakic/pseudophakic) (%) 48 (64.0)/27 (36.0) Extent of retinal detachment 1 quadrant 2 quadrants 3 quadrants 4 quadrants 19 (25.3) 34 (45.3) 8 (10.7) 14 (18.7) Surgery type (%) PPV PPV + scleral buckle PPV + CEIOL 47 (67.2) 22 (29.3) 6 (8.0) Endotamponde type (%) Silicone oil SF6 C3F8 52 (69.3) 1 (1.3) 22 (29.3) Silicone oil retention time 8.3 ± 4.73 months CEIOL: cataract extraction and intraocular lens implantation; C3F8: perfluoropropane; F: female; M: male; PPV: pars plana vitrectomy; SF6: Sulfur hexafluoride CEIOL: cataract extraction and intraocular lens implantation; C3F8: perfluoropropane; F: female; M: male; PPV: pars plana vitrectomy; SF6: Sulfur hexafluoride Preoperative Factors Affecting Visual Acuity The mean BCVA preoperatively was 1.35 ± 1.24 (Range: 0-3.7), at postoperative 12th month was 0.66 ± 0.5 (Range: 0-1.6), at last follow-up was 0.58 ± 0.5 (Range: 0-1.6) logMAR. There was a statistically significant increase in BCVA at postoperative 1st, 3rd, 6th, 12 months and last visit compared to preoperative BCVA (all p values < 0.001). Extent of detachment involved one quadrant in 19 (25.3%) patients, two quadrants in 34 (45.3%) patients, three quadrants in 8 (10.7%) patients, and four quadrants in 14 (18.7%) patients. Mean BCVA of these patients at postoperative 12th month visit was 0.41 ± 0.5, 0.67 ± 0.5, 0.55 ± 0.3, 0.96 ± 0.4 logMAR respectively (p = 0.03, post hoc analysis one quadrant detachment vs. four quadrant detachment p = 0.004). The mean postoperative BCVA was significantly higher in patients who were operated in the first week of their onset of symptoms (Table 2). Twelve patients (16.0%) had VH and 4 patients (5.3%) had PVR at preoperative visits. Patients who were found to have PVR in the initial examination had significantly lower preoperative and postoperative BCVA (Table 2). Preoperative OCT images showed that 34 patients (45.3%) had intact baseline ELM. Preoperative ELM integrity was Page 4/10 Page 4/10 significantly related to postoperative visual outcome at 12 months (p < 0.001). Preoperative factors affecting visual acuity at postoperative 12 months were shown in Table 2. Preoperative Factors Affecting Visual Acuity Table 2 Preoperative Factors Affecting Postoperative Visual Acuity Preoperative BCVA (SD) P value POM1 BCVA (SD) P value POM3 BCVA (SD) P value POM6 BCVA (SD) P value POM12 BCVA (SD) P value BCVA (n = 75) 1.35 (± 1.2) 0.79 (± 0.5) 0.73 (± 0.5) 0.71 (± 0.5) 0.66 (± 0.5) Duration of symptoms (Number of patients) < 7 days before surgery (n = 33) 1.22 (± 1.2) 0.231 0.68 (± 0.5) 0.03 0.58 (± 0.5) 0.03 0.55 (± 0.5) 0.01 0.53 (± 0.5) 0.03 ≥ 7 days before surgery (n = 42) 1.50 (± 1.3) 0.91 (± 0.4) 0.86 (± 0.5) 0.87 (± 0.5) 0.81 (± 0.5) Preoperative ELM integrity Intact (n = 34) 0.67 (± 0.9) < 0.01 0.55 (± 0.4) < 0.01 0.40 (± 0.4) < 0.01 0.40 (± 0.4) < 0.01 0.35 (± 0.4) < 0.01 Absent (n = 41) 1.91 (± 1.2) 0.99 (± 0.4) 0.97 (± 0.4) 0.93 (± 0.5) 0.87 (± 0.4) Preoperative VH Present (n = 12) 2.01 (± 0.8) 0.04 0.94 (± 0.4) 0.23 0.88 (± 0.5) 0.23 1.02 (± 0.5) 0.02 0.92 (± 0.5) 0.04 Absent (n = 63) 1.22 (± 1.3) 0.76 (± 0.5) 0.69 (± 0.5) 0.64 (± 0.5) 0.60 (± 0.5) Preoperative PVR Present (n = 4) 3.50 (± 0.2) < 0.01 1.45 (± 0.3) 0.01 1.60 (± 0) < 0.01 1.30 (± 0.3) 0.03 1.10 (± 0.2) 0.02 Absent (n = 71) 1.23 (± 1.2) 0.76 (± 0.4) 0.67 (± 0.5) 0.67 (± 0.5) 0.64 (± 0.5) BCVA: Best corrected visual acuity. SD: Standard deviation. ELM: External limiting membrane. VH: Vitreous hemorrhage, POM: postoperative month, PVR: Proliferative vitreoretinopathy. All the visual acuities are in LogMAR. Bold p values are statistically significant. BCVA: Best corrected visual acuity. SD: Standard deviation. ELM: External limiting membrane. VH: Vitreous hemorrhage, POM: postoperative month, PVR: Proliferative vitreoretinopathy. All the visual acuities are in LogMAR. Bold p values are statistically significant. In univariate model, preoperative ELM integrity (ß=0.512; p < 0.001), preoperative vision (ß=0.334; p = 0.007) and symptom duration (ß=0.312; p = 0.006) were significantly related to postoperative BCVA at 12 months. However, in multivariate model only preoperative ELM integrity (ß=0.419; p < 0.05) was related to post operative BCVA at 12 months. Postoperative Factors Affecting Visual Acuity In univariate model, postoperative 1st month ELM integrity (ß=0.455; p < 0.001), 1st month EZ formation (ß=0.446; p < 0.001), 1st month CIZ formation (ß=0.344; p = 0.005), 1st month foveal bulge formation (ß=0.312; p = 0.002) were significantly related to postoperative BCVA at 12 months. Page 5/10 Page 5/10 Postoperative Outer Retinal Changes Macular subretinal fluid (SRF) was observed in 13 patients (17.3%) at first postoperative visit and resolved in all patients at follow ups. Presence of SRF at first month did not significantly alter postoperative BCVA at 12 months (p = 0.74). Postoperative Outer Retinal Changes The hierarchical structure in ELM, EZ and CIZ restoration was observed in our study. The number of patients who had ELM restoration was the highest at first month postoperatively and was followed by patients who had EZ and CIZ restoration respectively. The numbers of patients with restoration of these outer retinal layers consecutively increased during the follow ups in the first year. ELM and EZ were absent in five (6.7%) patients at postoperative 12 months. These patients did not have an improvement in the outer retinal layers at last visit. CIZ restoration in one more patient and foveal bulge restoration in four more patients were seen at last visit after 12 months (Table 3). There was no significant BCVA increase at last visit compared to 12 months (p = 0.26). Table 3 Preoperative and Postoperative Optic Coherence Tomography (OCT) Features N: 75 Preoperative n (%) POM1 n (%) POM3 n (%) POM6 n (%) POM12 n (%) Last visit n (%) ELM Disrupted Intact - 34 (45.3) 16 (21.3) 37 (49.3) 14 (18.6) 48 (64.0) 11 (14.6) 54 (72.0) 10 (14.2) 60 (80.0) 9 (12.0) 61 (81.3) EZ Disrupted Intact - 20 (26.6) 23 (30.6) 14 (18.6) 38 (50.6) 17 (22.6) 42 (56.0) 13 (17.3) 57 (76.0) 8 (10.6) 57 (76.0) CIZ Disrupted Intact - 15 (20.0) 10 (13.3) 21 (28.0) 13 (17.3) 26 (34.6) 18 (24.0) 26 (34.6) 25 (33.3) 28 (37.3) 26 (34.6) Foveal bulge - 19 (25.3) 28 (37.3) 30 (40.0) 36 (48.0) 40 (53.3) CIZ: cone interdigitation zone; ELM: external limiting membrane; EZ: ellipsoid zone; POM: postoperative month, (1 0%) i d h f 3 d ll i l l d i 12 h i h Table 3 Twelve (16.0%) patients were under the age of 35, and all retinal layers were restored at postoperative 12 months in these patients. Epiretinal membrane was observed in 4 (5.3%) patients at postoperative last visit. Macular subretinal fluid (SRF) was observed in 13 patients (17.3%) at first postoperative visit and resolved in all patients at follow ups. Presence of SRF at first month did not significantly alter postoperative BCVA at 12 months (p = 0.74). Twelve (16.0%) patients were under the age of 35, and all retinal layers were restored at postoperative 12 months in these patients. Epiretinal membrane was observed in 4 (5.3%) patients at postoperative last visit. DISCUSSION It has been claimed that vitreous levels of transforming growth factor beta-3 (TGFB-3) and interleukin-8 (IL-8) were higher in patients with more extensive retinal detachment, suggesting inflammatory and apoptotic processes[15– 18]. Presence of preoperative VH and/or PVR was a poor prognostic factor for BCVA at 12 months in our study. VH and/or PVR negatively affect regeneration of the outer retinal layers in RRD[3, 6, 13, 19–21]. It has been claimed that this may occur due to some mediators secreted and subretinal gliosis during PVR, affecting photoreceptor healing[22]. In this study, multivariate analysis showed that preoperative ELM integrity was highly related to outcome vision. ELM together with Muller cells forms the connection points in photoreceptors, and maintaining these connections is thought to accelerate photoreceptor healing. ELM damage indicates that retinal damage not only affects photoreceptors, but also spreads to the Muller cell layer. Again, duration of macular detachment and extent of detachment affects ELM integrity and restoration[6, 7, 9, 23]. Dell’Omo et al. showed that preoperative ELM integrity is strongly associated with postoperative BCVA similar to our results and they observed ELM restoration in first 6 months after surgery[9]. In our study although most of patients had ELM restoration at their 6 postoperative months, we have also seen restoration after this period. Even though anatomical restoration of outer retinal layers was observed even after 12 months, BCVA did not significantly improve after 12 months. Molecular studies are needed in the future to evaluate outer retinal restoration and it is functional effect. In various studies, postoperative restoration of CIZ, EZ, ELM integrity and the restoration of foveal bulge positively affected the visual outcome[6–8, 10]. Our results were consistent with the literature. ELM, EZ and CIZ integrity may not be seen in OCT in the first postoperative month, but it was found that outer retinal layer could be formed in subsequent examinations. This shows that the regeneration and organization of the retinal layers may occur in the following months. Restoration of retinal layers can be visualized with OCT and this can be indicator of the functional prognosis. The hierarchical structure in ELM, EZ and CIZ restoration was observed in our study. The number of patients who had ELM restoration was the highest and was followed by patients who had EZ and CIZ restoration respectively. The numbers of patients with restoration of these outer retinal layers consecutively increased during the follow ups. DISCUSSION Anatomical success rates after surgery for RRD are as high as 90%, but unfortunately functional success might be lower in some patients[3, 4, 6, 9]. In this study, we investigated long term results of RRD surgery, preoperative and postoperative factors affecting visual outcome including SD-OCT features. SD-OCT provides in vivo visualization of retinal morphology in the macula with high resolution. It is helpful to monitor outer retinal changes in follow up of postoperative patients with RRD as it might affect prognosis. In our study, the mean follow-up period of the patients was 36.7 ± 16 months. Similar studies have shorter follow up periods after surgery[3, 10]. This enabled us to evaluate both functional and OCT results for a longer period of time. Page 6/10 In various studies, it was shown that early surgery (within the first 7 days) for retinal detachment has a positive effect on the visual acuity outcome. Kim et al showed that patients who were operated in 6 days of visual symptom onset had significantly higher postoperative mean BCVA[14]. Williamson et al. also reported that mean postoperative BCVA was higher in patients who were operated in 3 days of symptom onset[3]. Patients who were operated in their first 7 days of complaints had significantly Page 6/10 Page 6/10 higher 1st, 3rd, and 6th, 12th and last visit BCVA Similarly our study demonstrated that early surgical intervention (< 7 days) had beneficial effect on postoperative visual acuity. This could be explained by photoreceptor damage in retinal detachment after seven days. It was shown in histopathological studies that prolonged detachment causes atrophy and apoptosis in photoreceptors [7, 9, 14, 15]. higher 1st, 3rd, and 6th, 12th and last visit BCVA Similarly our study demonstrated that early surgical intervention (< 7 days) had beneficial effect on postoperative visual acuity. This could be explained by photoreceptor damage in retinal detachment after seven days. It was shown in histopathological studies that prolonged detachment causes atrophy and apoptosis in photoreceptors [7, 9, 14, 15]. It was previously shown that extent of retinal detachment was associated with postoperative visual acuity. Williamson et al. and Park et al. found a statistically significant difference between the extent of detachment and the final BCVA[3, 6]. In our study, patients with 4 quadrants of detachment had significantly lower visual acuity at 12 months compared to patients with 1 quadrant detachment. DISCUSSION The EZ shows the inner segments, while the CIZ shows the outer segments. In photoreceptor regeneration, first development of the inner segments and then the outer segments was observed[7, 8, 24]. On the other hand, although the integrity of the outer layers is complete, functional success cannot be achieved in some patients. This may be due to loss of function of photoreceptors. Other cells might replace photoreceptors in the process of healing of outer segments[9]. The integrity of the retinal outer layers in the early postoperative period may be a good indicator of foveal bulge restoration and visual outcome[7, 9]. Foveal bulge is thought to occur due to the inner segments of the cones being wider than the outer segments. The restoration of foveal bulge can be associated with the presence of healthy cone cells. Again, the absence of foveal bulge in the postoperative period can be associated with photoreceptor damage[25–30]. In our study foveal bulge restoration improved visual outcome. In the study of Hasegawa et al., at 7 months follow-up, the restoration of foveal bulge was 28.6%[10].In our study, it was found to be 40% at 6 month follow-up and 48% 12 month visit. All retinal layers had regenerated at the end of the first year in patients less than 35 years of age. The regeneration capacity of retinal layers are considered higher at a young age[31]. Page 7/10 Page 7/10 The important advantages of our study are the relatively high number of patients and longer postoperative follow-up time compared to similar studies. Preoperative demographic, clinical, imaging findings and postoperative OCT findings of relatively high number of patients were evaluated. Limitations of our study included retrospective design and the fact that symptom duration relied on patient recalling. The OCT findings of the patients were examined in a holistic way, the thickness of isolated retinal layers and the change of these thicknesses during follow-up could not be evaluated. It is known that in a small percentage of cases who underwent retinal tamponade with silicone oil, ganglion cells can suffer a direct damage either from particles of oil that migrate in the retina or from direct contact with silicone[32]. But this toxicity was never clearly associated with visual loss and none of our patients had obvious silicone oil maculopathy or optic neuropathy on exam. In conclusion, preoperative and postoperative factors can affect functional success in RRD surgery. DISCUSSION Retinal outer layer regeneration can continue up to one year postoperatively, sometimes even longer. This regeneration especially first year postoperatively can be related to better functional outcomes. Preoperative ELM integrity and postoperative ELM restoration are main predictors of functional outcome. 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Lai WW, Leung GY, Chan CW, et al (2010) Simultaneous spectral domain OCT and fundus autofluorescence imaging of the macula and microperimetric correspondence after successful repair of rhegmatogenous retinal detachment. Br J Ophthalmol 94:311–318. https://doi.org/10.1136/bjo.2009.163584 References Gharbiya M, Grandinetti F, Scavella V, et al (2012) Correlation between spectral-domain optical coherence tomography findings and visual outcome after primary rhegmatogenous retinal detachment repair. Retina 32:43–53. https://doi.org/10.1097/IAE.0b013e3182180114 26. Shimoda Y, Sano M, Hashimoto H, et al (2010) Restoration of photoreceptor outer segment after vitrectomy for retinal detachment. Am J Ophthalmol 149:284–290. https://doi.org/10.1016/j.ajo.2009.08.025 26. Shimoda Y, Sano M, Hashimoto H, et al (2010) Restoration of photoreceptor outer segment after vitrectomy for retinal detachment. Am J Ophthalmol 149:284–290. https://doi.org/10.1016/j.ajo.2009.08.025 27. 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https://openalex.org/W2969796069	http://pdf.blucher.com.br/chemicalengineeringproceedings/cobecic2019/PBIO27.pdf	Portuguese	null	ESTUDO DAS MOLÉCULAS MAJORITÁRIAS DO EXTRATO DA CASCA DE CINNAMOMUM VERUM POR MODELAGEM MOLECULAR	Blucher Chemical Engineering Proceedings	2,019	cc-by	2,212	A. L. S. CARVALHO1, E. MOCHIUTTI1, A. E. S. NASCIMENTO1, D. S. B. BRASIL1, M. C. MARTELLI1 1 Universidade Federal do Pará, Faculdade de Engenharia Química E-mail para contato: mrcarvalhoarthur@gmail.com RESUMO – Produtos naturais podem possuir inúmeras propriedades biológicas benéficas para o organismo humano em diversos aspectos. O trabalho teve como objetivo utilizar de técnicas de modelagem molecular para indicar essas proprie- dades nas estruturas majoritárias do extrato de casca de Cinnamomum Verum e es- tabelecer se as mesmas podem ter alguma aplicação na indústria farmacêutica. Es- se estudo teve como base o desenho das estruturas anteriormente isoladas e a apli- cação de banco de dados PreAdmet para obter propriedades farmacocinéticas e toxicológicas desses compostos, além de uma análise físico-química destes com- postos com o software Spartan. Como resultados, todas as moléculas demonstra- ram possibilidade de serem drogas de consumo oral por serem aprovadas da regra de Lipinski, além de possuírem boa capacidade de absorção no intestino. Porém, as mesmas apresentaram dificuldade de fixação nas células do organismo e duas estruturas (4-Vinylbenzoic acid e trans-Cinnamaldehyde) expressaram tendências cancerígenas em ratos e camundongos. A. L. S. CARVALHO1, E. MOCHIUTTI1, A. E. S. NASCIMENTO1, D. S. B. BRASIL1, M. C. MARTELLI1 A. L. S. CARVALHO1, E. MOCHIUTTI1, A. E. S. NASCIMENTO1, D. S. B. BRASI C. MARTELLI1 1. INTRODUÇÃO A espécie Cinnamomum Verum (canela) pertence à família Lauraceae, é bastante utili- zada em medicinas tradicionais, a casca é usada como condimento e agente aromatizante, sendo picante, amarga, aromática, adstringente e desodorizante. Além dos fatores sensoriais, essa espécie traz propriedades medicinais contra bronquite, asma, cefaléia (dor de cabeça), odontalgia (dor nos dentes), doenças cardíacas, entre outras. Por fim, ela também possui utili- dade para ações antioxidantes, antifúngicas e anti-inflamatórias. As moléculas mais presentes no extrato da casca da canela são: (E)-3-(2-Methoxyphenyl)-2- propenoic acid (40,41%), trans-Cinnamaldehyde (20,28%), 4-Vinyl benzoic acid (10,54%), coumarin (8,47%). (MARIAPPAN et al., 2013). Os produtos naturais vêm sendo usados na medicina tradicional durante toda a história da humanidade. Incluindo seus derivados, produtos naturais são cerca de um terço das NME (New Molecular Entities) aprovadas pela FDA (U.S. Food and Drug Administration). Alguns desafios são vinculados à produção de novas drogas utilizando esse tipo de matéria prima, como problemas com absorção, distribuição, metabolismo e excreção (ADME) no organismo para com essas estruturas (PRESS et al., 2019) Neste trabalho, foram utilizadas técnicas de modelagem molecular para fazer uma pre- dição de propriedades farmacocinéticas, quimiométricas e toxicológicas das moléculas majo- ritárias do extrato de casca de Cinnamomum Verum (canela) e indicar possíveis utilidades na indústria farmacêutica e problemas vinculados a essas estruturas. Neste trabalho, foram utilizadas técnicas de modelagem molecular para fazer uma pre- dição de propriedades farmacocinéticas, quimiométricas e toxicológicas das moléculas majo- ritárias do extrato de casca de Cinnamomum Verum (canela) e indicar possíveis utilidades na indústria farmacêutica e problemas vinculados a essas estruturas. Neste trabalho, foram utilizadas técnicas de modelagem molecular para fazer uma pre- dição de propriedades farmacocinéticas, quimiométricas e toxicológicas das moléculas majo- ritárias do extrato de casca de Cinnamomum Verum (canela) e indicar possíveis utilidades na indústria farmacêutica e problemas vinculados a essas estruturas. 2. MATERIAIS E MÉTODOS As estruturas analisadas nesse trabalho foram selecionadas a partir do estudo do extra- to da casca de Cinnamomum Verum feito por Mariappan (2013) e dispostas na Tabela 1, mos- trada abaixo. Após a seleção, essas moléculas foram desenhadas bidimensionalmente com o auxílio do software ChemSketch. Em seguida, a geometria foi otimizada para um modelo tri- dimensional pelo software Avogadro utilizando o campo de força MMF94s, com o algoritmo steepest descent com 10000 passos. Tabela 1 - Moléculas majoritárias da Cinnamomum Verum 3-2-Methoxyphenyl-2propenoic acid 4-Vinylbenzoic acid Coumarin Trans-Cinnamaldehyde Trans-Cinnamaldehyde Coumarin Coumarin A minimização de energia destes compostos foi calculada pelo software Gaussian a partir da metodologia DFT (Density Functional Theory), utilizando a função B3LYP com base 6-31G* para uma aproximação de energia. Para confirmar a validade das estruturas obti- das e certificar que não exista frequência imaginária foram realizados os cálculos de frequên- cias, pois, se ocorrer frequência imaginária, a estrutura não está no seu mínimo verdadeiro. (DAPPRICH et al., 1999). As moléculas foram submetidas ao cálculo de propriedades físico-químicas no softwa- re Spartan. Assim destacando os parâmetros de maior interesse: Energia HOMO (energia do maior orbital preenchido), Energia LUMO (energia do menor orbital não preenchido), GAP (diferença entre HOMO e LUMO), MWT (massa molar), PSA (área de superfície polar), LogP (lipossolubilidade), HBD e HBA (indicadores de ligações de hidrogênio). (diferença entre HOMO e LUMO), MWT (massa molar), PSA (área de superfície polar), LogP (lipossolubilidade), HBD e HBA (indicadores de ligações de hidrogênio). Em 90% dos fármacos oralmente ativos, os compostos seguem um padrão chamado “regra dos 5” (Rule Of Five – RO5), em que 4 parâmetros estão em uma faixa de aceitação relacionados à solubilidade em água e permeabilidade intestinal. Os valores destas proprieda- des são restritos à MWT ≤ 500 Dalton (g/mol), logP ≤ 5, HBD (Hydrogen Bond Donors) ≤ 5, HBA (Hydrogen Bond Acceptors) ≤ 10. Passar na RO5 não significa que o composto terá necessariamente utilidade como droga, afinal, a regra não especifica fatores estruturais destas substâncias. (LIPINSKI, 2004). As moléculas foram ainda comparadas às do banco de dados PreAdmet para a obten- ção dos resultados de ADME (Absorção, Distribuição, Metabolismo e Excreção): BBB, Ca- co2, HIA e PPB. E de Toxicidade: Mutagenicidade, Carcinogenicidade (ratos) e Carcinogeni- cidade (camundongos). 3. RESULTADOS E DISCUSSÃO A tabela 2 apresenta resultados de algumas características físico-químicas relevantes para esse estudo. Tabela 2 – Resultados de Propriedades Físico-Químicas obtidas pelo Spartan. Molécula HOMO (eV) LUMO (eV) GAP (eV) MWT (Da) PSA (Å2) HBD HBA LogP 3-2-Methoxyphenyl-2 propenoic acid -6,21 -1,84 4,37 178,187 38,972 1 2 0,33 4-Vinylbenzoic acid -6,59 -1,81 4,78 148,161 32,805 1 1 1,12 Coumarin -6,5 -1,88 4,62 146,145 21,371 0 1 0,51 Trans-Cinnamaldehyde -6,58 -2,09 4,49 132,162 14,942 0 1 1,04 Maiores valores de Energia HOMO e Energia LUMO acusam uma maior tendência a doar elétrons e a receber elétrons respectivamente. Com relação ao GAP, quanto menor o nú- mero, mais a molécula se torna polarizável e reativa. (SIVAJEYANTHI et al., 2017). Desta forma, o trans-Cinnamaldehyde e o 4-Vinylbenzoic acid têm mais possibilidades de serem doadores de elétrons. Assim como o mesmo trans-Cinnamaldehyde se mostra como um bom receptor de elétrons. O GAP HOMO-LUMO tem como maior resultado o 4,78 do 4- Vinylbenzoic acid, indicando este como o composto menos reativo. Diante disso, possivel- mente, a molécula mais estável é 4-Vinylbenzoic acid e a mais instável é 3-2-Methoxyphenyl- 2 propenoic acid. A área de superfície polar (PSA) está relacionada com a capacidade de absorção que o intestino terá quando em contato com estas estruturas: para PSA < 63 Å2 a estrutura será completamente absorvida (HIA > 90%), enquanto compostos com PSA > 139 Å2, praticamen- te não serão absorvidos (HIA < 10%). (PALM et al., 1997). Assim todas as estruturas possu- em boa absorção quando sendo utilizadas como drogas de consumo oral. Os resultados das 4 propriedades abordadas pela RO5 (MWT, HBD, HBA e LogP) mostraram que todos os compostos seguem as normas propostas por Lipinski. Tendo MWT um valor menor que 500 Da, HBD e LogP menores que 5 e HBA sendo menor que 10. O que significa que os compostos possuem propriedades dentro dos parâmetros estabelecidos para serem usados como drogas orais. Os resultados das 4 propriedades abordadas pela RO5 (MWT, HBD, HBA e LogP) mostraram que todos os compostos seguem as normas propostas por Lipinski. Tendo MWT um valor menor que 500 Da, HBD e LogP menores que 5 e HBA sendo menor que 10. O que significa que os compostos possuem propriedades dentro dos parâmetros estabelecidos para serem usados como drogas orais. A tabela 3 mostra as propriedades de ADME dos compostos. A tabela 3 mostra as propriedades de ADME dos compostos. Tabela 3 – Resultados de ADME obtidos pelo banco de dados PreADME/T. Molécula BBB Caco2 (nm/seg) HIA (%) PPB (%) 3-2-Methoxyphenyl-2 propenoic acid 1,74859 21,1225 96,72931 82,80881 4-Vinylbenzoic acid 1,89161 21,0421 97,8452 68,46717 Coumarin 1,56844 32,1202 100 43,39511 trans-Cinnamaldehyde 1,26197 23,784 100 52,89236 Para os resultados de BBB (índice de penetração na barreira hematoencefálica), se- gundo a escala proposta por Ma (2005), as 4 moléculas possuem esse índice de forma mode- rada, já que se encontram entre 0,1 e 2. Com base nesses resultados, o estudo não especifica se as estruturas estudadas podem ser danosas ao sistema nervoso, ou seja, faz-se necessária uma análise mais aprofundada dos efeitos dessas moléculas para julgar se esse índice é o ideal para tais substâncias. Com relação a Caco2, o PreAdmet utiliza uma análise feita por Yamashita (2000), em que menos de 4 nm/seg indica baixa permeabilidade em células intestinais, entre 4 e 70 nm/seg, uma permeabilidade mediana e mais de 70 nm/seg uma alta permeabilidade. Dessa forma, todos os compostos estão na faixa de permeabilidade moderada, indicando que não demorariam a ser absorvidos pelo intestino delgado. Quanto aos valores de HIA, todas as substâncias obtiveram resultados acima de 95%, sendo que, as moléculas Coumarin e trans-Cinnanmaldehyde apresentaram 100% de absor- ção. Esse parâmetro está relacionado à capacidade total de absorção pelo intestino. Os dados de PPB (Plasma Protein Binding) indicam a capacidade de se fixar em pro- teínas das células do organismo. A escala utilizada indica que qualquer resultado menor que 90% (como o das moléculas estudadas) traduz uma dificuldade de fixação, e, consequente- mente, uma menor capacidade de distribuição pelo corpo. Os parâmetros da tabela 4 avaliam a toxicidade das substâncias em questão no corpo humano Os parâmetros da tabela 4 avaliam a toxicidade das substâncias em questão no corpo humano. Os parâmetros da tabela 4 avaliam a toxicidade das substâncias em questão no corpo humano Tabela 4 - Resultados de Toxicidade obtidos pelo banco de dados PreADME/T. Molécula Mutagenicidade Carcinogenicidade (Ratos) Carcinogenicidade (Camundongos) 3-2-Methoxyphenyl-2 propenoic acid Mutagênico Não Não 4-Vinylbenzoic acid Mutagênico Sim Sim Coumarin Mutagênico Não Não Trans-Cinnamaldehyde Mutagênico Sim Sim A princípio, nota-se que todas apresentaram características mutagênicas. O que permi- te várias interpretações sobre o efeito (negativo ou positivo) delas em nível celular. A tabela 3 mostra as propriedades de ADME dos compostos. A partir do resultado positivo de mutagenicidade não se sabe se a molécula é de fato prejudicial para humanos, haja vista que não há um padrão de predição para relacionar esse fator com a carci- nogenicidade. Sendo assim, o banco de dados permite uma predição de carcinogenicidade para ratos e camundongos. Dessa forma, se o composto apresenta uma tendência cancerígena para estas espécies, pode indicar uma maior probabilidade dessa mesma tendência ser aplica- da para seres humanos. Os resultados mostraram que duas moléculas foram consideradas can- cerígenas para ambas as espécies (4-Vinylbenzoic acid e trans-Cinnamaldehyde) e duas obti- veram os resultados opostos (3-2-Methoxyphenyl-2 propenoic acid e Coumarin). 4. CONCLUSÃO Como conclusão para o estudo realizado, as estruturas em questão demonstraram uma possibilidade em serem drogas oralmente ativas devido aos seus resultados referentes à regra de Lipinski (características físico-químicas favoráveis). Em que as mesmas não violaram a regra. Além disso, ao analisar as propriedades farmacocinéticas, foi observado que as 4 substâncias obtiveram resultados em faixas semelhantes em todos os parâmetros. Porém, com relação às propriedades toxicológicas, duas moléculas se mostraram não cancerígenas para ratos e camundongos (apesar de todas apresentarem tendência mutagênica). Assim, conclui-se que essas duas estruturas (3-2-Methoxyphenyl-2 propenoic acid e Coumarin) são as mais promissoras em um âmbito farmacológico, sendo assim necessários estudos mais profundos para saber todos os seus efeitos no organismo. LIPINSKI, C. A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discovery Today: Technologies, v. 1, n. 4, p. 337–341, 2004. 5. REFERÊNCIAS DAPPRICH, S.; KOMÁROMI, I.; BYUN, K. S.; MOROKUMA, K.; FRISCH, M. J. A new ONIOM implementation in Gaussian98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. Journal of Molecular Structure: THEOCHEM, p. 461-462, 1999. LIPINSKI, C. A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discovery Today: Technologies, v. 1, n. 4, p. 337–341, 2004. 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Natural product drug delivery: A special challenge? Progress in Medicinal Chemistry, p. 2, 2019. SIVAJEYANTHI, P.; JEEVARAJ, M.; BALASUBRAMANI, K.; VISWANATHAN, V.; VELMURUGAN, D. Synthesis, crystal structure, Hirshfeld surface analysis and frontier molecular orbital analysis of 2-((2-hydroxynaphthalen-1- yl)methylene)hydrazinecarbothioamide. Chemical Data Collections, v. 11–12, p. 220– 231, 2017. YAMASHITA, S.; FURUBAYASHI, T.; KATAOKA, M.; SAKANE, T.; SEZAKI, H.; TOKUDA, H. Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells. Eur. J. Pharm. Sci, v. 10, p. 195-204, 2000.
https://openalex.org/W4281756976	https://www.frontiersin.org/articles/10.3389/feduc.2022.846998/pdf	English	null	T-MOOC for Initial Teacher Training in Digital Competences: Technology and Educational Innovation	Frontiers in education	2,022	cc-by	10,731	T-MOOC for Initial Teacher Training in Digital Competences: Technology and Educational Innovation Sandra Martínez-Pérez, Julio Cabero-Almenara, Julio Barroso-Osuna and Antonio Palacios-Rodríguez Department of Didactics and Educational Organization, Faculty of Education Sciences, University of Seville, Seville, Spain Massive open online courses (MOOCs) are perceived as emerging technologies for training and innovation in the educational context. They have become approaches for distance education in the face of the new challenges, changes, and crises experienced by the COVID-19 pandemic. They represent, in turn, new emerging opportunities as a response to the United Nations recommendations for open education and the development of sustainable goals. The presence of technologies in the development of educational tasks means that the acquisition of Digital Competences (DC) by teachers and students in training goes beyond the mere mastery of content and teaching methodologies. The research presented aims to analyze the educational possibilities of T-MOOCs for the development of DC in teachers, and as resources that favor autonomous and collaborative learning in innovative scenarios. The study sample is made up of 313 students of the Primary Education Degree at the University of Seville (Spain). For this purpose, two online questionnaires (Google Forms) were applied at the beginning of the course: the Digital Teaching Competence Questionnaire (DigCompEdu), and the Content Questionnaire: Digital Resources and Digital Pedagogy. The results obtained show that the students’ level of both digital competences and subject content is low to medium, so that training in educational technology is required for the acquisition of key digital competences. Based on the data obtained, the following actions are proposed: (a) The concretion of the contents structured by means of a learning guide and e-activities to be developed by the student body, taking into account the United Nations guidelines with regard to the Development of Sustainable Objectives; (b) The creation of a training and innovative environment under the T-MOOC architecture, based on open and distance learning due to the current health situation of COVID- 19, which, on the one hand, empowers students to use digital tools, and on the other hand, facilitates the acquisition of the SDGs; and (c) The evaluation of the T-MOOC designed as a resource for autonomous, collaborative, guided learning in emerging contexts in which technologies and educational innovation play an important role for sustainable development. Keywords: digital competences, T-MOOC, initial training, educational innovation, e-activities, undergraduate ORIGINAL RESEARCH published: 09 June 2022 doi: 10.3389/feduc.2022.846998 Edited by: Nam Ju Kim, University of Miami, United States Reviewed by: Erla Mariela Morales Morgado, Reviewed by: Erla Mariela Morales Morgado, University of Salamanca, Spain Miriam Agreda Montoro, University of Jaén, Spain Correspondence: Sandra Martínez-Pérez smartinezperez@us.es Specialty section: This article was submitted to Digital Learning Innovations, a section of the journal Frontiers in Education Received: 31 December 2021 Accepted: 19 April 2022 Published: 09 June 2022 INTRODUCTION In this line, Pilli and Admiraal (2016) performed a taxonomy of diﬀerent MOOCs according to two dimensions: massiveness (number of participants) and openness (degree of accessibility and ﬂexibility); classifying them into four classes according to these dimensions: (i) Small scale and less open, (ii) Small scale and more open, (iii) Large scale and less open, (iv) Large scale and more open. In this sense, MOOCs (Massive Open Online Courses) emerge from the open educational resources movement (Pilli and Admiraal, 2016), for lifelong learning, and can be seen as a disruptive innovation (Al-Imarah and Shields, 2019) and a technology that have been gaining ground, increasing their practices and transforming teaching and learning processes (Gordon and Wiltrout, 2021). They also emerge as a new pedagogical approach to address diversity and interculturality with the purpose of promoting an inclusion of opportunities for more active participation, and meeting learning needs in an open and distributed way (Boaler et al., 2018; Beltrán and Ramírez-Montoya, 2019; Khalid et al., 2020; Cabero- Almenara et al., 2021b). Moreover, they have the potential to contribute to innovation under pedagogical strategies, enabling co-creation, knowledge acquisition, and fostering professional and competence development (Gudmundsdottir and Hatlevic, 2018; Ruiz-Palmero et al., 2021). As proposed by Kady and Vadeboncoeur (2013), Watson et al. (2017), García-Peñalvo et al. (2018); Zawacki-Richer et al. (2018), Cornelius et al. (2019), and Deng et al. (2020), these can: (a) Generate global learning opportunities, where student participation and engagement are key, (b) Provide access to open and shared content, leading to emergent knowledge; (c) Have a signiﬁcant impact on Higher Education; and (d) Foster educational quality and instructional design. MOOCs therefore represent an impetus to enhance and promote the 2030 Agenda and the SDGs (Hueso, 2022). Among the MOOC typologies, the tMOOC is selected for this study. These are based on the transfer of learning, pedagogical transformation, and the development of diﬀerent tasks that students must perform to continue advancing in the course and to be able to demonstrate that they have mastered the competencies that are deployed in the tMOOC (Osuna-Acedo et al., 2018; Cabero-Almenara et al., 2021a). Along the same lines, Pilli and Admiraal (2016) aﬃrm that these types of MOOCs are supported by instructivism and constructivism, whose student body presents an active participation in the educational process. For their part, Albelbisi et al. (2018) point out that a key element of success of MOOCs is evaluation. INTRODUCTION (Escudero-Nahón and Núñez-Urbina, 2020). In this sense, the study by Albelbisi et al. (2018) highlights the relevance of taking into account 12 main factors for a successful implementation of MOOCs “earner characteristic with sub -factors (learner demographics, learner motivation, and interactivity), instructor, pedagogy, pattern of engagement, instructional design, assessment, credit, plagiarism, sustainability, learning analytics, student dropout rate, and MOOC quality” (p. 3006). The economic, political, technological, social, and educational changes raise the need to look for other ways of: relating, communicating and organizing, disseminating information, generating resources, creating alternative and innovative pedagogical models, with methodologies that deploy other methods that favor the teaching-learning processes. Creativity, knowledge, and technology are key to achieving the SDGs in all contexts. The incorporation of technologies in institutions in general, and in the educational system in particular, involves responding to current demands, requirements and trends. In these transformation processes, higher education institutions play an important role in the promotion of knowledge, the acquisition of competencies, the development of innovation and digital metamorphosis, which invite to adapt to new times, crossing time, and space boundaries (Tang, 2017; Gudmundsdottir and Hatlevic, 2018; Ithurburu, 2019; Martínez-Pérez and Rodríguez-Abitia, 2021). p q y (p ) Taking all these elements into account, it should be noted that MOOCs have resulted in the emergence of several variants: xMOOC (visualized as traditional courses, focused on the acquisition of content by students), cMOOC (referring to connectivism, to the connections that students are able to establish in training environments), hMOOC (hybrid models between xMOOCs and cMOOCs), bMOOCs (combining the advantages of online learning and face-to-face interaction), sMOOC (the “s” of social and seamless, enhancing interactions in learning and without breaks, are constantly accessible), tMOOC (transfer massive open online courses, the participants, through collaborative work, acquire competences to put into practice tools, learning methods, co-evaluations in relation to the theme chosen for their course), and SPOCs (small private on-line courses, maintaining the structure and methodology of MOOCs but with restrictions on the number of students and their access) (Aguayo and Bravo, 2017; García-Peñalvo et al., 2018; Osuna- Acedo et al., 2018; Zhao and Song, 2020; Cabero-Almenara et al., 2021b). Citation: Citation: Martínez-Pérez S, Cabero-Almenara J, Barroso-Osuna J and Palacios-Rodríguez A (2022) T-MOOC for Initial Teacher Training in Digital Competences: Technology and Educational Innovation. Front. Educ. 7:846998. doi: 10.3389/feduc.2022.846998 June 2022 \| Volume 7 \| Article 846998 1 Frontiers in Education \| www.frontiersin.org T-MOOC Initial Training Digital Competences Martínez-Pérez et al. Frontiers in Education \| www.frontiersin.org INTRODUCTION This assessment becomes a critical variable in this MOOC format so that the subject progresses in the training action (Cabero-Almenara et al., 2021b). Doherty et al. (2015), Drake et al. (2015), and Raposo-Rivas et al. (2017) pointed out that, for the development of MOOCs and to avoid possible dropout and abandonment, the pedagogical design (autonomy, diversity, openness, and interactivity), which in turn has to be attractive, and the principles by which they are governed (meaningful, engaging, measurable, accessible, and scalable) are key elements that pivot on the students and their learning process; especially when outlining materials, providing resources and planning activities, seeking a shared construction based on autonomous, self-regulated, rhizomatic, situated and collaborative mediated learning, and horizontal communication between peers and teachers Thus, taking into account the diﬀerent authors, MOOCs are an excellent strategy for the development of e-activities and the training of future teachers in digital competences under the Digital Teaching Competences Framework “DigCompEdu.” Thus, taking into account the diﬀerent authors, MOOCs are an excellent strategy for the development of e-activities and the training of future teachers in digital competences under the Digital Teaching Competences Framework “DigCompEdu.” UNESCO (2018) deﬁnes a key competence as the “combination of knowledge, skills, and attitudes adapted to the context” (p. 7). Being competent is related to everything that society requires overcoming the obstacles of the time in which it develops; one of the fundamental competencies of today’s society UNESCO (2018) deﬁnes a key competence as the “combination of knowledge, skills, and attitudes adapted to the context” (p. 7). Being competent is related to everything that society requires overcoming the obstacles of the time in which it develops; one of the fundamental competencies of today’s society June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 2 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. is digital competence. In this sense, teacher training is considered of great importance. For the European Union (2019), a Digital competence “involves the conﬁdent, critical and responsible use of, and engagement with, digital technologies for learning, at work, and for participation in society. It includes information and data literacy, communication and collaboration, media literacy, digital content creation (including programming), safety (including digital wellbeing and competences related to cybersecurity), intellectual property related questions, problem solving, and critical thinking” (p. 10). learners, and (vi) Facilitating learners’ digital competence. INTRODUCTION Each area has a series of competencies that “teachers must have in order to promote eﬀective, inclusive and innovative learning strategies, using digital tools” (Redecker and Punie, 2017, p. 4). In addition, the DigCompEdu framework proposes six progressive levels of competence: A1 (newcomer), A2 (explorers), B1 (integrators), B2 (experts), C1 (leaders), and C2 (pioneer). Focusing on the development of tasks by students to continue advancing in the course and to be able to demonstrate that they have mastered the competencies that are deployed in the tMOOC, Cabero-Almenara and Palacios-Rodríguez (2021), taking into account the above, emphasize the need to perform e-activities, deﬁning them as all the tasks developed by the student individually or collectively in a digital environment, whose purpose is the acquisition of speciﬁc learning. The diﬀerence between virtual and face-to-face activities lies in the possibilities oﬀered by virtual environments, since these can be more motivating and less frustrating, to promote an interactive context between information and participants (students and teachers) (Gómez-Rey et al., 2018); and, in turn, promote reﬂective and collaborative learning, and acquire the competence of learning to learn (Luo et al., 2017). To assess the importance of a Digital Competences Framework for teachers, in the studies conducted by Cabero- Almenara and Palacios-Rodríguez (2020) and Cabero-Almenara et al. (2020) to 179 national and international experts on digital competences, it was concluded that the DigCompEdu Framework was the most highly valued, making it the most suitable for use in the university context, hence its importance and having taken it as the object of study in this research. The study values very positively its pedagogical component, the main advantage over other frameworks. In contrast, the other frameworks analyzed pay special attention to the technological dimension of digital competence, leaving aside the pedagogical competence. Furthermore, Gros (2018) points out the importance of the pedagogical design of the tasks, and states that the success of e-activities will depend on “the student’s ability to direct and manage their own learning process, establishing objectives, and appropriate strategies to achieve their goals” (p. 74). In this sense, Maina (2020) lists diﬀerent types of e-activities to be deployed in MOOCs: (i) Analysis and synthesis, (ii) Research and/or problem solving, (iii) Interaction and communication, The DigCompEdu framework (Redecker and Punie, 2017; Ghomi and Redecker, 2019) focuses, as shown in Figure 1, on three broad dimensions of competencies: educators’ professional, educators’ pedagogical, and student competences. INTRODUCTION DigCompEdu is a digital competence model with six diﬀerentiated competence areas: (i) Professional engagement, (ii) Digital resources, (iii) Teaching and learning, (iv) Assessment, (v) Empowering FIGURE 1 \| The European Framework for the digital competence of educators (DigCompEdu). FIGURE 1 \| The European Framework for the digital competence of educators (DigCompEdu). June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 3 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. (iv) Collaborative construction of knowledge, and (v) Reﬂection. In addition, Cabero-Almenara and Palacios-Rodríguez (2021) emphasize the relevance of incorporating meaningful elements for all students, with quality e-activities, designed with technical and pedagogical criteria, adapted to the context. normality criteria (Ruiz et al., 2010). For the latter procedure, the AMOS software has been used, capable of revealing hypothetical complex relationships between variables, using structural equation modeling (SEM). At the same time, it has been veriﬁed that the data are not normally distributed through a descriptive study in which skewness and kurtosis have been taken into account. The Kolmogorov-Smirnov goodness-of-ﬁt test conﬁrmed this ﬁnding, with signiﬁcance (p-value) equal to 0.000 for all items, a non-normal distribution according to Siegel (1976). Consequently, in response to the ﬁrst research objective, the means and standard deviations of the questionnaire items, dimensions, and total values are presented. This paper aims to analyze the educational possibilities of T-MOOCs for the formation of digital competences of teachers, as resources that favor the concretion of the contents structured in e-activities, and the autonomous and collaborative learning in innovative scenarios under the T-MOOC architecture. Participants The sample was made up of 313 students (23,3%, f = 73 were male and 76,7%, f = 240 were female) of the Primary Education Degree at the University of Seville (Spain), of the basic training course “Information and Communication Technologies Applied to Education,” which is taught in the ﬁrst year of the Degree, second quarter (February–June). The average age of the students was 20 years old. The instrument lacked analyses to conﬁrm exploratory and conﬁrmatory validity, because this was performed and checked. The exploratory factor analysis (EFA) was used under the maximum likelihood method with varimax rotation. The KMO test (Kaiser–Meyer–Olkin) was 0.924 and Bartlett’s test was signiﬁcant (p < 0.05). The ﬁnal version explained 85.65% of the true variance of it. On the other hand, the conﬁrmatory factor analysis (CFA) showed that the teachers’ data ﬁtted correctly to the theoretical model proposed by Cabero-Almenara and Palacios-Rodríguez (2020). The coeﬃcients were correct and respected the thresholds established by Schumacker and Lomax (2004) and Bentler (2006). This model supported the factorial structure formulated in the CFA, formed by two correlated latent variables. The structural equation model was performed with AMOS V.24 software. In addition, the reliability of the selected items was examined through Cronbach’s Alpha (α = 0.949) and McDonald’s Omega coeﬃcient ( = 0.945), for each of the instrument’s scales. Both coeﬃcients obtained very satisfactory values. Research Aims The data collection instruments are Digital Teaching Competence Questionnaire “DigCompEdu” (Cabero-Almenara and Palacios-Rodríguez, 2020) and the Content Questionnaire: Digital Resources and Digital Pedagogy. Regarding the ﬁrst questionnaire, it is an adaptation of the DigCompEdu European Framework for Digital Teaching Competence analysis instrument validated by Ghomi and Redecker (2019). This competency framework is selected as the most appropriate for assessing the Digital Teaching Competence of university faculty by means of expert judgment (Cabero-Almenara et al., 2020). In order to analyze the educational possibilities of T-MOOCs for the development of Digital Competences in teachers, and as resources that favor autonomous and collaborative learning in innovative scenarios. The research objectives being pursued are as follows: – To analyze the level of Digital Teaching Competence in initial teacher training. – To elaborate a training proposal to improve the level of Di i l T hi C f h i h – To elaborate a training proposal to improve the level of Digital Teaching Competence of the trainee teachers. Digital Teaching Competence of the trainee teachers. The ﬁrst questionnaire is composed of 7 items/dimensions, which refer to the 2 competency areas worked in the subject: digital resources (3 items) and digital pedagogy (4 items). Each of the items measures the diﬀerent competencies that make up the competency framework: B1–selecting digital resources; B2–creating and modifying digital resources; B3–managing, protecting and sharing digital resources; C1–teaching; C2– guiding; C3–collaborative learning; C4–self-directed learning. Frontiers in Education \| www.frontiersin.org Data Analysis Procedure A cross-sectional descriptive research design is proposed that takes into account the participation of the students of the Primary Education Degree. The reliability, discriminate validity, and convergent validity of the Digital Teaching Competence questionnaire (DigCompEdu Check-In) were calculated using the coeﬃcients: Cronbach’s Alpha, McDonald’s Omega, Composite Reliability (CR), Average Variance Extracted (AVE), and Maximum Shared Variance (MSV). The construct validity of the test was obtained by means of an exploratory factor analysis (EFA). The method used for factor selection is the principal components method. The factors obtained are orthogonally rotated using the Varimax method with Kaiser Normalization. Once the number of factors has been determined, a conﬁrmatory factor analysis (CFA) is performed. Conﬁrmatory factor analysis is used to check whether the theoretical measures of the model are consistent through the modeling of diagrams and the use of structural equations (Ruiz et al., 2010). In other words, it is tested whether the data ﬁt the hypothetical measurement model yielded by the exploratory factor analysis. The method used to test the theoretical model was weighted least squares (WLS), which provides consistent estimates in samples that do not ﬁt The values for the diﬀerent dimensions analyzed through the instrument were also obtained; presenting the results of both Cronbach’s Alpha and McDonald’s Omega remained suﬃciently high and signiﬁcant. All coeﬃcients are shown in Table 1. The second questionnaire consists of 20 a multiple-choice question (Table 2) in which only one option is correct (test). June 2022 \| Volume 7 \| Article 846998 4 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. TABLE 1 \| Exploratory and conﬁrmatory factorial results and reliability of the instrument. Model ﬁt summary χ2 p CFI TLI IFI NFI RMR RMSEA 3.014 0.001 0.925 0.942 0.926 0.936 0.049 0.077 Dimensions Dim. 1 Dim. 2 Validity analysis CR 0.919 0.929 AVE 0.798 0.812 MSV 0.502 0.552 Test reliability α 0.919 0.901 0.929 0.908 TABLE 1 \| Exploratory and conﬁrmatory factorial results and reliability of the instrument. Both instruments were administered online, through the Google Forms platform. The anonymity of the participants is assured at all times. The following links show the general structure of the data collection instruments: https://cutt.ly/ IUnEyCg (DigCompEdu Check-In) and https://cutt.ly/5UnEssX (questionnaire content). RESULTS As regards the second dimension, create my own digital resources and modify existing ones to adapt them to my needs as a future teacher, the item: “I create digital slideshows. For example: Power Point, Prezi.” with 56.5% (f = 177); as opposed to 3.5% (f = 11) and 4.5% (f = 14) of the items: “I create activity sheets with the computer and then print them out” and “I conﬁgure and adapt complex and interactive resources,” respectively. The third and last dimension within the area “digital resources,” able to securely protect sensitive content, we ﬁnd, as signiﬁcant data regarding the secure protection of sensitive content, that 31.3% (f = 98) and 31.9% (f = 100), respectively, protect their personal data and their own passwords; only 2.2% (f = 7) indicate that they do not need to do so. This fact leads us to think about the little importance that some students give to pedagogical competencies as future teachers, competencies such as protection, creation and collaboration, and protection, management and exchange of digital content. The results obtained from the two questionnaires: DigCompEdu Check-In and questionnaire content are shown below. Results that subsequently help, on the one hand, to demonstrate the knowledge, content, and skills acquired by the students of the Primary Education Degree of the University of Seville; and subsequently, to respond to the second objective of this study, to design a training proposal to improve the level of Digital Teaching Competence of teachers in training under the architecture of the T-MOOC. Data Analysis Procedure in the ﬁrst dimension, “use diﬀerent internet sites (web pages) and search strategies to ﬁnd and select a wide range of digital resources”, 38% (f = 119) of the students indicate that they use search engines (e.g., Google) and/or educational platforms to ﬁnd educational resources; followed by 30% (f = 94) who state that they evaluate and select the digital resources I ﬁnd based on their suitability for my needs as a student and future teacher. The most signiﬁcant data is found in the item: “rarely use the Internet to ﬁnd resources,” with only 0.6% (f = 2) of the participants. Frontiers in Education \| www.frontiersin.org DigCompEdu Check-In Among the different possibilities that the teacher has for the use of technological resources, the one that best adapts to the characteristics of the material produced and the needs of the students is the following. 3. What is NOT an emerging educational strategy? 4. If I detect a security gap in my context, how should I act? 5. Taking into account your knowledge about the possibilities of technologies and their correct integration in educational contexts, it is interesting that: 6. Among the following programs, which would be the best option if we want to make a collective presentation? - Google Slides. - Bing. - Bing. - Microsoft Teams. - Edmodo. 7. The TLK are. . . 7. The TLK are. . . - Information and Communication Technologies. - Technologies for Learning and Knowledge. - Technologies for Cooperative Learning. - Technologies for Continuous Learning. 8. In order to carry out the tutorial function, the teacher must rely on different synchronous and asynchronous communication tools. 8. In order to carry out the tutorial function, the teacher must rely on different synchronous and asynchronous communication tools. 9. What is NOT an online collaborative learning environment? - True. 8. In order to carry out the tutorial function, the teacher must rely on different synchronous and asynchronous communication tools. 9. What is NOT an online collaborative learning environment? - False. 9. What is NOT an online collaborative learning - Moodle. - Blackboard. environment? - Google Classroom. - Mentimeter. 10. Among the basic rules that can help us to mitigate the risks of identity theft are: 10. Among the basic rules that can help us to mitigate the risks of identity theft are: - Knowing with whom information is shared, personally investigating the identity of the person with whom I share information, storing and disposing of information securely. - Store and delete information securely, know with whom information is shared, ask questions before deciding to share information, and maintain an appropriate level of security on our devices. - Knowing with whom information is shared, personally investigating the identity of the person with whom I share information, storing and disposing of information securely. p , g p g y - Store and delete information securely, know with whom information is shared, ask questions before deciding to share information, and maintain an appropriate level of security on our devices. June 2022 \| Volume 7 \| Article 846998 DigCompEdu Check-In The results obtained after administration of the DigCompEdu Check-In questionnaire provide the frequencies and percentages (valid and cumulative) (Table 3) for each of the items comprising the seven key dimensions: (i) Use diﬀerent internet sites (web pages) and search strategies to ﬁnd and select a wide range of digital resources; (ii) Create my own digital resources and modify existing ones to adapt them to my needs as a future teacher; (iii) Able to securely protect sensitive content. For example: photographs, videos, ﬁles, exams, grades, personal data.; (iv) Consider how, when, and why to use digital technologies in the teaching-learning process, to ensure that their added value is exploited; (v) Consider the supervision of the activities and interactions of my future students with ICT in my educational proposals; (vi) Consider cooperative work with ICT to acquire and document knowledge in my educational proposals; and (vii) Consider the use of digital technologies to allow my future students to plan, document, and evaluate their learning by themselves. In relation to the second area “teaching and learning,” the three dimensions to be analyzed are. First, the dimension: carefully consider how, when and why to use digital technologies in the teaching-learning process, to ensure that their added value is exploited, it is striking how only 0.6% (f = 2) do not consider the use or rarely use technology in future teaching-learning strategies; that in contrast, 30.7% (f = 96) and 26.5% (f = 83) consider the use of digital tools as an opportunity to implement innovative pedagogical strategies in their teaching practices, and as key elements to systematically improve their own educational proposals. The results converge with the “teaching” competency of the DigCompEdu framework, “program and implement digital devices and resources in the teaching process to improve the eﬀectiveness of teaching interventions; manage and coordinate adequately digital didactic interventions; experiment with and develop new formats and pedagogical methods for teaching” (Redecker and Punie, 2017; Ghomi and Redecker, 2019). It should be noted that the ﬁrst three dimensions are included in area 2 “digital resources/content”; and the remaining four dimensions in area 3 “teaching and learning” of the “Digital competence of teachers in training (DigCompeEdu)” questionnaire. As can be seen in Table 3, the results show that June 2022 \| Volume 7 \| Article 846998 5 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. 2. 14. Which is NOT one of the chat planning stages? DigCompEdu Check-In - Personal Development Environment. 20. What evaluation strategy requires a ﬁnal version of the program: - Consultation with experts. - Evaluation “by” and “from” the users. - Illuminative evaluation. In second place, the dimension “consider the supervision of the activities and interactions of my future students with ICT in my educational proposals,” the most signiﬁcant data is found in the item: “regularly consider the intervention with comments to motivate or correct the activity proposed online” with 44.1% (f = 138), compared to the 1.6% (f = 5) found in the item “do not oﬀer educational proposals that contemplate the use of ICT” in contrast to the competence “guidance and support in learning, oriented to: “use digital technologies and services to improve individual and collective interaction with students inside and outside the teaching sessions; use digital technologies to provide relevant and speciﬁc guidance and assistance; and experiment with and develop new ways and formats to oﬀer guidance and support” (Redecker and Punie, 2017; Ghomi and Redecker, 2019). the items: “my educational proposals do not contemplate group work” and “I do not feel able to integrate digital technologies in group work.” Both participants consider or do not contemplate the importance of group work and, consequently, the added value of collaborative learning; perhaps a ﬁrst reading could be found in the lack of training of some students to use technologies as part of collaborative tasks and the joint creation of knowledge. Finally, the fourth dimension, consider the use of digital technologies to allow my future students to plan, document and evaluate their learning by themselves, shows data of 75.4% (f = 236) in relation to the competence “self-regulated learning,” in which the importance of using digital technologies to promote and encourage learning processes, where students can plan, document, and reﬂect on their own learning, is expressed. In this sense, the student body apparently presents certain abilities to share ideas and creative solutions through the use of digital tools. Only 3.5% (f = 11) do not feel trained or qualiﬁed to deploy the variety of digital tools available to them. Frontiers in Education \| www.frontiersin.org 20. What evaluation strategy requires a ﬁnal version of the program: DigCompEdu Check-In - Do not share information as a general rule and know with whom the information is shared. - None of the above options is correct. 11. In order to achieve an effective search, it is advisable to contemplate some rules such as, for example: - Do not use more than 10 words because some search engines do not consider them. - Do not use more than 10 words because some sea - Do not use more than 10 words because some search engines do not consider them. - The order in which you put the words is important. - The order in which you put the words is important. - Generally, search engines do not identify short words, except for “AND” and “OR.” - All options are correct. 12. If you intend to search for information on the web about the rankings of soccer teams in the 1979 and 2019 league championships. What term would you place to reﬁne your search? - Soccer league standings 1979–2019. - Soccer league standings 1979 2019. - Soccer league standings 1979 OR 2019. - Soccer league standings 1979 AND 2019. 13. Which is NOT a blogging tool? - Blogger. - Blogia. - Weebly. - Blogly. 14. Which is NOT one of the chat planning stages? - Planning. - Production. - Development. - Completion. (Continued) June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 6 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. Martínez Pérez et al. T MOOC Initial Training Digital Competences TABLE 2 \| (Continued) Item/question Multiple-choice 15. What is a characteristic of collaborative learning? - Individual responsibility of the person in the participation in the project, as well as group responsibility in the acquisition of the objectives and in the conﬁguration of quality educational actions. - Individual responsibility of the person in the participation in the project. - Group responsibility in the acquisition of the objectives and in the conﬁguration of quality educational actions. - Learning is only achieved through interaction. 16. Among the general principles to be taken into account for the selection and use of ICT in education we ﬁnd: - The learner is a passive processor of information. - ICTs work in the same way in any context and are not conditioned by it. - ICTs are vicarious transformers of reality. DigCompEdu Check-In - The main task of the teacher is to ﬁnd the supertechnology that will help him/her to solve his/her educational problems. 17. When it comes to the curricular integration of any resource, we have to consider: - Learning objectives, context, pedagogical approach and characteristics of the group of students. - Technical characteristics of the resource, learning objectives, context, pedagogical approach, and characteristics of the learner group. - Learning objectives and technical characteristics of the resource. - Context, pedagogical approach, characteristics of the learner group and technical characteristics of the resource. 18. Bauman (2010) points out that we live in a society that is. - Modern. - Post-modern. - Liquid. - Liberal. 19. A PLE is. - Personal Learning Environment. - Personal Development Environment. - Virtual Learning Environment. - Online Learning Environment. 20. What evaluation strategy requires a ﬁnal version of the program: - Self-assessment by producers. - Consultation with experts. - Evaluation “by” and “from” the users. - Illuminative evaluation. Multiple-choice Multiple-choice - Individual responsibility of the person in the participation in the project, as well as group responsibility in the acquisition of the objectives and in the conﬁguration of quality educational actions. dividual responsibility of the person in the participation in the proje - Group responsibility in the acquisition of the objectives and in the conﬁguration of quality educational actions. - Learning is only achieved through interaction. 16. Among the general principles to be taken into account for the selection and use of ICT in education we ﬁnd: - The learner is a passive processor of information. - ICTs work in the same way in any context and are not conditioned by it. - ICTs are vicarious transformers of reality. - The main task of the teacher is to ﬁnd the supertechnology that will help him/her to solve his/her educational problems. 17. When it comes to the curricular integration of any resource, we have to consider: - Learning objectives, context, pedagogical approach and characteristics of the group of students. - Technical characteristics of the resource, learning objectives, context, pedagogical approach, g p - Learning objectives and technical characteristics of the resource. - Context, pedagogical approach, characteristics of the learner group and technical characteristics of the resource. 18. Bauman (2010) points out that we live in a society that is. - Modern. - Post-modern. - Liquid. - Liberal. 19. A PLE is. 19. A PLE is. - Personal Learning Environment. DigCompEdu Check-In In third place, the dimension “consider cooperative work with ICT to acquire and document knowledge in my educational proposals,” yields signiﬁcant data showing high values for the items “collaborative work proposals, I always contemplate the use of the Internet to ﬁnd information and present the results in digital format” and “consider the exchange and creation of group knowledge in diﬀerent online collaborative spaces, e.g., class blog, virtual platform, wiki” with 41.2% (f = 129) and 37.4 (f = 117), respectively. At the other extreme, low values can be found in the response of two students with 0.3% (f = 1), respectively, to Table 4 shows the average (m) and deviation (SD) achieved for each of the dimensions analyzed. The values range from 1.96 (basic level) to 3.15 (intermediate level). Speciﬁcally, the students present a basic level in the use diﬀerent internet sites (web pages) and search strategies to ﬁnd and select a wide range of digital resources; a fact that leads us to think about the relevance of promoting and enhancing competences oriented to June 2022 \| Volume 7 \| Article 846998 7 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. TABLE 3 \| Digital teaching competence questionnaire check-in item results (response percentage). Frequency Percentage Valid percentage Cumulative percentage I use different internet sites (web pages) and search strategies to ﬁnd and select a wide range of digital resources. - I advise colleagues on appropriate digital resources and search strategies. 14 4,5 4,5 4,5 - I compare resources using a series of criteria relevant to my needs as a student and my future educational practice. For example: quality, pedagogical ﬁt, design and interactivity. 84 26,8 26,8 31,3 - I evaluate and select the digital resources I ﬁnd based on their suitability for my needs as a student and future teacher. 94 30,0 30,0 61,3 - I rarely use the Internet to ﬁnd resources. 2 6 6 62,0 - I use search engines (e.g., Google) and/or educational platforms to ﬁnd educational resources. 119 38,0 38,0 100,0 Total 313 100,0 100,0 I create my own digital resources and modify existing ones to adapt them to my needs as a future teacher. - I conﬁgure and adapt complex and interactive resources. 14 4,5 4,5 4,5 - I create activity sheets with the computer and then print them out. 11 3,5 3,5 8,0 - I create digital slideshows. For example: Power Point, Prezi. DigCompEdu Check-In 177 56,5 56,5 64,5 - I create and modify different types of digital resources. 74 23,6 23,6 88,2 - I do not create my own digital resources. 37 11,8 11,8 100,0 Total 313 100,0 100,0 I am able to securely protect sensitive content. For example: photographs, videos, ﬁles, exams, grades, personal data. - I avoid storing personal data electronically. 61 19,5 19,5 19,5 - I don’t need to do that. 7 2,2 2,2 21,7 - I protect some personal data. 98 31,3 31,3 53,0 - I password protect ﬁles with personal data. 100 31,9 31,9 85,0 - I protect personal data thoroughly. For example: combining hard-to-guess passwords, encrypting ﬁles, performing frequent software updates. 47 15,0 15,0 100,0 Total 313 100,0 100,0 I carefully consider how, when and why to use digital technologies in the teaching-learning process, to ensure that their added value is exploited. - I consider the basic use of the equipment available in the classroom. For example: audio equipment, television, projector, digital whiteboard. 60 19,2 19,2 19,2 - I consider the use of digital tools to systematically improve my educational proposals. 83 26,5 26,5 45,7 - I consider the use of digital tools to implement innovative pedagogical strategies in my future educational proposals. 96 30,7 30,7 76,4 - I consider a wide variety of digital strategies in my future educational proposals. 72 23,0 23,0 99,4 - I do not consider the use or rarely use technology in future teaching-learning strategies. 2 6 6 100,0 Total 313 100,0 100,0 I consider the supervision of the activities and interactions of my future students with ICT in my educational proposals. - I regularly consider the intervention with comments to motivate or correct the activity proposed online. 138 44,1 44,1 44,1 - I occasionally consider the review and keep in mind. 54 17,3 17,3 61,3 - I do not consider monitoring student activity in the online environments we use. 11 3,5 3,5 64,9 - I do not offer educational proposals that contemplate the use of ICT. 5 1,6 1,6 66,5 - I regularly consider the supervision and analyze the online activity of my students. 105 33,5 33,5 100,0 Total 313 100,0 100,0 I consider cooperative work with ICT to acquire and document knowledge in my educational proposals. - I consider the exchange and creation of group knowledge in different online collaborative spaces. For example: class blog, virtual platform, wiki. DigCompEdu Check-In 117 37,4 37,4 37,4 - I consider searching for information online or presenting results in digital format in my cooperative work proposals. 65 20,8 20,8 58,1 - In my collaborative work proposals, I always contemplate the use of the Internet to ﬁnd information and present the results in digital format. 129 41,2 41,2 99,4 - My educational proposals do not contemplate group work. 1 3 3 99,7 - I do not feel able to integrate digital technologies in group work. 1 3 3 100,0 Total 313 100,0 100,0 I consider the use of digital technologies to allow my future students to plan, document, and evaluate their learning by themselves. For example: self-assessment tests, digital portfolios, blogs, forums. Sometimes I use, for example, tests for self-evaluation, blog, portfolio. . . 103 32,9 32,9 32,9 - I systematically integrate different digital tools to plan and reﬂect on progress. 54 17,3 17,3 50,2 - I don’t feel qualiﬁed to use these kinds of digital tools. 11 3,5 3,5 53,7 - They reﬂect on their learning, but not with digital technologies. 12 3,8 3,8 57,5 - I use a variety of digital tools to plan, document or reﬂect on learning. 133 42,5 42,5 100,0 Total 313 100,0 100,0 TABLE 3 \| Digital teaching competence questionnaire check-in item results (response percentage). TABLE 3 \| Digital teaching competence questionnaire check-in item results (response percentage). Frequency - I create activity sheets with the computer and then print them out - I create digital slideshows. For example: Power Point, Prezi. - I create and modify different types of digital resources. - I do not create my own digital resources. - I don’t need to do that. - I protect some personal data. - I password protect ﬁles with personal data. - My educational proposals do not contemplate group work. - I don’t feel qualiﬁed to use these kinds of digital tools. - They reﬂect on their learning, but not with digital technologies June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 8 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. TABLE 4 \| Digital teaching competence questionnaire check-in items results (Likert scale 0–4). Item Average Deviation - I use different internet sites (web pages) and search strategies to ﬁnd and select a wide range of digital resources. 1,96 0,924 - I create my own digital resources and modify existing ones to adapt them to my needs as a future teacher. 2,05 0,961 - I am able to securely protect sensitive content. For example: photographs, videos, ﬁles, exams, grades, personal data. 2,38 1,031 - I carefully consider how, when and why to use digital technologies in the teaching-learning process, to ensure that their added value is exploited. 2,67 1,122 - I consider the supervision of my future students’ activities and interactions with ICT in my educational proposals. 3,15 0,937 - I consider cooperative work with ICT to acquire and document knowledge in my educational proposals. 3,15 0,776 - I consider the use of digital technologies to allow my future students to plan, document and evaluate their learning by themselves. For example: self-assessment tests, digital portfolios, blogs, forums. 2,66 0,927 The scale of values is between 0 and 4 points, where the values between 0 and 1 represent a low level of competence, 2 and 3 points an intermediate level, and 4 a high level. TABLE 4 \| Digital teaching competence questionnaire check-in items results (Likert scale 0–4). TABLE 4 \| Digital teaching competence questionnaire check-in items results (Likert scale 0–4). The scale of values is between 0 and 4 points, where the values between 0 and 1 represent a low level of competence, 2 and 3 points an intermediate level, and 4 a high level. Frequency The answers obtained are shown as a percentage of correct answers (% hits) to the 20 questions asked (Table 6). the selection, creation, protection, management, and exchange of digital resources and contents. As for the competencies that stand out (intermediate level), they are focused on teaching and learning, and mainly refer to orientation and support in learning, whether autonomous, self-regulated, or collaborative. That is to say, the student body indicates (m = 3.15) that as teachers they have to contemplate and carry out educational proposals using technologies in their teaching-learning processes. A high percentage of correct answers was observed in the questions/items: 8 “To carry out the tutorial function, the teacher must have diﬀerent synchronous and asynchronous communication tools,” 95.9% answered correctly; 4 “If I detect a security gap in my context, how should I act?” with 87.6%; 9 “What is NOT an online collaborative learning environment?” with 80.7%; and 11 “To achieve an eﬀective search, it is advisable to contemplate some rules” with 77.9%. In contrast, the questions/items with the lowest percentage of correct answers were 17 (15.5%) “When integrating any resource into the curriculum, it is necessary to consider”; 3 (19.7%) “Which is NOT an emerging educational strategy?”; 2 (28.6%) “Among the diﬀerent possibilities that the teacher has for the use of technological resources, the one that best suits the characteristics of the material produced and the needs of the students is the following”; 14 (32.1%) “Which is NOT one of the planning stages of the lecture?”; 13 (32.4%) “What is NOT a blogging tool?”; 1 (35.9%) “What is NOT a tool used in gamiﬁcation?” and 12 “If you intend to search for information on the web about the rankings of soccer teams in the 1979 and 2019 league championships. What term would you place to reﬁne your search?” (36,2%).” The percentage of correct answers for the rest of the items/questions answered by the students ranged from 44.1 to 66.2%. In closing, Table 5 shows the average and deviation with respect to the level of digital competence in initial teacher training with respect to two key axes: resources and the pedagogical nature of all training. Content Questionnaire The purpose of this questionnaire was to inquire about the students’ knowledge of digital resources, emerging educational strategies, and the use and possibilities of technologies in educational contexts in accordance with the contents of the subject “Information and Communication Technologies Applied to Education.” And in this way, to design the contents of the T-MOOC for the development of digital competence in teaching, which is presented in the following section. Frequency The table shows that the student body is at an intermediate level in terms of resources (m = 2.13; D = 0.972) and pedagogy (m = 2.91; D = 0.941), Through the implementation of the T-MOOC, the objective would be for students to reach all levels, until they reach a high level (leaders or pioneers) in terms of digital competencies. TABLE 6 \| Test results content (% hits). TABLE 6 \| Test results content (% hits). Item/question % hits 1. What is NOT a tool used in gamiﬁcation? 35,9 2. Among the different possibilities that the teacher has for the use of technological resources, the one that best adapts to the characteristics of the material produced and the needs of the students is the following. 28,6 3. What is NOT an emerging educational strategy? 19,7 4. If I detect a security gap in my context, how should I act? 87,6 5. Taking into account your knowledge about the possibilities of technologies and their correct integration in educational contexts, it is interesting that: 51 6. Among the following programs, which would be the best option if we want to make a collective presentation? 54,1 7. The TLK are. . . 66,2 8. In order to carry out the tutorial function, the teacher must rely on different synchronous and asynchronous communication tools. 95,9 9. What is NOT an online collaborative learning environment? 80,7 10. Among the basic rules that can help us to mitigate the risks of identity theft are: 64,8 11. In order to achieve an effective search, it is advisable to contemplate some rules. 77,9 12. If you intend to search for information on the web about the rankings of soccer teams in the 1979 and 2019 league championships. What term would you place to reﬁne your search? 36,2 13. Which is NOT a blogging tool? 32,4 14. Which is NOT one of the chat planning stages? 32,1 15. What is a characteristic of collaborative learning? 64,8 16. Among the general principles to be taken into account for the selection and use of ICT in education we ﬁnd: 44,1 17. When it comes to the curricular integration of any resource, we have to consider: 15,5 18. Bauman (2010) points out that we live in a society that is. 52,4 19. A PLE is. 65,5 20. What evaluation strategy requires a ﬁnal version of the program: 44,8 T-MOOC Design TABLE 5 \| Digital teaching competence questionnaire check-in dimensions and total results (Likert scale 0–4). Digital kind Average Deviation Digital resources 2,13 0,972 Digital pedagogy 2,91 0,941 Total 2,52 0,957 Frontiers in Education \| www frontiersin org 9 TABLE 5 \| Digital teaching competence questionnaire check-in dimensions and total results (Likert scale 0–4). After obtaining the results from the students of the DigCompEdu Check-In and content questionnaires, and taking into account the responses of the students, we proceed to create a training and innovative environment under the tMOOC architecture. The purpose is to promote the acquisition of digital competences by the teachers, in our case, for the initial training of the teachers of June 2022 \| Volume 7 \| Article 846998 9 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. Item/question 1. What is NOT a tool used in gamiﬁcation? 2. Among the different possibilities that the teacher has for the use of technological resources, the one that best adapts to the characteristics of the material produced and the needs of the students is the following. 9. What is NOT an online collaborative learning environment? 20. What evaluation strategy requires a ﬁnal version of the program: FIGURE 2 \| Transfer massive open online courses for the acquisition of digital teaching competencies. FIGURE 2 \| Transfer massive open online courses for the acquisition of digital teaching competencies. To access the T-MOOC, each user is assigned an identiﬁer and a password. Once inside, students are presented with the structure of the course. First, there is a presentation of the course To access the T-MOOC, each user is assigned an identiﬁer and a password. Once inside, students are presented with the structure of the course. First, there is a presentation of the course the University of Seville. For this purpose, the platform chosen for the design and development of the t-MOOC was Moodle (Figure 2). June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 10 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. and the DigCompEdu Framework through two animations: one with instructions on how to proceed through the course, and the other with the DigCompEdu model with its diﬀerent areas and competencies. After viewing the videos, the diﬀerent areas are shown (Figure 3). Each competency area is composed by its respective competencies and each competency by its corresponding level (beginner, intermediate, and advanced). Each competency with its corresponding presentation for its correct procedure, levels, tasks and forums (Figure 4). navigation instructions and use of the t-MOOC, and the other on DigCompEdu); 22 animations speciﬁc to each DigcompEdu competency; 16 animations integrated in the diﬀerent learning modules; 66 learning modules, 230 e-activities distributed in the diﬀerent modules; 24 infographics and 11 multimedia, both resources integrated in the diﬀerent learning modules. The presentation of the tasks (e-activities) is done through a guide that incorporates aspects such as: their identiﬁcation, recommendations for their completion, a checklist for the user to check the quality of the delivery, and an evaluation rubric that is used by t-MOOC tutors. DISCUSSION AND CONCLUSION Based on the results, we can corroborate those presented by Luo et al. (2017) and Gudmundsdottir and Hatlevic (2018), and which highlight the concern on the part of teachers about their training in digital competencies. The presented research implies a transformation in traditional training and educational structures, methods, and assumptions. This is why, as Cabero-Almenara and Palacios-Rodríguez (2020) pointed out in their research, there is a need to rethink other ways of approaching teacher training in order to promote authentic competence development for the current demands of society. Based on the results, we can corroborate those presented by Luo et al. (2017) and Gudmundsdottir and Hatlevic (2018), and which highlight the concern on the part of teachers about their training in digital competencies. The presented research implies a transformation in traditional training and educational structures, methods, and assumptions. In view of the above, it is considered that the present research adds value to the ﬁeld of educational innovation and technologies, as it opens new perspectives for further research in future studies related to the T-MOOC phenomenon in terms of the acquisition of digital teaching skills, both for teachers who are currently working as well as for those who are undergoing initial training (students). It may also be of interest to educational administrations in order to structure and evaluate training plans and improve the level of digital competencies of teachers. This is why, as Cabero-Almenara and Palacios-Rodríguez (2020) pointed out in their research, there is a need to rethink other ways of approaching teacher training in order to promote authentic competence development for the current demands of society. It should be noted that the period of data collection and the results obtained present an overview of the initial training of future teachers in reference to digital competencies at a time when the pandemic situation generated by COVID-19 led us to teach in virtual mode (February–June 2021). These results are similar to those of another study (Cabero-Almenara et al., 2021a), showing teachers in training with a moderate level (basic- intermediate) in terms of digital competencies (Redecker and Punie, 2017; Ghomi and Redecker, 2019). Item/question The T-MOOC has a diversity of programs (ExeLearning, VYOND, Genially, Photoshop, Adobe Premiere, and Audacity), distributed as follows: two general animations (one with FIGURE 3 \| Areas and competencies. FIGURE 4 \| Area and competencies development. FIGURE 3 \| Areas and competencies. FIGURE 4 \| Area and competencies development. FIGURE 3 \| Areas and competencies. FIGURE 4 \| Area and competencies development. FIGURE 4 \| Area and competencies development. June 2022 \| Volume 7 \| Article 846998 11 Frontiers in Education \| www.frontiersin.org T-MOOC Initial Training Digital Competences Martínez-Pérez et al. It should be noted that the e-activities proposed are of various types: making concept maps, participating in forums, building a blog, creating a PLE with certain tools, creating learning communities, among others. As for the resources used in the learning modules: didactic animations, polimedia recordings, videos, infographics, web addresses, and complementary documents. In addition, several forums have been designed: for general doubts about how t-MOOC works, for doubts about each competency area and speciﬁc forums for activities. training, as pointed out by Cornelius et al. (2019) in their study; not to mention the entire organizational structure and pedagogical design, as pointed out by Raposo-Rivas et al. (2017) and Gros (2018) in their research. It is hoped that with the implementation of the T-MOOC presented in initial teacher education, the inclusion of opportunities for more active participation will be promoted. It is understood that the conclusion presented should be interpreted with caution. The type of non-experimental design and the size of the sample imply some restrictions for the generalization and application of the results. Future research could consider larger samples and carry them out in other subjects and university careers. Therefore, the purpose is to continue improving and expanding the characteristics of this study, in order to contract results. ETHICS STATEMENT Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The datasets generated during the current study are not publicly available because the identities of some participants are visible, undermining privacy protection. Requests to access the datasets should be directed to SM-P, smartinezperez@us.es; JC-A, cabero@us.es; JB-O, jbarroso@us.es; AP-R, aprodriguez@us.es. In order to analyze the educational possibilities of T-MOOCs for initial teacher training in digital competencies, and as resources that favor autonomous and collaborative learning in innovative scenarios, the results of the diﬀerent analyses carried out provide answers to the two objectives presented: (i) To analyze the level of Digital Teaching Competence in initial teacher training through the DigCompEdu Check- In and content questionnaires; (ii) To develop a training proposal, under the innovative architecture of the T-MOOC, to improve the level of Digital Teaching Competence of teachers in training. AUTHOR CONTRIBUTIONS SM-P and AP-R wrote the ﬁrst draft. All authors listed have made a substantial, direct, and intellectual contribution to the work, read and edited each draft, and approved it for publication. In relation to the implementation of online courses for teachers, and taking into account the research of Drake et al. (2015), Boaler et al. (2018), Beltrán and Ramírez-Montoya (2019), and Escudero-Nahón and Núñez-Urbina (2020), we consider, after the results obtained from the participating students, that for the acquisition of digital competencies, a change of mentality, methodologies, strategies and pedagogical resources is important; whose principles are the use of the Internet in order to access digital resources and content, networked learning and horizontal communication. And, in turn, they are envisioned as a means of opportunities for eﬀective teaching and for the involvement of teachers in Frontiers in Education \| www.frontiersin.org REFERENCES education. Telemat. 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Technol. 7, 223–240. Raposo-Rivas, M., Sarmiento, J., and Martínez-Figueira, M. (2017). ACKNOWLEDGMENTS I + D + I. In addition, it was also the result of the research project: “Design, Production and Evaluation of T-MOOCs for the Acquisition of Digital Competencies of University Professors” (US-1260616), funded by Junta de Andalucía (Consejería de Economía y Conocimiento). I + D + I. In addition, it was also the result of the research project: “Design, Production and Evaluation of T-MOOCs for the Acquisition of Digital Competencies of University Professors” (US-1260616), funded by Junta de Andalucía (Consejería de Economía y Conocimiento). We thank the teachers, managers, and students of the Primary Education Degree of the Faculty of Education Sciences of the University of Seville, Spain. FUNDING This study was the result of R + D + i research project: “Design, Production and Evaluation of T-MOOC for the Acquisition by Teachers of Digital Competences for Teachers” (RTI2018- 097214-B-C31), funded by Ministerio de Ciencia, Innovación y Universidades, Plan Estatal 2017–2020 Retos - Proyectos June 2022 \| Volume 7 \| Article 846998 12 T-MOOC Initial Training Digital Competences Martínez-Pérez et al. REFERENCES El perﬁl pedagógico de los MOOC a partir de un estudio exploratorio. Estud. Pedagóg. XLIII, 277–292. doi: 10.4067/S0718-07052017000200015 Doherty, I., Harbutt, D., and Sharma, N. (2015). Designing and developing a MOOC. Med. Sci. Educ. 25, 177–181. doi: 10.1007/s40670-015-0123-9 Redecker, C., and Punie, Y. (2017). Digital Competence of Educators DigCompEdu. Brussels: Publications Oﬃce of the European Union. Drake, J. R., O’Hara, M., and Seeman, E. (2015). Five principles for MOOC design: with a case study. J. Inf. Technol. 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June 2022 \| Volume 7 \| Article 846998 Frontiers in Education \| www.frontiersin.org 13 Martínez-Pérez et al. T-MOOC Initial Training Digital Competences Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Siegel, S. (1976). Estadística no Paramétrica Aplicada a las Ciencias de la Conducta. Mexico City: Editorial Trilla. Tang, S. (2017). Learning mechanism and function characteristics of MOOC in the process of higher education. Eurasia J. Math. Sci. Technol. Educ. 13, 8067–8072. doi: 10.12973/ejmste/80769 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. j UNESCO (2018). UNESCO ICT Competency Framework for Teachers. Paris: United Nations Educational, Scientiﬁc and Cultural Organization. Watson, S. L., Watson, W. R., Yu, J. H., Alamri, H., and Mueller, C. (2017). Frontiers in Education \| www.frontiersin.org June 2022 \| Volume 7 \| Article 846998 REFERENCES Learner proﬁles of attitudinal learning in a MOOC: an explanatory sequential mixed methods study. Comput. Educ. 114, 274–285. doi: 10.1016/j.compedu.2017.07. 005 Copyright © 2022 Martínez-Pérez, Cabero-Almenara, Barroso-Osuna and Palacios- Rodríguez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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https://openalex.org/W3005198600	http://www.scielo.br/pdf/sausoc/v29n1/1984-0470-sausoc-29-01-e180824.pdf	Portuguese	null	Saúde pública e inovações tecnológicas para abastecimento público	Saúde e Sociedade	2,020	cc-by	4,689	Public health and technological innovations for pu Nathalie Cruza https://orcid.org/0000-0002-5711-2770 E-mail: nathaliecruz@usp.br José Carlos Mierzwaa https://orcid.org/0000-0003-0377-563X E-mail: mierzwa@usp.br aUniversidade de São Paulo. Escola Politécnica. Departamento de Engenharia Hidráulica e Ambiental. São Paulo, SP, Brasil. Nathalie Cruza https://orcid.org/0000-0002-5711-2770 E-mail: nathaliecruz@usp.br José Carlos Mierzwaa https://orcid.org/0000-0003-0377-563X E-mail: mierzwa@usp.br aUniversidade de São Paulo. Escola Politécnica. Departamento de Engenharia Hidráulica e Ambiental. São Paulo, SP, Brasil. Artigos originais Artigos originais Resumo A escassez de água em metrópoles brasileiras tem se agravado em função das características de seu próprio desenvolvimento. A expansão urbana desordenada e próxima a mananciais, junto com a falta de infraestrutura para estes novos núcleos habitacionais, tem favorecido a degradação dos mananciais utilizados para abastecimento público. Em razão disto, a população fica mais suscetível às doenças de veiculação hídrica. Independentemente dos tipos de contaminantes presentes na água, busca-se aprimorar as tecnologias tradicionalmente empregadas nos processos de tratamento hídrico a fim de garantir uma água segura para o abastecimento da população, sem considerar as inovações tecnológicas neste setor. Assim, o presente estudo busca evidenciar a necessidade de melhorar as ações de tratamento de água e esgotos realizadas atualmente no país, em função dos riscos associados à saúde pública. Busca-se também analisar o reúso potável direto e como esta prática pode ser uma solução para promover uma água de qualidade e suprir a demanda de grandes centros urbanos em situação de escassez hídrica. A escassez de água em metrópoles brasileiras tem se agravado em função das características de seu próprio desenvolvimento. A expansão urbana desordenada e próxima a mananciais, junto com a falta de infraestrutura para estes novos núcleos habitacionais, tem favorecido a degradação dos mananciais utilizados para abastecimento público. Palavras-chave: Reúso Urbano; Inovação Tecnológica; Saúde Pública. Correspondência Nathalie Cruz Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Hidráulica e Ambiental. Av. Prof. Almeida Prado, travessa 2, 83, Cidade Universitária. São Paulo, SP, Brasil. CEP 05508-900. Saúde pública e inovações tecnológicas para abastecimento público Public health and technological innovations for public supply Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 1 Abstract Abstract Há alguns anos as principais regiões metropolitanas do Brasil vêm sofrendo escassez de água, principalmente no Sudeste. O problema se agrava tanto pelas mudanças climáticas quanto por questões pontuais de grandes centros urbanos, como crescimento populacional, ocupação desordenada na área urbana e perto de mananciais, mudanças no ambiente hidrológico (diminuição do tempo de concentração) e a falta de planejamento das concessionárias de saneamento (falta de estudos de demanda e oferta de água, bem como de infraestrutura e fornecimentos intermitentes), intensificando a situação (Britto; Maiello; Quintslr, 2019; Rodrigues; Villela, 2016). Water scarcity in Brazilian metropolises has been aggravated by the characteristics of their very development. The disordered urban expansion near fountainheads and the lack of infrastructure for new housing units has contributed to the degradation of water sources used for public supply. This causes the population to be more susceptible to waterborne diseases. Regardless of the various types of contaminants present in the water, the improvement of traditional technologies used in the water treatment process and wastewater treatment is sought after to promote safe water supply to the population, without considering technological innovations on the sector. This study aims to demonstrate the ineffectiveness of treatment processes currently used in Brazil and the risks to public health. We also analyze the direct potable reuse and how this practice can be a solution to promote water quality and meet the demand of large urban centers in situations of water scarcity. Apesar da notória disponibilidade de água no país e da melhora nos índices de atendimento com os serviços de saneamento, segundo o último senso do Instituto Brasileiro de Geografia e Estatística (IBGE, 2008), o Brasil ainda não chegou na universalização destes serviços, pois 99,4% dos municípios possuem rede de distribuição de água e apenas 55,2% contam com rede coletora de esgotos. Apesar da notória disponibilidade de água no país e da melhora nos índices de atendimento com os serviços de saneamento, segundo o último senso do Instituto Brasileiro de Geografia e Estatística (IBGE, 2008), o Brasil ainda não chegou na universalização destes serviços, pois 99,4% dos municípios possuem rede de distribuição de água e apenas 55,2% contam com rede coletora de esgotos. Dados mais recentes mostram que apenas 51,92% dos municípios atendidos com água possuem rede coletora de esgotos, e que somente 74,87% do esgoto coletado segue para tratamento (Brasil, 2018). Correspondência Nathalie Cruz Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Hidráulica e Ambiental. Av. Prof. Almeida Prado, travessa 2, 83, Cidade Universitária. São Paulo, SP, Brasil. CEP 05508-900. Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 1 Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 2 Abstract Isto posto, pode-se inferir que a maior parte dos esgotos domésticos e industriais gerados chega aos corpos receptores sem tratamento adequado, favorecendo o aumento de matéria orgânica, nutrientes e de contaminantes como metais tóxicos, compostos orgânicos persistentes e poluentes emergentes (fármacos, agrotóxicos, detergentes e hormônios). A questão da seca intensifica esta situação, já que a vazão natural dos mananciais é reduzida e, com isso, o lançamento contínuo de águas residuais passa a ser a maior contribuição na deterioração dos mananciais utilizados para abastecimento, além de inflacionar os custos com tratamento de água e aumentar os casos de doença por veiculação hídrica (Tran; Jassby; Schwabe, 2017). Keywords: Urban Reuse; Technological Innovation; Public Health. Dados mais recentes mostram que apenas 51,92% dos municípios atendidos com água possuem rede coletora de esgotos, e que somente 74,87% do esgoto coletado segue para tratamento (Brasil, 2018). Isto posto, pode-se inferir que a maior parte dos esgotos domésticos e industriais gerados chega aos corpos receptores sem tratamento adequado, favorecendo o aumento de matéria orgânica, nutrientes e de contaminantes como metais tóxicos, compostos orgânicos persistentes e poluentes emergentes (fármacos, agrotóxicos, detergentes e hormônios). A questão da seca intensifica esta situação, já que a vazão natural dos mananciais é reduzida e, com isso, o lançamento contínuo de águas residuais passa a ser a maior contribuição na deterioração dos mananciais utilizados para abastecimento, além de inflacionar os custos com tratamento de água e aumentar os casos de doença por veiculação hídrica (Tran; Jassby; Schwabe, 2017). Atualmente os processos de tratamento de água e esgoto são baseados em seus custos, e não na implantação da melhor tecnologia que promova uma água segura. No Brasil, as estações de tratamento de esgoto (ETE) atuam na remoção de nutrientes e sólidos em suspensão, não sendo eficientes na remoção de íons divalentes e monovalentes. Já as estações de tratamento de água (ETA) são responsáveis por retirar material em suspensão e coloidal, patógenos e algas, porém são ineficazes com os micropoluentes. Por outro lado, processos avançados de tratamento são capazes de remover essas substâncias e promovem um efluente com melhor qualidade, podendo ser incorporados nas etapas de tratamento de águas residuais e em sistemas de tratamento de água potável. Abstract Por conta dessa grande variabilidade de contaminantes presentes nas águas utilizadas para o abastecimento, se faz necessário aprimorar os processos de tratamento com a melhor tecnologia disponível para promover uma água segura (Mierzwa, 2017; Simões, 2016; Tran; Jassby; Schwabe, 2017). contrário: estudos já detectaram a presença de um grupo desse tipo de contaminantes, denominados de disruptores endócrinos, em afluentes e efluentes de ETE por lodos ativados na Itália; em São Paulo constatou-se a presença de hormônios naturais e sintéticos no reservatório Guarapiranga, em amostra tomada da ETA Alto da Boa Vista (Hespanhol, 2015a). Essa prática deve ocorrer quando se tem mananciais protegidos e livres de efluentes com contaminantes que os sistemas de tratamento empregados são incapazes de remover. Porém, tal forma de reúso tem ocorrido em mananciais poluídos, como em São Paulo, ao longo do rio Tietê e do Paraíba do Sul, e na reversão da Represa Billings para a Guarapiranga, já que as águas desta seguem para ETA por meio de sistema convencional (Hespanhol, 2015a). Segundo estudo de Giatti (2007), este tipo de reúso também tem sido observado no estado do Amazonas, demonstrando que a situação é a mesma em diversas regiões do país. Por falta de coleta e tratamento, os igarapés recebem efluentes que chegam ao Rio Negro, à montante do ponto de captação para abastecimento público, e apesar de o Rio Negro ter grande capacidade de autodepuração de efluentes, ele já não suporta a carga recebida e está poluído. Somado a isso, os hábitos regionais favorecem o aumento das doenças de veiculação hídrica. No Norte do país os ribeirinhos utilizam a água do Rio Negro para a maior parte de suas atividades diárias, desde lavagem de roupas e utensílios até higiene pessoal. Assim, o presente trabalho aborda o sistema de reúso potável indireto que vem ocorrendo nos mananciais de abastecimento e os riscos associados à saúde pública. Em seguida, analisa-se o reúso potável direto e como esta prática pode ser uma solução para as metrópoles brasileiras. Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 3 Reúso potável indireto e saúde pública Reúso potável indireto e saúde pública Por conta dos desequilíbrios ecológico- ambientais, junto com os desarranjos gerados pela urbanização e a falta de saneamento, algumas práticas utilizadas nos sistemas de tratamento de água e esgoto colocam em risco a população, favorecendo o surgimento de doenças. Uma dessas práticas é o reúso potável indireto, comumente utilizado no país, quando os esgotos, após tratamento ou não, são dispostos em mananciais superficiais ou subterrâneos para diluição e na cidade à jusante, posteriormente captados, tratados e distribuídos para os diversos fins. Embora se acredite que em metrópoles a situação seja diferente, em alguns bairros de São Paulo que beiram o reservatório Billings, por falta de infraestrutura de abastecimento, os moradores utilizam-se de poços artesianos para consumo, cujas águas estão contaminadas por metais pesados, afetando a saúde da população local (Santana, 2018). O reúso potável indireto, planejado ou não, tem sido empregado porque acreditava-se que um manancial poderia receber efluentes domésticos, por estes estarem livres de contaminantes como micropoluentes complexos, orgânicos e inorgânicos, característicos de efluentes industriais. Porém, o cenário atual mostra o A pouca infraestrutura de saneamento em regiões normalmente mais pobres e afastadas favorece o aparecimento de doenças de veiculação hídrica. Segundo o Ministério da Saúde (Brasil, 2009), o índice de doenças infecciosas e parasitárias no país foi de 8,3%. O índice de cada região é apresentado no Gráfico 1. Gráfico 1 – Índice de doenças infeciosas e parasitárias por região do país 16,0 13,80 12,40 7,50 5,20 6,00 14,0 12,0 10,0 8,0 6,0 4,0 2,0 - Norte Nordeste Centro-Oeste Sudeste Sul Fonte: Brasil (2009) Gráfico 1 – Índice de doenças infeciosas e parasitárias por região do país Gráfico 1 – Índice de doenças infeciosas e parasitárias por região do país A grande questão é que saúde não equivale a mera ausência de doenças, mas à erradicação delas na sociedade, já que os determinantes patogênicos residem em todos os setores sociais e não apenas no da saúde. Desta forma, o saneamento possui duas perspectivas: prevenir enfermidades ou promover a saúde. A prevenção antecede o surgimento ou o agravamento de uma doença, criando uma barreira de contato entre seu vetor e os indivíduos, de forma a reduzir sua incidência ou garantir que sua manifestação seja amena, comprometendo fortemente o ciclo dos agentes biológicos, físicos e químicos. Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 4 Reúso potável indireto e saúde pública Neste ponto de vista, o saneamento seria parceiro do setor de saúde, e as doenças sinalizadoras estariam vinculadas à falta ou insuficiência de abastecimento de água de boa qualidade, à ausência de coleta e tratamento de águas residuais, dentre outros fatores, como manejo de resíduos sólidos, drenagem urbana e poluição do ar. Além de doenças infecciosas e parasitárias, os contaminantes orgânicos e inorgânicos chamados de disruptores endócrinos, já mencionados, são capazes de gerar disfunções endócrinas e perturbação hormonal em seres saudáveis. Neste grupo estão substâncias como fármacos, pesticidas, hormônios e toxinas, muitos dos quais não são biodegradáveis ou removidos pelos sistemas de tratamento de esgoto, causando incertezas quanto aos seus efeitos a longo prazo (Hespanhol, 2015a; Rezende, 2010). Quando seres de vida selvagem são expostos a disruptores endócrinos, sofrem impactos no sistema reprodutor e imunológico, reduzindo severamente o número de populações. Em humanos, há efeitos relacionados ao sistema reprodutivo: pesquisas demonstraram o declínio da qualidade de esperma e aumento de abortos espontâneos. Além disso, o desenvolvimento neurológico e comportamental pode ser prejudicado quando o feto é exposto a alguns químicos na fase pré-natal. O contato com pesticidas e organoclorados também é capaz de ocasionar doenças como câncer de próstata. Outra preocupação, detectada na Austrália em rios na região de South East Queensland, é que apesar de alguns antibióticos não causarem efeito cíclico em humanos, por estarem em baixas concentrações nas águas superficiais, as descargas no ambiente acabaram tornando determinadas bactérias resistentes a estes fármacos (Rezende, 2010). Já a promoção da saúde busca erradicar ou eliminar por longo tempo determinadas doenças, cujo aparecimento sinalizaria que algo não está bem e, a partir disto, definir ações assertivas e necessárias para intermediar a situação. Neste caso, os projetos de saneamento têm como objetivo implantar sistemas de engenharia, mas a preocupação maior seria com o seu funcionamento pleno, duradouro e acessível à toda população, não com a implantação propriamente dita (Souza, 2007). Diante disso, para melhorar a saúde pública é preciso uma abordagem interdisciplinar, possibilitando a gestão dos recursos hídricos e a implementação de infraestrutura e tecnologias de saneamento, de forma a promover a saúde e erradicar as doenças entregando à população uma água segura. que reage negativamente diante dessa fonte alternativa. Mas, valendo-se de tecnologia confiável e de campanhas educativas, é possível mitigar os riscos e aumentar a aceitação da população a essa prática (Rezende, 2010). Reúso potável direto e segurança no abastecimento O cenário dos mananciais de abastecimento, os sistemas de tratamento ineficazes na remoção de poluentes e a escassez de água em metrópoles apontam o reúso potável direto como solução para promover a saúde. Reúso potável indireto e saúde pública O reúso potável direto é seguro por contar com técnicas de certificação da qualidade hídrica que permitem produzir água a partir de esgotos domésticos, respeitando critérios econômicos e de saúde pública. A seguir, serão descritas resumidamente algumas tecnologias empregadas em sistemas desse tipo (Hespanhol, 2015b). Reúso potável direto e segurança no abastecimento Carvão ativado biológico ser aplicado em diversos países, como Austrália, África do Sul, Estados Unidos e na Namíbia, que desde 1968 não tem problemas de saúde pública associados à prática (Cunha, 2008; Hespanhol, 2015b). O carvão ativado biológico (CAB) atua por adsorção, degradação e filtração de substâncias, formando um biofilme que consome a matéria orgânica e gera subprodutos como água, dióxido de carbono e biomassa. No Brasil, a equipe de Hespanhol (2017) desenvolveu um estudo com uma estação piloto de reúso potável direto na ETE Capivari II, localizada no município de Campinas. Esta estação é composta por membranas de OR, POA, carvão ativado granular (CAG) e CAB, seguido de processos de desinfecção (PD). O processo se inicia pela ação de oxidante forte aplicado na entrada da unidade filtrante. Dessa maneira são removidos materiais orgânicos (geralmente biodegradáveis), não orgânicos (compostos estáveis e de difícil degradação) e organismos patogênicos contidos em águas superficiais ou subterrâneas. O processo se inicia pela ação de oxidante forte aplicado na entrada da unidade filtrante. Dessa maneira são removidos materiais orgânicos (geralmente biodegradáveis), não orgânicos (compostos estáveis e de difícil degradação) e organismos patogênicos contidos em águas superficiais ou subterrâneas. por membranas de OR, POA, carvão ativado granular (CAG) e CAB, seguido de processos de desinfecção (PD). Após as análises, os resultados obtidos demonstram que a qualidade do efluente final está de acordo com a maior parte das variáveis da Portaria do Ministério da Saúde nº 2.914, de 12 de dezembro de 2011, que trata dos padrões de qualidade da água para abastecimento público, com remoção de cor, coliformes e Escherichia coli, ferro, manganês, sulfato e nitratos. Além disso, as unidades complementares dos arranjos tiveram desempenho significativo na remoção de bactérias heterotróficas (100%), sulfato (99%), manganês (>98%), total de sólidos dissolvidos (95%), sódio (94%), dureza total (>93%), cor aparente (>88%), alumínio (42%), níquel (>37%) e zinco (28%). Neste estudo, além das variáveis recomendadas pela Portaria nº 2.914/2011, foram realizadas análises de toxicidade, mutagenicidade, vírus e hormônios, de forma a caracterizar melhor o efluente final da estação de reúso. Os resultados indicaram toxicidade na maioria das amostras após a cloração, mas não no arranjo com OR/POA/PD. Em relação às análises de mutagenicidade, não foi indicado nenhum potencial mutagênico nas amostras analisadas, como também não foram encontrados vírus entéricos. Membranas No reúso potável direto utilizando membranas, estas são classificadas quanto ao tamanho de seus poros, podendo ser de microfiltração, ultrafiltração, nanofiltração e osmose reversa (OR). As membranas são responsáveis pela remoção de poluentes químicos tradicionais e emergentes, mesmo os de baixa massa molecular, como os disruptores endócrinos, e de organismos patogênicos de dimensões muito pequenas, por exemplo, os oocistos de Cryptosporidium spp. A Figura 1 exemplifica qual membrana atua em determinada faixa de remoção de contaminantes, em comparação com o sistema convencional de tratamento (coagulação e floculação). Esta prática consiste no tratamento avançado de efluentes domésticos seguido por tratamento convencional ou avançado em ETA. Ou seja, traduz-se em tratar e reusar o esgoto gerado em áreas urbanas, favorecendo a redução do estresse hídrico nas metrópoles, complementando o abastecimento público e oferecendo uma água de qualidade à população. Contudo, como não há um manancial que intermedeie os tratamentos, pois o esgoto tratado em ETE segue diretamente para uma ETA, existem algumas implicações no processo, entre elas, o aumento dos riscos à saúde humana e a resistência da população, Figura 1 – Faixas de remoção de substâncias por processos de tratamento de água Microscopia eletrônica Microscopia ótica Visível a olho nu Íons Micrometros (μm) 10−3 10−2 10−1 1 10 100 10 102 103 104 105 106 Angstrons (Å) Moléculas Macromoléculas Micropartículas Macropartículas Emulsão de látex Açúcares Emulsão de óleo Negro de fumo Pigmentos de tintas Endotoxinas (pirogênios) Células de leveduras Íons metálicos Vírus Bactérias Sais dissolvidos Coloides Areia Osmose reversa Microfiltração Evaporação Troca iônica Ultrafiltração Coagulação/floculação e filtração Nota: 1Å=10−10m=10−4μm Fonte: Mierzwa (2017) Figura 1 – Faixas de remoção de substâncias por processos de tratamento de água Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 5 Carvão ativado biológico Após as análises, os resultados obtidos demonstram que a qualidade do efluente final está de acordo com a maior parte das variáveis da Portaria do Ministério da Saúde nº 2.914, de 12 de dezembro de 2011, que trata dos padrões de qualidade da água para abastecimento público, com remoção de cor, coliformes e Escherichia coli, ferro, manganês, sulfato e nitratos. Além disso, as unidades complementares dos arranjos tiveram desempenho significativo na remoção de bactérias heterotróficas (100%), sulfato (99%), manganês (>98%), total de sólidos dissolvidos (95%), sódio (94%), dureza total (>93%), cor aparente (>88%), alumínio (42%), níquel (>37%) e zinco (28%). Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 6 Processos oxidativos avançados Diário Oficial da União, Brasília, DF, 14 dez. 2011. Disponível em: <http:// bit.ly/2OjUPeU>. Acesso em: 18 abr. 2018. procedimentos de controle e de vigilância da qualidade da água para consumo humano e seu padrão de potabilidade. Diário Oficial da União, Brasília, DF, 14 dez. 2011. Disponível em: <http:// bit.ly/2OjUPeU>. Acesso em: 18 abr. 2018. substâncias) de acordo com a realidade dos mananciais brasileiros facilita a identificação, o monitoramento e o aprimoramento das tecnologias empregadas nos sistemas de saneamento. Considerações finais 134-144, 2007. HESPANHOL, I. Reúso potável direto e o desafio dos poluentes emergentes. Revista USP, São Paulo, n. 106, p. 79-94, 2015a. Conforme demonstrado, o reúso potável direto pode ser uma solução tanto para a crise hídrica, suprindo a demanda local sem a necessidade de buscar fontes distantes, como para fornecer uma água segura à população. HESPANHOL, I. A inexorabilidade do reúso potável direto. Revista Dae, São Paulo, v. 63, n. 198, p. 63-82, 2015b. HESPANHOL, I. Direct potable reuse in Brazil: a pilot plant study. In: INTERNATIONAL DESALINATION ASSOCIATION WORLD CONGRESS, 2017, São Paulo. Anais… São Paulo: IDA, 2017. p. 1-13. HESPANHOL, I. Direct potable reuse in Brazil: a pilot plant study. In: INTERNATIONAL Considerações finais BRASIL. Ministério das Cidades. Secretaria Nacional de Saneamento Ambiental (Org.). Sistema nacional de informações sobre saneamento: diagnóstico dos serviços de água e esgotos 2016. Brasília, DF, 2018. Disponível em: <http://bit.ly/2sbqOoZ>. Acesso em: 18 abr. 2018. A escassez, a distância e a poluição de mananciais tornam pouco viável a prática do reúso potável indireto. Os sistemas de tratamento tradicionais, tanto para águas residuárias como para água potável, já não são suficientes para promover uma água segura à população. Por isso, é necessário aprimorar as tecnologias capazes de remover contaminantes orgânicos, inorgânicos e organismos patogênicos que os processos comumente utilizados não são. Em paralelo, a fim de atender à realidade dos mananciais, é essencial aprimorar instrumentos legais para monitorar os processos utilizados. BRITTO, A. L.; MAIELLO, A.; QUINTSLR, S. Water supply system in the Rio de Janeiro Metropolitan Region: open issues, contradictions, and challenges for water access in an emerging megacity. Journal of Hydrology, Amsterdam, v. 573, p. 1007-1020, 2019. BRITTO, A. L.; MAIELLO, A.; QUINTSLR, S. Water supply system in the Rio de Janeiro Metropolitan Region: open issues, contradictions, and challenges for water access in an emerging megacity. Journal of Hydrology, Amsterdam, v. 573, p. 1007-1020, 2019. CUNHA, V. D. Estudo para proposta de critérios de qualidade da água para reúso urbano. 2008. Dissertação (Mestrado em Engenharia Hidráulica e Saneamento Ambiental) – Universidade de São Paulo, São Paulo, 2008. Disponível em: <http://bit.ly/2OFjzNm>. Acesso em: 11 jul. 2018. CUNHA, V. D. Estudo para proposta de critérios de qualidade da água para reúso urbano. 2008. Dissertação (Mestrado em Engenharia Hidráulica e Saneamento Ambiental) – Universidade de São Paulo, São Paulo, 2008. Disponível em: <http://bit.ly/2OFjzNm>. Acesso em: 11 jul. 2018. Este estudo demonstra que o saneamento é uma medida de promoção à saúde, através da implantação de sistemas de engenharia, e também é uma medida de prevenção por criar múltiplas barreiras à contaminação, de forma a garantir a segurança da água através de mananciais protegidos, com coleta e tratamento de esgotos, uso e ocupação do solo de forma ordenada e distante dos mananciais utilizados para abastecimento. Estas medidas são recomendadas pela Organização Mundial da Saúde (WHO, 2017), por meio do Plano de Segurança da Água, que abrange questões sobre tecnologias e medidas de conservação hídrica. GIATTI, L. L. Reflexões sobre água de abastecimento e saúde pública: um estudo de caso na Amazônia brasileira. Saúde e Sociedade, São Paulo, v. 16, n. 1, p. Processos oxidativos avançados Utilizados em efluentes que contenham traços de compostos orgânicos naturais ou sintéticos, os processos oxidativos avançados (POA) são responsáveis pela geração do radical livre hidroxila (OHº), um oxidante forte com capacidade de oxidar compostos não afetados por oxidantes convencionais, como oxigênio, ozônio e cloro, reduzindo a toxicidade por mineralizar completamente os contaminantes. Neste estudo, além das variáveis recomendadas pela Portaria nº 2.914/2011, foram realizadas análises de toxicidade, mutagenicidade, vírus e hormônios, de forma a caracterizar melhor o efluente final da estação de reúso. Os resultados indicaram toxicidade na maioria das amostras após a cloração, mas não no arranjo com OR/POA/PD. Em relação às análises de mutagenicidade, não foi indicado nenhum potencial mutagênico nas amostras analisadas, como também não foram encontrados vírus entéricos. Os sistemas são estudados com base nas características do efluente a ser tratado, e, dependendo da qualidade deste, ainda se faz necessário empregar tecnologias avançadas na ETA, como membranas ou POA. Desta forma, após a caracterização do efluente é importante implantar uma estação piloto para identificar quaisquer problemas no processo e subsidiar o projeto a ser desenvolvido, tomando parâmetros de projetos reais e avaliando os custos associados. Isto demonstra uma defasagem nos padrões utilizados no país, em função da grande variabilidade de compostos existentes, sendo necessário que a legislação contemple um maior grupo de contaminantes. A lei não deve utilizar os parâmetros de forma pontual, o que torna o processo complexo, exigindo a análise de muitos parâmetros individualmente. É importante que os dispositivos legais recorram a parâmetros que identifiquem grupos de contaminantes e seus efeitos quando substâncias estão em sinergia no meio, facilitando a identificação destes grupos e abrangendo novos contaminantes. Desta forma, utilizar variáveis de controle (indicadores que abrangem diversas Uma grande barreira à implantação de sistemas avançados no país tem sido de caráter econômico. Atualmente, pensa-se no custo da tecnologia a ser empregada e não no da produção de uma água segura. Segundo estudo de Mierzwa (2009), apesar de os sistemas convencionais custarem muito menos que um processo de tratamento com membranas, quando a qualidade da água de um manancial está comprometida e é necessário aprimorar o sistema convencional (por exemplo, com carvão ativado) esses custos chegam a se equiparar. Conforme exposto, a segurança da prática e a escassez hídrica global fizeram o reúso potável direto procedimentos de controle e de vigilância da qualidade da água para consumo humano e seu padrão de potabilidade. Referências DESALINATION ASSOCIATION WORLD CONGRESS, 2017, São Paulo. Anais… São Paulo: IDA, 2017. p. 1-13. BRASIL. Ministério da Saúde. Departamento de Informática do Sistema Único de Saúde (Org.). Cadernos de informação de saúde. Brasília, DF, 2009. Disponível em: <http://bit.ly/348GrLM>. Acesso em: 30 jul. 2018. BRASIL. Ministério da Saúde. Departamento de Informática do Sistema Único de Saúde (Org.). Cadernos de informação de saúde. Brasília, DF, 2009. Disponível em: <http://bit.ly/348GrLM>. Acesso em: 30 jul. 2018. IBGE – INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Pesquisa nacional de saneamento básico. Rio de Janeiro, 2008. Disponível em: <http://bit.ly/2D4UUN3>. Acesso em: 18 abr. 2018. IBGE – INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Pesquisa nacional de saneamento básico. Rio de Janeiro, 2008. BRASIL. Ministério da Saúde. Portaria nº 2.914, de 12 de dezembro de 2011. Dispõe sobre os Disponível em: <http://bit.ly/2D4UUN3>. Acesso em: 18 abr. 2018. Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 7 jul. 2018. Disponível em: <https://glo.bo/34eC8i3>. Acesso em: 21 ago. 2018. MIERZWA, J. C. Desafio para o tratamento de água de abastecimento e o potencial de aplicação do processo de ultrafiltração. 2009. Tese (Livre- Docência em Engenharia Hidráulica e Sanitária) – Universidade de São Paulo, São Paulo, 2009. SIMÕES, C. P. P. Avaliação operacional e remoção de bisfenol-a no tratamento de água por diferentes tipos de membranas: avaliação em escala piloto. 2016. Dissertação (Mestrado em Tecnologia Ambiental e Recursos Hídricos) – Universidade de Brasília, Brasília, DF, 2016. Disponível em: <http://bit.ly/2DaSn3G>. Acesso em: 13 maio 2017. MIERZWA, J. C. Processos de separação por membranas de água e efluentes. São Paulo: Vídeo, 2017. 48 slides, color. REZENDE, C. C. S. Reúso potável de esgoto sanitário: possibilidades e riscos. 2010. REZENDE, C. C. S. Reúso potável de esgoto sanitário: possibilidades e riscos. 2010. Dissertação (Mestrado em Ciências) – Universidade de São Paulo, São Carlos, 2010. Disponível em: <http://bit.ly/35rRD6o>. Acesso em: 18 jun. 2018. SOUZA, C. M. N. Relação saneamento-saúde- ambiente: os discursos preventivista e da promoção da saúde. Saúde e Sociedade, São Paulo, v. 16, n. 3, p. 125-137, 2007. Dissertação (Mestrado em Ciências) – Universidade de São Paulo, São Carlos, 2010. Disponível em: <http://bit.ly/35rRD6o>. Acesso em: 18 jun. 2018. TRAN, Q. K.; JASSBY, D.; SCHWABE, K. A. The implications of drought and water conservation on the reuse of municipal wastewater: recognizing impacts and identifying mitigation possibilities. Water Research, Amsterdam, v. 124, p. 472-481, 2017. RODRIGUES, C.; VILLELA, F. N. J. Referências Disponibilidade e escassez de água na Grande São Paulo: elementos-chave para compreender a origem da atual crise de abastecimento. Geousp, São Paulo, v. 19, n. 3, p. 399-421, 2016. WHO – WORLD HEALTH ORGANIZATION. Guidelines for drinking water quality. 4. ed. Genebra, 2017. SANTANA, F. São Paulo e as cidades do Cabo e do México vivem uma grave crise hídrica. G1, [s.l.], 22 SANTANA, F. São Paulo e as cidades do Cabo e do México vivem uma grave crise hídrica. G1, [s.l.], 22 Contribuição dos autores Contribuição dos autores Cruz concebeu o estudo e redigiu o artigo. Mierzwa orientou e revisou o artigo. Contribuição dos autores Cruz concebeu o estudo e redigiu o artigo. Mierzwa orientou e revisou o artigo. Recebido: 12/03/2019 Aprovado: 26/09/2019 Recebido: 12/03/2019 Aprovado: 26/09/2019 Recebido: 12/03/2019 Aprovado: 26/09/2019 Aprovado: 26/09/2019 Saúde Soc. São Paulo, v.29, n.1, e180824, 2020 8
https://openalex.org/W3209979741	https://www.dora.lib4ri.ch/eawag/islandora/object/eawag%3A23731/datastream/PDF/Schelbert-2021-Quality_determinants_of_shared_sanitation-%28published_version%29.pdf	English	null	Quality determinants of shared sanitation facilities in low-income urban settlements	null	2,021	cc-by-sa	4,053	QUISS staff on a site visit to inspect shared sanitation facilities in the low-income area of Manyatta, located in the city of Kisumu, Kenya (Image: Sandec) nspect shared sanitation facilities in the low-income area of Manyatta, located in the city of Kisumu, Kenya (Image: Sandec QUISS staff on a site visit to inspect shared sanitation facilities in the low-income area of Manyatta, located in the city Quality determinants of shared sanitation facilities in low- income urban settlements Vasco Schelbert1, Dario Meili2, Sheillah Simiyu3, Mahbub-Ul Alam4, Prince Antwi-Agyei5, Christoph Lüthi1 corresponding author: vasco.schelbert@eawag.ch 1Eawag – Swiss Federal Institute of Aquatic Science and Technology, Department Sanitation, Water and Solid Waste for Development, Überlandstrasse 133, 8600 Dübendorf, Switzerland; 2ETH Zürich, Nadel Center for Development and Cooperation, Clausiusstrasse 37, 8092 Zürich, Switzerland; 3African Population and Health Research Center, P.O Box 10787-00100, Nairobi, Kenya; 4Environmental Interventions Unit, Infectious Disease Division, icddrb, 68, Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka-1212, Bangladesh; 5Department of Civil and Environmental Engineering, University of Energy and Natural Resources (UENR), Regional Centre of Energy and Environmental Sustainability (RCEES), P.O. Box 214, Sunyani BS0061, Ghana THREE POLICY RECOMMENDATIONS:  Acknowledge shared sanitation as an adequate (intermediate) sanitation solution and include shared sanitation as a basic sanitation solution for low-income urban settings provided quality standards are met.  Acknowledge shared sanitation as an adequate (intermediate) sanitation solution and include shared sanitation as a basic sanitation solution for low-income urban settings provided quality standards are met. OVERVIEW AND APPROACH QUISS project results include quality indicators from a large- scale quantitative assessment as well as qualitatively evaluated criteria from a user perspective, including gender differences. In 2019, 17 focus group discussions, a survey of 3,600 households and 2,026 observational spot-checks of SSF and private household toilets were completed in Ghana, Kenya and Bangladesh. A detailed description of QUISS is presented in (Schelbert et al., 2020), and (Schelbert et al., 2021). Country respective findings and recommendations can be found in Alam et al., (2021), Simiyu et al., (2021) and Antwi-Agyei et al. (2021). EXECUTIVE SUMMARY QUISS staff on a site visit to inspect shared sanitation facilities in Bhasantek, a low-income area located in the city of Dhaka, Bangladesh (Image: Sandec). Shared sanitation has immensely contributed to sanitation access in urban areas. However, due to the lack of quality standards within Sustainable Development Goal #6 and the WHO/UNICEF Joint Monitoring Programme (JMP), which evaluates progress on the SDGs, shared sanitation is at best considered a ‘limited’ solution. This policy brief presents the main results of the Quality Indicators of Shared Sanitation (QUISS) project: a three-country comparative mixed- methods study that identifies the key criteria of what constitutes ‘acceptable quality’ shared toilets in low- income urban contexts and provides recommendations for strengthening the acceptability, functionality and sustainability of shared sanitation facilities. BACKGROUND LAST VERSION In recent years, shared sanitation facilities (SSF) have substantially contributed to sanitation access in low-income urban areas. The global percentage of users has increased from 5.4% in 2000 to 8.3% in 2017 (WHO, 2020). However, while SSF are often the only viable option in densely populated low-income urban areas, within Sustainable Development Goal (SDG) #6 and the WHO/UNICEF Joint Monitoring Programme (JMP), which evaluates progress on the SDGs, they are only considered to be a ‘limited’ solution (WHO, 2018a). Depending on how excreta are managed, improved sanitation facilities, which are those designed to hygienically separate excreta from human contact, are divided into three categories: limited, basic, and safely managed (JMP sanitation service levels). Private household toilets are categorised as either basic or safely managed services. In contrast, SSF are at best classified as a limited solution because they are shared by more than one household – irrespective of use and how the excreta are managed. The reason is that ‘[it is] extremely difficult – for global monitoring purposes – to differentiate between shared toilets that are hygienic, accessible and safe, and the more common ones, which are poorly designed and managed’ (Evans et al., 2017). To address this shortcoming, QUISS identified key criteria of what constitutes ‘acceptable quality’ of SSF in urban low-income contexts, using a mixed- methods approach. QUISS staff on a site visit to inspect shared sanitation facilities in Bhasantek, a low-income area located in the city of Dhaka, Bangladesh (Image: Sandec). Reviewed and edited by: Ashley Webb and William Hickey Received: 28 June 2021  Establish and apply (contextualised) quality indicators tailored to shared sanitation facilities (SSF) and enable monitoring to ensure user acceptance and support the success of sanitation interventions to improve public health. Citation: Schelbert, V. et al. (2021) 'Quality determinants of shared sanitation facilities in low- income urban settlements' Water Science Policy. doi: https://dx.doi.org/10.53014/HZGG5241 Citation: Schelbert, V. et al. (2021) 'Quality determinants of shared sanitation facilities in low- income urban settlements' Water Science Policy. doi: https://dx.doi.org/10.53014/HZGG5241  Adopt minimal quality standards for ‘basic’ shared sanitation facilities including the following factors: (i) the toilet technology using flush or pour-flush toilet technology where water is available and, if not available, construct improved toilets; (ii) a maximum number of three households per facility; (iii) good accessibility (toilet located inside dwelling/inside compound/on plot, no restrictions of use, eg reported use 24/7, including at night); (iv) safety/security: solid floor and superstructure without cracks/holes, and functional lighting; (v) availability of gender-separate toilets and lockable/functional doors; (vi) acceptable cleanliness (eg no solid waste, no visible faeces, bloodstains, sputum, no insects); and (vii) functional handwashing stations (soap and water). Publisher’s Note: Water Science Policy stays neutral with regards to jurisdictional claims in published maps and institutional affiliations. Publisher’s Note: Water Science Policy stays neutral with regards to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution- ShareAlike 4.0 International (CC BY-SA 4.0). Indicators for assessing and monitoring SSF quality In the second phase, the study investigated the current quality standards and monitoring guidelines (JMP sanitation service levels) to ascertain whether they adequately reflect the situation on the ground and if other/additional informative indicators could enhance the usefulness of urban sanitation quality assessments, particularly concerning SSF. A survey of 3,600 HHs and 2,026 observational spot-checks of SSF and private household toilets were done using a combination of systematic and purposive sampling (Meili et al., 2021). Collecting gender-specific quality criteria for adequate shared sanitation from a user perspective with residents of Adam-Ali Tek, a low-income urban area of Dhaka, Bangladesh (Images: Sandec). To identify potential indicators to measure urban sanitation quality, the Sanitation Quality Index (SQI) was developed. It comprises three quality dimensions: the hygiene, safety, and privacy of sanitation facilities. 1 Variables for each quality dimension were identified based on user priorities (Schelbert et al., 2020) and the WHO guidelines on sanitation and health (WHO, 2018b). The SQI only contains observable indicators due to validity and reliability concerns of reported household data (Meili et al., 2021). The empirical approach followed three steps. First, the SQI was aggregated based on the three dimensions using eight variables. Second, the relationship between the SQI as a proxy for toilet quality, currently used sanitation indicators (eg technology and sharing), and additional variables2 were analysed using regression analysis. Third, the findings were Results are presented according to the two research phases and respective findings. 1 Hygiene [no solid waste inside the cubicle; no visible faeces in or around the manhole/pan; no insects inside the cubicle, handwashing facility with soap; not clogged in the case of a flush toilet or full in the case of a pit latrine]. Safety [solid roof (without holes): The roof protects the user from external (environmental) factors such as rain; solid floor (without cracks/holes): The floor separates the user from excreta and is, therefore, a gatekeeper for health hazards through both direct contact and indirect contact, eg insects]. User perspectives on acceptable sanitation and quality criteria We must consider users and their perspectives on sanitation priorities in order to meet their needs with public investments, ensure user acceptance, and achieve the success of sanitation interventions. To evaluate user perspectives, in a first phase, we used a qualitative approach and analysed 17 focus group discussions, eight of which were women-only, five were mixed and four were men-only. Each had eight to twelve participants between 18 and 65 years of age (Schelbert et al., 2020). incorporated into the current JMP framework to determine the implications of new quality indicators for assessing sanitation service levels. In the first evaluation step, users themselves determined the criteria for ‘acceptable quality’ of adequate sanitation. User quality criteria are those aspects that affect the user’s experience in a positive or negative way when using or avoiding SSF. This includes factors that make an experience (in-)convenient or (dis-)comforting or lead to adverse or beneficial health or well-being effects. User quality criteria are factors that users discussed in at least two types of focus group discussions (women-only, mixed, or men-only) in every country. In the second step, we evaluated gender differences regarding the user quality criteria, since women and men have different hygiene practices and needs. DISCUSSION Collecting gender-specific quality criteria for adequate shared sanitation from a user perspective with residents of Adam-Ali Tek, a low-income urban area of Dhaka, Bangladesh (Images: Sandec). p Safety [solid roof (without holes): The roof protects the user from external (environmental) factors such as rain; solid floor (without cracks/holes): The floor separates the user from excreta and is, therefore, a gatekeeper for health hazards through both direct contact and indirect contact, eg insects]. 1 Hygiene [no solid waste inside the cubicle; no visible faeces in or around the manhole/pan; no insects inside the cubicle, handwashing facility with soap; not clogged in the case of a flush toilet or full in the case of a pit latrine]. Indicators for assessing and monitoring SSF quality Regarding the quantitative assessment, descriptive statistics from the household survey and the toilet spot- checks reveal that the majority of the toilets observed (65%) were improved, of which 37% were connected to a sewer/septic tank and 63% were improved pit latrines (with slab). Among the households interviewed, 93% shared their toilet with at least one other household. The majority (96%) of the toilet facilities were located on the compound, and most had solid walls (89%), roofs (79%) and floors (91%) without holes. Although two thirds had an improved water source onsite (68%), only 11% of the toilets had a handwashing facility with soap available. Over two-thirds (67%) had a door that was lockable from the inside and outside, and 40% of the facilities provided functional lighting. Conversely, only 3% of the toilets were gender- separated. There were resident landlords on half of the compounds (52%). Less than half (43%) of the respondents reported that there was a cleaning arrangement in place. Based on observable indicators, only 41% were categorised as clean (no visible faeces, no insects, and no solid waste). Overall, the users prioritised water availability and cleanliness. These two are closely related; users perceive water availability (in close proximity) as essential to keeping the facility clean (Schelbert et al., 2020). With water being immediately available for flush WCs, participants expect cleanliness to increase with concomitant beneficial effects, such as decreasing odour/smell and vermin (Rheinländer et al., 2013). Further, users prioritise lighting, lockable/functional door and tiling for reasons of cleanliness, privacy and/or safety/security (Cardone et al., 2018; Simiyu et al., 2017; Tidwell et al., 2018). Users prioritise handwashing stations for personal hygiene motives (Tidwell et al., 2019). Regarding gender particularities, women expressed a higher concern for almost all the quality criteria, except for two (flush technology and tiling). Women prioritised lighting and a lockable/functional door for privacy and safety/security reasons. Men prioritised lighting and tiling for cleanliness reasons because tiles are easy to (keep) clean. Regarding gender-separated toilets, women prefer these for privacy, whereas men for cleanliness reasons. Women feel safer having a private toilet cubicle reserved for them, while men complained about visible bloodstains. Both motives indicate inadequate menstrual health management provisions. This includes a lack of or inadequate personal hygiene facilities for women, leading to unhygienic and potentially humiliating conditions (Hueso et al., 2018; Mitlin, 2011). User perspectives including a gender-lens on acceptable sanitation and quality criteria According to the designated evaluation criteria from the focus group discussions, we identified nine user quality priorities. In descending priority based on their score, these are: 1. Water availability in close proximity 2. Cleanliness 3. A gender-separated toilet Privacy [solid wall: The wall must be of solid material and have no holes that would allow a person to peek through]. 2 The additional variables were: the toilet’s location, water on the premises, a handwashing facility with soap, functional lighting, a lockable door, floor tiling, gender-separate cubicles, a cleaning arrangement, the degree of user relationship, the toilet’s age, the landlord living on the same plot, and a bin inside the toilet cubicle. Privacy [solid wall: The wall must be of solid material and have no holes that would allow a person to peek through]. 4. Flush Water Closet (WC) (pour/cistern flush) 4. Flush Water Closet (WC) (pour/cistern flush) 5. Lighting 6. A lockable/functional door 7. Tiling 8. A handwashing station 9. Privacy 4. Flush Water Closet (WC) (pour/cistern flush) 5. Lighting 6. A lockable/functional door 7. Tiling 8. A handwashing station 9. Privacy 4. Flush Water Closet (WC) (p 5. Lighting 6. A lockable/functional door 7. Tiling 8. A handwashing station 9. Privacy develop sooner than men (Kwiringira et al., 2014; Simiyu, 2015). develop sooner than men (Kwiringira et al., 2014; Simiyu, 2015). REFERENCES (EDITED BY AUTHOR, INCL. URL)  ALAM, M.-U., SCHELBERT, V., MEILI, D., FERDOUS, S., & LÜTHI, C. (2021). SHARED SANITATION IN LOW-INCOME URBAN SETTLEMENTS IN BANGLADESH. RESEARCH FOR POLICY, EAWAG, SWITZERLAND. HTTPS://WWW.EAWAG.CH/FILEADMIN/DOMAIN1/ABTEILUNG EN/SANDEC/SCHWERPUNKTE/SESP/QUISS/QUISS_POLICY_B RIEF_BANGLADESH.PDF These indicators should then be applied to distinguish between adequate (defined as available and accessible, safe and secure, private and hygienic) and non-adequate SSF in low-income urban settings as part of the efforts to distinguish between “basic” and “limited” SSF. Indicators for assessing and monitoring SSF quality In this sense, gender separated toilets are linked via menstrual health management to privacy and cleanliness. Regression analysis tested the relationship between the observable SQI and the quality indicators. The results suggest that relying on improved technologies (ie at least a pit latrine with slab) as a single distinguishing indicator for toilet quality is inadequate in urban settings – even for private household toilets. SQI scores of pit latrines with slab are significantly lower compared to flush toilets, even though WHO JMP considers both types improved technologies (WHO, 2018b). Therefore, classifying pit latrines with a slab as unimproved sanitation improves the prediction of sanitation quality as defined by the SQI (Meili et al., 2021). Regarding sharing, toilets shared by two to three HHs are mostly cleaner, safer and more private than toilets shared by four or more HHs, which is similar to the findings of Günther et al. (2012). However, the results vary considerably across countries (Alam et al., 2021; Antwi- Agyei et al., 2021; Simiyu et al. 2021). Other strongly significant indicators that correlated with quality included the toilet’s location, lighting, and a lockable door (from the inside and outside). Further, the presence of a cleaning list and floor tiling display a moderate positive correlation. The availability of water on the premises, gender-separate cubicles, the sharing users’ relationships to one another, Overall, users mention the insufficient number of toilets to lead to queuing and increased waiting times (Hutton, 2015), which impedes toilet availability and has adverse effects on privacy, safety/security, and cleanliness. Long waiting times also occur where toilets simultaneously serve as a shower/bathing area and as places for women to manage their menstrual health. Gender-separate toilets and – where possible – designated shower/bathing areas could enhance user privacy and increase toilet availability. Inadequate conditions force users to develop coping mechanisms (eg accompanying at night), which women Use a range of technical solutions, designed and implemented through an adaptive and incremental approach with full consideration of the entire sanitation service chain and integration of complementary urban services. These services include water supply, drainage, greywater management, and solid waste management. Develop legislative and regulative frameworks with policies guiding sanitation at the national level, which feed into the policies at the district or county government level. Plan with secure budgets for both capital and operational expenses and allocate funding for non- infrastructural aspects of service delivery, such as capacity building, household engagement and outreach, and sanitation marketing.  Technology: Flush or pour-flush toilet technology where water is available or improved toilet technology where water is not available;  Numbers of users: Up to three households per facility;  Accessibility/Availability: Toilet located inside dwelling/inside compound/on plot, 24/7 access;  Safety/Security: Solid floor and superstructure without cracks/holes with functional lighting;  Adequate privacy: Availability of gender-separate toilets (whenever multiple cubicles are feasible/available) and lockable/functional doors;  Acceptable Cleanliness: No solid waste, no visible faeces/blood stains/sputum, no insects and  Functional handwashing stations: soap and water. the toilet facility’s age, and a landlord living on the same plot did not significantly correlate with toilet quality. 2. Establish government support and develop national guidelines and bylaws that embrace essential quality standards and indicators for adequate SSF. Clearly define and assign the roles and responsibilities of state and non-state actors to enhance collaboration, including financing with strategic direction for sharing capital and operational costs among poor and non- poor users and enforcing mechanisms to promote and enforce standards. 2. Establish government support and develop national guidelines and bylaws that embrace essential quality standards and indicators for adequate SSF. Clearly define and assign the roles and responsibilities of state and non-state actors to enhance collaboration, including financing with strategic direction for sharing capital and operational costs among poor and non- poor users and enforcing mechanisms to promote and enforce standards. QUISS showed that the current JMP sanitation service levels for SSF, which are exclusively based on (improved) technology and sharing, provide insufficient information regarding sanitation quality. These service levels should be revised and new indicators for determining adequate quality established. According to our findings, quality standards for shared sanitation refer to facilities with the following characteristics: QUISS showed that the current JMP sanitation service levels for SSF, which are exclusively based on (improved) technology and sharing, provide insufficient information regarding sanitation quality. These service levels should be revised and new indicators for determining adequate quality established. According to our findings, quality standards for shared sanitation refer to facilities with the following characteristics: 3. Establish an enabling environment for adequate sanitation service delivery. Use a range of technical solutions, designed and implemented through an adaptive and incremental approach with full consideration of the entire sanitation service chain and integration of complementary urban services. These services include water supply, drainage, greywater management, and solid waste management. Develop legislative and regulative frameworks with policies guiding sanitation at the national level, which feed into the policies at the district or county government level. Plan with secure budgets for both capital and operational expenses and allocate funding for non- infrastructural aspects of service delivery, such as capacity building, household engagement and outreach, and sanitation marketing. 3. Establish an enabling environment for adequate sanitation service delivery. THREE MAIN RECOMMENDATIONS FOR POLICY REFORM  ANTWI-AGYEI, P., DWUMFOUR-ASARE, B., ADJEI, K. A., SCHELBERT, V., MEILI, D., & LÜTHI, C. (2021). SHARED SANITATION IN LOW-INCOME URBAN SETTLEMENTS IN GHANA. RESEARCH FOR POLICY, EAWAG, SWITZERLAND. HTTPS://WWW.EAWAG.CH/FILEADMIN/DOMAIN1/ABTEILUNG EN/SANDEC/SCHWERPUNKTE/SESP/QUISS/QUISS_POLICY_B RIEF_GHANA.PDF 1. Foster global acknowledgement of SSF as a basic sanitation solution by major sector players for low- income urban settings, provided quality standards are met. The exclusion of SSF from the WHO/UNICEF JMP framework as a basic sanitation solution might produce unintended incentives to donor agencies and NGOs not to improve such facilities. Setting goals, identifying targets and developing monitoring frameworks have far-reaching effects. Good quality monitoring and benchmarking can often create strong incentives to improve performance.  CARDONE, R., SCHRECONGOST, A., & GILSDORF, R. (2018). SHARED AND PUBLIC TOILETS: CHAMPIONING DELIVERY MODELS THAT WORK. IN: WORLD BANK. HTTPS://OPENKNOWLEDGE.WORLDBANK.ORG/BITSTREAM/HAN DLE/10986/30296/W18035.PDF  EVANS, B., HUESO, A., JOHNSTON, R., NORMAN, G., PÉREZ, E., SLAYMAKER, T., & TRÉMOLET, S. (2017). LIMITED SERVICES? THE ROLE OF SHARED SANITATION IN THE 2030 AGENDA FOR SUSTAINABLE DEVELOPMENT. JOURNAL OF WATER, SANITATION AND HYGIENE FOR DEVELOPMENT, 7(3), 349-351. HTTPS://DOI.ORG/10.2166/WASHDEV.2017.023  SIMIYU, S. (2015). SOCIO-ECONOMIC DYNAMICS IN SLUMS AND IMPLICATIONS FOR SANITATION SUSTAINABILITY IN KISUMU, KENYA. DEVELOPMENT IN PRACTICE, 25(7), 986-996. HTTPS://DOI.ORG/10.1080/09614524.2015.1073223  SIMIYU, S. (2015). SOCIO-ECONOMIC DYNAMICS IN SLUMS AND IMPLICATIONS FOR SANITATION SUSTAINABILITY IN KISUMU, KENYA. DEVELOPMENT IN PRACTICE, 25(7), 986-996. HTTPS://DOI.ORG/10.1080/09614524.2015.1073223  GÜNTHER, I., NIWAGABA, C., LÜTHI, C., HORST, A., MOSLER, H.-J., & TUMWEBAZE, I. K. (2012). WHEN IS SHARED SANITATION IMPROVED SANITATION. THE CORRELATION BETWEEN NUMBER OF USERS AND TOILET HYGIENE. RESEARCH FOR POLICY, EAWAG, SWITZERLAND. HTTPS://MPRA.UB.UNI- MUENCHEN.DE/45830/1/MPRA_PAPER_45830.PDF  SIMIYU, S., SWILLING, M., CAIRNCROSS, S., & RHEINGANS, R. (2017). DETERMINANTS OF QUALITY OF SHARED SANITATION FACILITIES IN INFORMAL SETTLEMENTS: CASE STUDY OF KISUMU, KENYA. BMC PUBLIC HEALTH, 17(1), 68. HTTPS://RESEARCHONLINE.LSHTM.AC.UK/ID/EPRINT/3348539  HUESO, A., MALHOTRA, M., SHIPRA, P., & NATH, P. (2018). FEMALE-FRIENDLY PUBLIC AND COMMUNITY TOILETS: A GUIDE FOR PLANNERS AND DECISION MAKERS. IN (PP. 56). NEW YORK, NY; LONDON, UK: UNICEF, WSUP, WATERAID. HTTPS://WASHMATTERS.WATERAID.ORG/SITES/G/FILES/JKXOO F256/FILES/FEMALE-FRIENDLY-PUBLIC-AND-COMMUNITY- TOILETS-A-GUIDE.PDF  SIMIYU, S., SCHELBERT, V., MEILI, D., & LÜTHI, C. (2021). SHARED SANITATION IN LOW-INCOME URBAN SETTLEMENTS IN KENYA. RESEARCH FOR POLICY, EAWAG, SWITZERLAND. HTTPS://WWW.EAWAG.CH/FILEADMIN/DOMAIN1/ABTEILUNG EN/SANDEC/SCHWERPUNKTE/SESP/QUISS/QUISS_POLICY_B RIEF_KENYA.PDF  TIDWELL, J. B., CHIPUNGU, J., CHILENGI, R., & AUNGER, R. (2018). ASSESSING PERI-URBAN SANITATION QUALITY USING A THEORETICALLY DERIVED COMPOSITE MEASURE IN LUSAKA, ZAMBIA. JOURNAL OF WATER, SANITATION AND HYGIENE FOR DEVELOPMENT, 8(4), 668-678. HTTPS://DOI.ORG/10.2166/WASHDEV.2018.029  HUTTON, G. (2015). BENEFITS AND COSTS OF THE WATER SANITATION AND HYGIENE TARGETS FOR THE POST-2015 DEVELOPMENT AGENDA: COPENHAGEN CONSENSUS CENTER. HTTP://WWW.COPENHAGENCONSENSUS.COM/SITES/DEFAULT/ FILES/WATER_SANITATION_ASSESSMENT_-_HUTTON.PDF  KWIRINGIRA, J., ATEKYEREZA, P., NIWAGABA, C., & GÜNTHER, I. (2014). GENDER VARIATIONS IN ACCESS, CHOICE TO USE AND CLEANING OF SHARED LATRINES; EXPERIENCES FROM KAMPALA SLUMS, UGANDA. BMC PUBLIC HEALTH, 14(1), 1180. HTTPS://DOI.ORG/10.1186/1471-2458-14-1180  TIDWELL, J. B., CHIPUNGU, J., CHILENGI, R., CURTIS, V., & AUNGER, R. (2019). THEORY-DRIVEN FORMATIVE RESEARCH ON ON-SITE, SHARED SANITATION QUALITY IMPROVEMENT AMONG LANDLORDS AND TENANTS IN PERI-URBAN LUSAKA, ZAMBIA. INTERNATIONAL JOURNAL OF ENVIRONMENTAL HEALTH RESEARCH, 29(3), 312-325. HTTPS://DOI.ORG/10.1080/09603123.2018.1543798  MEILI, D., SCHELBERT, V., ALAM, M.-U., SIMIYU, S., ANTWI- AGYEI, P., ADJEI, K. A., DWUMFOUR-ASARE, B., RAHMAN, M., FERDOUS, S., SARKER, S., GÜNTHER, I., LÜTHI, C. (2021). INDICATORS FOR SANITATION QUALITY IN LOW-INCOME URBAN SETTLEMENTS: EVIDENCE FROM KENYA, GHANA, AND BANGLADESH. (SUBMITTED TO SOCIAL INDICATORS RESEARCH).  WHO. (2018A). CORE QUESTIONS ON DRINKING WATER, SANITATION AND HYGIENE FOR HOUSEHOLD SURVEYS: 2018 UPDATE. WORLD HEALTH ORGANIZATION, GENEVA, SWITZERLAND. HTTPS://WASHDATA.ORG/REPORT/JMP-2018- CORE-QUESTIONS-HOUSEHOLD-SURVEYS  MITLIN, D. (2011). PATHFINDER PAPER: EQUITY AND GENDER 52.HTTPS://ASSETS.PUBLISHING.SERVICE.GOV.UK/MEDIA/57A 08AEEED915D3CFD000A10/SHAREPATHFINDEREQUITYAND GENDER_FINAL_AUGUST2011.PDF  WHO. (2018B). AND HYGIENE FOR DEVELOPMENT, 7(3), 349-351. HTTPS://DOI.ORG/10.2166/WASHDEV.2017.023 GUIDELINES ON SANITATION AND HEALTH. WORLD HEALTH ORGANIZATION, GENEVA, SWITZERLAND. HTTPS://APPS.WHO.INT/IRIS/BITSTREAM/HANDLE/10665/27 4939/9789241514705-ENG.PDF  RHEINLÄNDER, T., KERAITA, B., KONRADSEN, F., SAMUELSEN, H., & DALSGAARD, A. (2013). SMELL: AN OVERLOOKED FACTOR IN SANITATION PROMOTION. WATERLINES, 32(2), 106-112. HTTPS://WWW.JSTOR.ORG/STABLE/24686882  WHO. (2020). WHO/UNICEF JMP GLOBAL WASH DATABASE. WORLD HEALTH ORGANIZATION, GENEVA, SWITZERLAND. AVAILABLE FROM: HTTPS://WASHDATA.ORG/DATA/HOUSEHOLD#!/DASHBOARD/ NEW  WHO. (2020). WHO/UNICEF JMP GLOBAL WASH DATABASE. WORLD HEALTH ORGANIZATION, GENEVA, SWITZERLAND. AVAILABLE FROM: HTTPS://WASHDATA.ORG/DATA/HOUSEHOLD#!/DASHBOARD/ NEW  SCHELBERT, V., MEILI, D., ALAM, M.-U., SIMIYU, S., ANTWI- AGYEI, P., ADJEI, K. A., DWUMFOUR-ASARE, B., RAHMAN, M., FERDOUS, S., SARKER, S., GÜNTHER, I., LÜTHI, C. (2020). WHEN IS SHARED SANITATION ACCEPTABLE IN LOW-INCOME URBAN SETTLEMENTS? A USER PERSPECTIVE ON SHARED SANITATION QUALITY IN KUMASI, KISUMU AND DHAKA. JOURNAL OF WATER, SANITATION AND HYGIENE FOR DEVELOPMENT, 10(4), 959-968. HTTPS://DOI.ORG/10.2166/WASHDEV.2020.084  SCHELBERT, V., MEILI, D., ALAM, M.-U., SIMIYU, S., ANTWI- AGYEI, P., & LÜTHI, C. (2021). SHARED SANITATION IN LOW- INCOME URBAN SETTLEMENTS. EVIDENCE FROM GHANA, KENYA AND BANGLADESH. RESEARCH FOR POLICY, EAWAG, SWITZERLAND. HTTPS://WWW.EAWAG.CH/FILEADMIN/DOMAIN1/ABTEILUNG EN/SANDEC/SCHWERPUNKTE/SESP/QUISS/QUISS_POLICY_B RIEF_OVERALL_RESULTS.PDF About Water Science Policy  Water Science Policy is a global, multilingual platform for water journalism, thought leadership, engagement, and solutions. We aim to create a world where water is embedded across public policies. Since its launch in 2020, it has worked through a global decentralized network of water enthusiasts. WSP’s goal is to ensure that water- curious readers - regardless of their financial or technical resources - can access thought- provoking analyses to form their own opinions about the biggest challenges the world faces today. WSP’s briefs help inform better decision-making today for a water-secure tomorrow. Join the conversation at http://www.watersciencepolicy.com/ Contact us: contact@watersciencepolicy.com
https://openalex.org/W3006065562	https://www.nature.com/articles/s41598-020-66092-9.pdf	English	null	Cell Atlas of the Human Fovea and Peripheral Retina	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	14,600	OPEN Wenjun Yan1,7, Yi-Rong Peng1,2,7, Tavé van Zyl3,7, Aviv Regev4, Karthik Shekhar1,4,5, Dejan Juric6 & Joshua R. Sanes1 ✉ Most irreversible blindness results from retinal disease. To advance our understanding of the etiology of blinding diseases, we used single-cell RNA-sequencing (scRNA-seq) to analyze the transcriptomes of ~85,000 cells from the fovea and peripheral retina of seven adult human donors. Utilizing computational methods, we identified 58 cell types within 6 classes: photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and non-neuronal cells. Nearly all types are shared between the two retinal regions, but there are notable differences in gene expression and proportions between foveal and peripheral cohorts of shared types. We then used the human retinal atlas to map expression of 636 genes implicated as causes of or risk factors for blinding diseases. Many are expressed in striking cell class-, type-, or region- specific patterns. Finally, we compared gene expression signatures of cell types between human and the cynomolgus macaque monkey, Macaca fascicularis. We show that over 90% of human types correspond transcriptomically to those previously identified in macaque, and that expression of disease-related genes is largely conserved between the two species. These results validate the use of the macaque for modeling blinding disease, and provide a foundation for investigating molecular mechanisms underlying visual processing. The three leading causes of irreversible blindness can be classified as neurodegenerative retinal diseases: age-related macular degeneration, glaucoma and diabetic retinopathy; photoreceptors are lost in age-related macular degeneration and diabetic retinopathy, and retinal ganglion cells (RGCs) are lost in glaucoma1–3. These three groups of diseases affect over 300 million people world-wide, greatly outnumbering those affected by other neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Genetic contributors have been discov- ered for all of these retinal diseases, largely through genome-wide association studies (GWAS)4–6. In few cases, however, do we understand the role of the implicated gene in disease pathogenesis. p g p g One main obstacle to gaining such understanding is lack of knowledge about where the implicated genes are expressed; lacking such information, it is difficult to determine mechanisms by which they affect visual function. Another is that substantial differences in structure and gene expression between human and rodent retina have made it difficult to study these genes in animal models. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 OPEN For example, among mammals, only primates have a fovea, the small central region responsible for high acuity vision as well as most chromatic vision – and the region selectively affected in macular degeneration, diabetic macular edema and hereditary maculopathies7,8. As a first step toward addressing these issues, we recently used high throughput single cell RNA-seq (scRNA-seq) to gen- erate a retinal cell atlas from cynomolgus macaque (Macaca fascicularis), a non-human primate that is closely related to humans and frequently used in preclinical ophthalmological studies9–11. We separately profiled periph- eral retina and the fovea (Fig. 1a). In each region, we characterized the six classes of retinal cells – photoreceptors, horizontal cells, bipolar cells, amacrine cells, RGCs and Müller glial cells (Fig. 1b) and found molecular signatures that divided them into a total of 62 (fovea) or 70 (periphery) cell types12. Here, to extend this work, we used the macaque atlas as a foundation to generate a comprehensive cell atlas of the adult human retina. We expected that the similarity of macaque to human would aid in identifying cell types, 1Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, MA, 02138, USA. 2Department of Ophthalmology, Stein Eye Institute, University of California Los Angeles, Los Angeles, CA, 90095, USA. 3Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear, Boston, MA, 02114, USA. 4Howard Hughes Medical Institute, Koch Institute of Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140; and Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA. 5Department of Chemical and Biomolecular Engineering and Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, 94720, USA. 6Massachusetts General Hospital Cancer Center, Department of Medicine, Harvard Medical School, Boston, USA. 7These authors contributed equally: Wenjun Yan, Yi-Rong Peng and Tavé van Zyl. ✉e-mail: sanesj@mcb.harvard.edu Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 1. Cell classes in human retina. (a) (Top) Sketch of a human eye showing positions of retina and fovea. (Bottom) Sketch of a flat mounted retina showing foveal and peripheral regions. Circle in center is position at which optic nerve exits the eye. OPEN Finally, we mapped the expression of 636 genes implicated in blinding diseases by GWAS studies or as highly penetrant Mendelian mutations underlying a variety of inherited retinal degenerations, each rare but substantial in aggregate. We show that many of the genes queried are selectively expressed in particular retinal cell classes, in particular cell types within a class, or in foveal or peripheral cohorts of shared types. These results provide new insights into mechanisms underlying retinal disease. and this was indeed the case. By analyzing a total of 84,982 single cell transcriptomes, we identified 58 cell types in human fovea and 57 types in peripheral retina, nearly all of which were shared between the two regions. For many of these types, however, we documented substantial regional differences in gene expression and proportions. By comparing human and macaque atlases, we found 1:1 matches for >90% of cell types, supporting the use of Macaca fascicularis as a preclinical model. Finally, we mapped the expression of 636 genes implicated in blinding diseases by GWAS studies or as highly penetrant Mendelian mutations underlying a variety of inherited retinal degenerations, each rare but substantial in aggregate. We show that many of the genes queried are selectively expressed in particular retinal cell classes, in particular cell types within a class, or in foveal or peripheral cohorts of shared types. These results provide new insights into mechanisms underlying retinal disease. OPEN (b) Sketch of a peripheral retinal section showing its major cell classes— photoreceptors (PRs), horizontal cells (HCs), bipolar cells (BCs), amacrine cells (ACs), retinal ganglion cells (RGCs) and Müller glia (MG), outer and inner plexiform (synaptic) layers (OPL and IPL), outer and inner nuclear layers (ONL and INL), and ganglion cell layer (GCL). (c) scRNA-seq workflow. Foveal cells were dissociated from <1.5 mm-diameter punches and collected without further processing. Peripheral cells were dissociated from all four quadrants of peripheral retinas, and depleted of rods (CD73+) or enriched for RGCs (CD90+) with magnetic columns before processing. (d) Expression patterns of class-specific markers (rows) in individual cells (columns). Cells are grouped by their classes (color bars, top). Plot shows data from a maximum Figure 1. Cell classes in human retina. (a) (Top) Sketch of a human eye showing positions of retina and fovea. (Bottom) Sketch of a flat mounted retina showing foveal and peripheral regions. Circle in center is position at which optic nerve exits the eye. (b) Sketch of a peripheral retinal section showing its major cell classes— photoreceptors (PRs), horizontal cells (HCs), bipolar cells (BCs), amacrine cells (ACs), retinal ganglion cells (RGCs) and Müller glia (MG), outer and inner plexiform (synaptic) layers (OPL and IPL), outer and inner nuclear layers (ONL and INL), and ganglion cell layer (GCL). (c) scRNA-seq workflow. Foveal cells were dissociated from <1.5 mm-diameter punches and collected without further processing. Peripheral cells were dissociated from all four quadrants of peripheral retinas, and depleted of rods (CD73+) or enriched for RGCs (CD90+) with magnetic columns before processing. (d) Expression patterns of class-specific markers (rows) in individual cells (columns). Cells are grouped by their classes (color bars, top). Plot shows data from a maximum of 500 randomly selected cells per class. and this was indeed the case. By analyzing a total of 84,982 single cell transcriptomes, we identified 58 cell types in human fovea and 57 types in peripheral retina, nearly all of which were shared between the two regions. For many of these types, however, we documented substantial regional differences in gene expression and proportions. By comparing human and macaque atlases, we found 1:1 matches for >90% of cell types, supporting the use of Macaca fascicularis as a preclinical model. www.nature.com/scientificreports/ Humans and many old world monkeys, such as macaques, are trichro- mats, with three cone types, each expressing a single opsin (S-, M- or L-opsin) tuned to short-, medium- or long-wavelengths, respectively. We found three clear photoreceptor clusters: rods, which selectively express rho- dopsin; S-cones, which selectively express S-opsin; and M and L cones, which selectively express and M or L-opsin (Fig. 2a–c). The inability to distinguish M from L opsin results from their nearly identical coding sequences (98% nucleotide identity), the presence of multiple copies of the M-opsin gene in some individuals, and the high fre- quency of recombination between these two closely linked genes (see Peng et al., 2019 for discussion). The three human photoreceptor types mapped to their macaque counterparts with high confidence (Fig. 2b). Horizontal cells. Most primates, including macaques, have two horizontal cell types, H1 and H215. Based on morphological criteria, Kolb and colleagues argued for a third horizontal cell type in human retina16,17, with H3 differing from H1 in having larger somata and dendritic arbors. We identified two horizontal cell types (Fig. 2d), which corresponded to the macaque H1 and H2 types, respectively (Fig. 2e). Attempts to further subdivide the two types by increasing the sensitivity of the clustering algorithm failed to reveal a third type with a distinguishing molecular signature. Similar to macaque H1 and H2, human H1 and H2 were distinguished from each other by selectively expressing transcription factors LHX1 and ISL1, respectively (Fig. 2f). Bipolar cells. Bipolar cells are divided into three subclasses: ON and OFF cone bipolars, which release neu- rotransmitter in response to increases and decreases in illumination of cones, respectively; and rod bipolars, which generate ON responses to stimulation of rods18,19. In macaques, ON and OFF bipolars are characterized by expression of genes that encode the metabotropic glutamate receptor 6, GRM6, and the kainate-type glutamate receptor, GRIK1, respectively; rod bipolars are distinguished from cone bipolars by expression of PKCα, encoding protein kinase Cα12. These expression patterns were conserved in human bipolars, allowing us to divide 12 bipo- lar clusters into 1 ON rod, 5 ON cone, and 6 OFF cone types (Fig. 2g–i). The counterparts of all 12 macaque types were found in human retina and named based on this correspondence (Fig. 2h). www.nature.com/scientificreports/ www.nature.com/scientificreports/ four quadrants. Foveal samples were dissociated into single cells, which were profiled without further processing using high-throughput droplet sequencing13. For peripheral samples, in which rod photoreceptors and RGC com- prise ~80% and <2% of total cells respectively, we depleted rods using magnetic beads conjugated to anti-CD73 or enriched RGCs using anti-CD90-conjugated beads prior to collection (Fig. 1c), using protocols established in our study on macaque retina12. Libraries were prepared from foveal and peripheral samples, and sequenced. Altogether, we obtained 84,982 high-quality transcriptomes, 55,736 from fovea and 29,246 from peripheral retina. The median number of unique transcripts captured per cell was 2,577 and the median number of genes detected was 1,314 (Table S3). To maximize statistical power, we pooled data from fovea and periphery for initial analysis. Using methods adapted from12, we divided the cells into 9 groups based on expression of canonical markers, which were common to both retinal regions (Fig. 1d). We identified the five neuronal classes (9,070 photoreceptors, 2,868 horizontal cells, 25,908 bipolar cells, 13,607 amacrine cells and 11,404 RGCs) as well as four types of non-neuronal cells: 19,896 Müller glia, 1,149 astrocytes, 671 microglia and 409 vascular endothelial cells. Classification and identification of retinal cell types. We next re-clustered each neuronal class sepa- rately to discriminate cell types. We obtained a total of 54 clusters, each corresponding to a putative cell type or possibly a small group of closely related types: 3 photoreceptor, 2 horizontal cell, 12 bipolar cell, 25 amacrine cell, and 12 RGC types. Thus, including the 4 non-neuronal types, we detected a total of 58 cell types in human retina. Of them, 49 contained cells from at least 6 of the 7 donors (Supplemental Fig. 1), indicating that the heterogene- ity does not result from individual variations or batch effects. We took advantage of the evolutionary proximity between humans and macaques and utilized previously defined macaque retina cell types12 to train a multi-class supervised classification algorithm14. This enabled us to relate most human clusters to macaque types, based on their expression patterns of orthologous genes. Many of the human types were further characterized by assessing their expression of key genes reported previously. Photoreceptors. The two subclasses of photoreceptor cells in vertebrate retinas are rods, specialized for high-sensitivity vision at low light levels, and cones, which mediate chromatic vision. Rods and cones express rhodopsin and cone opsins, respectively. Results Cell classes in human retina. To generate a comprehensive cell atlas of human retina, we obtained eight retinas from seven genetically unrelated human donors with no clinical history of ocular disease (Table S1). We dissected foveal (~1.5 mm diameter centered on the foveal pit, which was visible under a dissecting micro- scope) and peripheral samples (>5 mm from the fovea) from whole retina, pooling peripheral pieces from all Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Notably, the provisionally named “OFFx” type, first identified and named in our analysis of macaque retina, was also present in human retina as a distinct cluster (Fig. 2h,i). Amacrine cells. Most amacrine cells are inhibitory neurons utilizing GABA or glycine as neurotransmitters. By assessing the expression of marker genes for GABAergic (glutamate carboxylase, GAD1 and GAD2) and gly- cinergic (SLC6A9, encoding the high affinity glycine transporter GLYT1) amacrines20, we identified 16 putative GABAergic and 8 putative glycinergic amacrine cell types among a total of 25 types (Fig. 3a,b). One type (C14) expressed none of these three genes at high levels, and might correspond to a non-GABAergic non-Glycinergic (nGnG) type identified in mouse2122 . One of the glycinergic types (C17) also expressed GAD2, raising the pos- sibility that it uses both transmitters. Several known amacrine types were detected based on key marker genes (Fig. 3d), including SLC17A8 for VG3 amacrine (an excitatory type that co-releases glycine and glutamate), SLC18A3 for cholinergic starburst amacrines, TH for catecholaminergic CAI/CAII amacrines, and GJD2 for AII amacrines, which mediate transmission of rod signals to RGCs. Those and many other AC types mapped faith- fully to macaque types (Fig. 3c). Many AC types also expressed neuropeptides (bold in Fig. 3d), with some pre- dominantly in single types (e.g. NPW in C7 and VIP in C24, and others expressed by multiple types (e.g. CARTPT and PENK). In several instances, more than one neuropeptide was detected in the same AC type – for example, thyrotropin-releasing hormone (TRH) and Natriuretic Peptide B (NPPB) in C9, and Proenkephalin (PENK) and Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 2. Photoreceptor, horizontal and bipolar cell types. (a–c). Photoreceptors. (a) Cell clusters visualized using t-distributed stochastic neighbor embedding (t-SNE). Dots represent individual cells and are color coded by their cluster assignments (text labels). (b) Transcriptional correspondence between human and macaque cell types summarized as a “confusion matrix.” In this and subsequent confusion matrices, the color and size of each dot reflect the percentage of cells in a given human cluster (columns) mapped to a corresponding macaque type (rows). (c) Dot plots showing expression of select marker genes. In this and subsequent gene expression plots, dot size represents the percentage of cells in a cluster with non-zero expression of a select gene; and color intensity represents the average expression of a gene within expressing cells. (d–f) Horizontal cells. www.nature.com/scientificreports/ Clusters (d), correspondence to macaque types (e) and expression of key genes (f) shown as in (a–c). (g–i) Bipolar cells. Clusters (g), correspondence to macaque types (h) and expression of key genes (i) shown as in (a–c). igure 2. Photoreceptor, horizontal and bipolar cell types. (a–c). Photoreceptors. (a) Cell clusters visualized Figure 2. Photoreceptor, horizontal and bipolar cell types. (a–c). Photoreceptors. (a) Cell clusters visualized using t-distributed stochastic neighbor embedding (t-SNE). Dots represent individual cells and are color coded by their cluster assignments (text labels). (b) Transcriptional correspondence between human and macaque cell types summarized as a “confusion matrix.” In this and subsequent confusion matrices, the color and size of each dot reflect the percentage of cells in a given human cluster (columns) mapped to a corresponding macaque type (rows). (c) Dot plots showing expression of select marker genes. In this and subsequent gene expression plots, dot size represents the percentage of cells in a cluster with non-zero expression of a select gene; and color intensity represents the average expression of a gene within expressing cells. (d–f) Horizontal cells. Clusters (d), correspondence to macaque types (e) and expression of key genes (f) shown as in (a–c). (g–i) Bipolar cells. Clusters (g), correspondence to macaque types (h) and expression of key genes (i) shown as in (a–c). Figure 2. Photoreceptor, horizontal and bipolar cell types. (a–c). Photoreceptors. (a) Cell clusters visualized using t-distributed stochastic neighbor embedding (t-SNE). Dots represent individual cells and are color coded by their cluster assignments (text labels). (b) Transcriptional correspondence between human and macaque cell types summarized as a “confusion matrix.” In this and subsequent confusion matrices, the color and size of each dot reflect the percentage of cells in a given human cluster (columns) mapped to a corresponding macaque type (rows). (c) Dot plots showing expression of select marker genes. In this and subsequent gene expression plots, dot size represents the percentage of cells in a cluster with non-zero expression of a select gene; and color intensity represents the average expression of a gene within expressing cells. (d–f) Horizontal cells. Clusters (d), correspondence to macaque types (e) and expression of key genes (f) shown as in (a–c). (g–i) Bipolar cells. Clusters (g), correspondence to macaque types (h) and expression of key genes (i) shown as in (a–c). Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Retinal ganglion cells. The predominant ganglion cell types in primate retina are ON and OFF midget RGCs, together accounting for >80% of RGCs in human (by morphological criteria) and macaque retina (by morpho- logical and molecular criteria)12,23. Next most abundant in both species are ON and OFF parasol RGCs, totaling ~10% of all RGCs. Based on abundance, four RGC clusters appeared likely to correspond to these types (Fig. 3e). Mapping to the macaque atlas confirmed their identities (Fig. 3f). The midget and parasol RGCs comprised 86% (44% ON and 42% OFF) and 10% (4% ON and 6% OFF) of all RGCs in our dataset, respectively.hh ( ) ( ) p y The remaining 8 clusters ranged in abundance from 0.1% to 1.6% of all RGCs. They included two types that expressed the transcription factor, FOXP2 (hRGC6 and 7), one of which also expressed FOXP1 (Fig. 3g); these might be related to mouse FoxP2 + FoxP1- and FoxP2 + FoxP1 + F-RGCs24. We also detected two RGC clusters that expressed melanopsin (OPN4), the canonical marker of intrinsically photosensitive RGCs (ipRGCs; hRGC5 and hRGC12; Fig. 3g). Recent morphological and physiological studies have demonstrated 2–4 human ipRGC types25,26. We speculate that hRGC12, which expressed the highest level of OPN4 (Fig. S2) corresponds to M1, which expresses highest levels of OPN4 in mice27; others could be included in hRGC5 or be too rare to detect. Non-neuronal cells. Four clusters of non-neuronal cells were identified as Müller glia, astrocytes, microglia, and endothelial cells based on expression of known markers (Fig. 1d). The Müller cell, the intrinsic glial cell of the retina, was the most abundant type among them (Fig. 4a). Astrocytes, which are largely confined to the ganglion Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ www.nature.com/scientificreports ture.com/scientificreports/ cell and nerve fiber layers were transcriptomically similar to Müller glia (Fig 1d) but the two types were readily Figure 3. Amacrine and retinal ganglion cell types. (a–d) Amacrine cells. Clusters visualized by tSNE (a), correspondence to macaque types (c) and expression of key genes (d) shown as in Fig. 2. Known amacrine types (SAC, VG3-AC, Aii, SEG) are conserved between macaque and human. Genes encoding neuropeptides are shown in bold in d. Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . (b) Top, dendrogram showing transcriptomic relationships among AC clusters; Bottom, violin plots representing the distribution of expression of GABAergic (GAD1, GAD2) and Glycinergic (SLC6A9) in each AC cluster. (e–g) Retinal ganglion cell clusters visualized by tSNE (e), correspondence to macaque types (f) and expression of key genes (g) shown as in Fig. 2. es. (a–d) Amacrine cells. Clusters visualized by tSNE (a) Figure 3. Amacrine and retinal ganglion cell types. (a–d) Amacrine cells. Clusters visualized by tSNE (a), correspondence to macaque types (c) and expression of key genes (d) shown as in Fig. 2. Known amacrine types (SAC, VG3-AC, Aii, SEG) are conserved between macaque and human. Genes encoding neuropeptides are shown in bold in d. (b) Top, dendrogram showing transcriptomic relationships among AC clusters; Bottom, violin plots representing the distribution of expression of GABAergic (GAD1, GAD2) and Glycinergic (SLC6A9 in each AC cluster. (e–g) Retinal ganglion cell clusters visualized by tSNE (e), correspondence to macaque types (f) and expression of key genes (g) shown as in Fig. 2. Figure 3. Amacrine and retinal ganglion cell types. (a–d) Amacrine cells. Clusters visualized by tSNE (a), d ( ) d i f k (d) h i Fi 2 K i Figure 3. Amacrine and retinal ganglion cell types. (a–d) Amacrine cells. Clusters visualized by tSNE (a), correspondence to macaque types (c) and expression of key genes (d) shown as in Fig. 2. Known amacrine types (SAC, VG3-AC, Aii, SEG) are conserved between macaque and human. Genes encoding neuropeptides are shown in bold in d. (b) Top, dendrogram showing transcriptomic relationships among AC clusters; Bottom, violin plots representing the distribution of expression of GABAergic (GAD1, GAD2) and Glycinergic (SLC6A9) in each AC cluster. (e–g) Retinal ganglion cell clusters visualized by tSNE (e), correspondence to macaque types (f) and expression of key genes (g) shown as in Fig. 2. cell and nerve fiber layers, were transcriptomically similar to Müller glia (Fig. 1d), but the two types were readily distinguished by selective expression of multiple genes (Fig. 4b). Comparison of human and macaque retinal cell types. As noted above, the evolutionary proximity of human and macaque enabled us to name most human clusters based on their striking transcriptional corre- spondence with types characterized in macaque. We next assessed the extent to which gene expression are con- served among corresponding types between the two species. Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . We compared the expression of type-specific marker genes in 34 corresponding types : 3 photoreceptor, 2 horizontal cell, 12 bipolar cell, 7 amacrine cell, 7 RGC, and 3 non-neuronal types (Fig. 5a–f, see Methods for details). As expected, all corresponding types expressed at least some common type-specific “marker” genes.i i In some cases, however, type-specific genes were expressed selectively and at high levels in only one of the two species. Examples of such genes include: (a) EPHX2 by macaque but not human cones; (b) GPATCH1 and CRHBP by human but not macaque cones; (c) CA8 by macaque but not human OFF parasol RGCs; (d) FABP4 by human but not macaque OFF parasol RGCs; (e) SCGN by macaque but not human bipolar types DB1 and Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 4. Non-neuronal cell types. (a) Visualization of four non-neuronal types using t-SNE. (b) Genes differentially expressed between Müller glia and astrocytes. Genes shown exhibited >1.5 log fold change. Figure 4. Non-neuronal cell types. (a) Visualization of four non-neuronal types using t-SNE. (b) Genes differentially expressed between Müller glia and astrocytes. Genes shown exhibited >1.5 log fold change. DB6; (f) RBPMS2 by human but not macaque midget RGCs; and (g) RGR by human but not macaque Müller glia (Fig. 5a–f). As another metric of similarity, we identified genes differentially expressed by each shared human and macaque type (log fold change ≥0.5, <0.001 adjusted p value for each type compared to other types within the class). We then calculated the proportion of DE genes in human that were also DE genes in macaque. The five pairs with the largest proportion of shared DE genes were 2 photoreceptor types, 2 non-neuronal types, and one bipolar type (Fig. 5g).f p yp ( g g) We used histological methods to validate some of these differences. Labeling with anti-secretagogin (SCGN) plus anti-VSX2 (CHX10), which labels all bipolar cells, confirmed that SCGN is expressed by bipolar cells in macaque retina but not in human retina (Fig. 6a). Conversely, RBPMS2 was expressed by human but not macaque midget RGCs, while the canonical markers, RBPMS and SLC17A6 (VGLUT2) were expressed by most or all RGCs in both species (Fig. 6b). Together, these comparisons demonstrate predominant but not complete conservation of gene expression by corresponding cell types in human and macaque retina. Comparison of foveal and peripheral retinal cells. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . For analyses presented so far, we pooled data from fovea and periphery. We next compared the regions with each other. Nearly all (57/58) cell types were present in both regions. One GABAergic amacrine type, C18, was found only in the fovea.f For all corresponding types, however, some genes were differentially expressed between foveal and peripheral cohorts. Of 47 types for which there were enough cells in both regions (>20) to enable a comparison, the number of differentially expressed genes ranged from 5 to 100 (log fold change >1; adjusted p-value < 0.001). The types with the most differences by these criteria were RGCs (5 types), non-neuronal cells (3 types) and M/L cones (Fig. 7a). Examples include EPB41L2 and VTN expressed by foveal, but not peripheral cones; TTR expressed at higher levels by foveal than peripheral bipolar type DB3b and DB4; TULP1 expressed by peripheral but not foveal bipolar type FMB and DB2; and RND3 expressed by peripheral but not foveal ON parasol RGCs (Fig. 7b).ff p yp p y p p p g In many cases, proportions of cell types also differed between fovea and periphery. Several differences were consistent with previous reports, such as the relatively lower proportion of S cones among all cones in the fovea compared to the periphery28,29; the depletion of astrocytes from fovea30,31 (0.9% of all non-neuronal cells in fovea and 12% in periphery); and the higher ratio of cone bipolar to rod bipolar cells in the fovea compared to the periphery32 (rod BCs were 35% of peripheral BCs but only 3% of foveal BCs, Fig. 7c). Other differences have not, to our knowledge, been noted previously. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 5. Comparison of gene expression between corresponding human and macaque cell types. (a–f) Dot plots showing similarities and differences in type-specific DE gene expression among corresponding human and macaque types (columns) in PRs (a), HCs (b), BCs (c), ACs (d), RGCs (e), and non-neuronal types (f). (g) Conservation of type-specific marker genes between human and macaque. Graph shows the proportion of DE genes in human (log fold change >0.5 and adjusted p < 0.001 for each type compared to all other types within the class) that are also DE in macaque within shared types (columns). Figure 5. Comparison of gene expression between corresponding human and macaque cell types. Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Human RGCs are labeled with SLC17A6 (magenta). Figures were generated using Zeiss ZEN (https://www.zeiss.com/microscopy/ en_us/products/microscope-software/zen.html), and ImageJ (https://imagej.nih.gov/ij/). Scale bars are 20 μm in a (top panels) , and b; 10 μm in a (bottom panels) . Figure 6. Histological validation of gene expression difference between human and macaque. (a) SCGN (green) does not label any CHX10-positive BCs (magenta) in human retina (top two panels), but does label a subset of BCs in macaque retina (bottom two panels). Circles outline the somatic positions of SCGN-positive cells. Boxed areas in top panels are shown at higher magnification in lower panels . The SCGN-positive CHX- 10 negative cells in human retina are likely to be amacrine cells. (b) RBPMS detected immunohistochemically (top two rows) is similar in human and macaque GCL, while the expression of RBPMS2 detected by in situ hybridization (bottom two rows) is unique to human RGCs, but not found in macaque RGCs. Human RGCs are labeled with SLC17A6 (magenta). Figures were generated using Zeiss ZEN (https://www.zeiss.com/microscopy/ en_us/products/microscope-software/zen.html), and ImageJ (https://imagej.nih.gov/ij/). Scale bars are 20 μm in a (top panels) , and b; 10 μm in a (bottom panels) . Telangiectasia type 2. Of these, 624 genes showed robust expression (detected in more than 20% cells of any class, foveal or peripheral, with average expression level >0.5). We evaluated these, as well as some that fell below threshold but are clinically interesting, further. y g We assessed expression of these genes in 9 cell classes: rods, cones, HCs, BCs, ACs, RGCs, Müller glia, astro- cytes, microglia and endothelial cells. All genes are listed in Fig. S3, and examples are shown in Fig. 8. Note that the retinal pigment epithelium, which plays a critical role in the pathogenesis of many retinal diseases was not included in our atlas (see Discussion). We summarize these groups here, beginning with diseases for which monogenic high penetrance causes have been identified. We then discuss disorders for which few monogenic causes are known, but numerous susceptibility factors have been implicated primarily through genome-wide association studies (GWAS). Retinitis Pigmentosa (RP) is a diffuse photoreceptor dystrophy that also affects the pigment epithelium. It manifests as night blindness with progressive visual field loss33. Clinical features in the macula often include loss of foveal reflex, abnormalities at the vitreoretinal interface, and cystoid macular edema. Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . (a–f) Dot plots showing similarities and differences in type-specific DE gene expression among corresponding human and macaque types (columns) in PRs (a), HCs (b), BCs (c), ACs (d), RGCs (e), and non-neuronal types (f). (g) Conservation of type-specific marker genes between human and macaque. Graph shows the proportion of DE genes in human (log fold change >0.5 and adjusted p < 0.001 for each type compared to all other types within the class) that are also DE in macaque within shared types (columns). The H1:H2 ratio was nearly ~4-fold higher in the fovea (7.3:1) than in peripheral retina, 1.9:1; Fig. 7d). The ratio of GABAergic to Glycinergic AC types was higher in fovea (1.8:1) than in the periphery (1.1:1). Several AC clusters showed enrichment in either fovea (e.g., C8, 12 and 23) or peripheral retina (e.g., C1, 2, and 4) (Fig. 7e). The OFF parasol is the only RGC type enriched in fovea using the same criteria, while 5 out of the 8 rare RGC types (hRGC cluster 7, 8, 9, 10, 12) were more abundant in peripheral retina than in fovea (Fig. 7f). Foveal enrich- ment of H1 horizontal cells and OFF parasol RGCs was also observed in the cynomolgus macaque12. Expression of genes implicated in retinal disease. We used the cell atlas to assess retinal expression of 1,756 genes associated with diseases in which vision loss results primarily from death or dysfunction of retinal cells. They include retinitis pigmentosa, cone-rod dystrophy, Leber congenital amaurosis, congenital station- ary night blindness, hereditary maculopathy, Leber hereditary optic neuropathy, dominant optic atrophy, open angle glaucoma, age-related macular degeneration, diabetic retinopathy, diabetic macular edema and Macular Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 6. Histological validation of gene expression difference between human and macaque. (a) SCGN (green) does not label any CHX10-positive BCs (magenta) in human retina (top two panels), but does label a subset of BCs in macaque retina (bottom two panels). Circles outline the somatic positions of SCGN-positive cells. Boxed areas in top panels are shown at higher magnification in lower panels . The SCGN-positive CHX- 10 negative cells in human retina are likely to be amacrine cells. (b) RBPMS detected immunohistochemically (top two rows) is similar in human and macaque GCL, while the expression of RBPMS2 detected by in situ hybridization (bottom two rows) is unique to human RGCs, but not found in macaque RGCs. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Black horizontal line, median; bars, interquartile range; vertical lines, minimum and maximum, dots are values from individual donors; n = 6 foveal, and 2 peripheral samples. Figure 7. Differences between corresponding cell types in fovea and periphery. (a) Bar plot showing the number of differential expression (DE) genes (log fold change >1 and adjusted p value < 0.001) per matched cell types between fovea and periphery (x-axis). (b) Violin plots showing the expression of select DE genes in relevant foveal and peripheral cell types. (c–f) Box-and-whisker plots showing proportions of cell types in fov and peripheral retina for BCs (c), HCs (d), ACs(e), RGCs (f). Black horizontal line, median; bars, interquartil range; vertical lines, minimum and maximum, dots are values from individual donors; n = 6 foveal, and 2 peripheral samples. Figure 7. Differences between corresponding cell types in fovea and periphery. (a) Bar plot showing the number of differential expression (DE) genes (log fold change >1 and adjusted p value < 0.001) per matched cell types between fovea and periphery (x-axis). (b) Violin plots showing the expression of select DE genes in relevant foveal and peripheral cell types. (c–f) Box-and-whisker plots showing proportions of cell types in fovea and peripheral retina for BCs (c), HCs (d), ACs(e), RGCs (f). Black horizontal line, median; bars, interquartile range; vertical lines, minimum and maximum, dots are values from individual donors; n = 6 foveal, and 2 peripheral samples. most cases, cell loss is minimal. Genes implicated in CSNB were generally expressed either in rods (e.g., GNAT1, SLC24A1) or bipolar cells (e.g., GRM6, TRPM1). CACNA1F, which harbors mutations in the majority of cases of incomplete X-linked CSNB, was expressed in both photoreceptor and bipolar cells, consistent with its wider phe- notypic spectrum encompassing X-linked progressive cone-rod dystrophy, optic atrophy, and Åland Island eye disease (AIED) (Fig. 8d). Finally, although, NYX and LRIT3, which harbor CSNB mutations36,37 were expressed at levels too low to meet our set screening threshold, they were preferentially expressed in bipolar cells and pho- toreceptors, respectively. p p y Macular dystrophies, including Stargardt Disease, Vitelliform degenerations, Pattern Dystrophies, Sorsby Macular Dystrophy and Familial drusen, are slowly progressive retinal degenerations that account for a sig- nificant proportion of cases of central vision loss among adults under the age of 5038. Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Other typical find- ings include arteriolar narrowing, waxy pallor to the optic disc, and variable amounts of bone-spicule pigment changes. Consistent with the predominant functional deficits of night blindness, most genes implicated as mono- genic causes of RP were predominantly expressed in rods (Figs. 8a, S3). Potentially consistent with other clinical findings of RP, some genes were also expressed in vascular endothelium and RGCs (e.g., RPGR and TOPORS), while others were expressed at highest levels in RGCs (e.g., SLC25A46, SLC7A14 and RP9) or Müller glia (e.g., RGR and RLBP1). As noted above, some genes in this and other disease groups (e.g., RPE65) are likely to act in and be expressed at higher levels by retinal pigment epithelial cells, which were not included in our dataset.f p g y p g p Cone-rod dystrophy affects both photoreceptor classes and patients with this condition demonstrate expand- ing central scotomas often leading to severe visual impairment34. Consistent with this pathology, causative genes were expressed in both rods and cones (e.g., CRX, RAX2), with expression often higher in the latter (e.g., GNAT2, PDE6H) (Fig. 8b). g Lebers Congenital Amaurosis (LCA) is a severe group of inherited retinal dystrophies characterized by nys- tagmus, sluggish or absent pupillary light reflexes and blindness, often in the first year of life35. Genes mutated in the most prevalent forms of LCA were expressed in both rod and cone photoreceptors, consistent with the char- acteristic early absence of retina-wide rod and cone photoreceptor function demonstrable by electroretinogram (ERG). Several (CEP290, GUCY2D and CRB1) were also expressed in RGCs (Fig. 8c). ( ) ( ) p ( g ) Congenital Stationary Night Blindness (CSNB), a lifelong, nonprogressive abnormality of scotopic vision, disrupts transmission through the rod pathway by disabling neurotransmission from rods to rod bipolar cells33. In Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 7. Differences between corresponding cell types in fovea and periphery. (a) Bar plot showing the number of differential expression (DE) genes (log fold change >1 and adjusted p value < 0.001) per matched cell types between fovea and periphery (x-axis). (b) Violin plots showing the expression of select DE genes in relevant foveal and peripheral cell types. (c–f) Box-and-whisker plots showing proportions of cell types in fovea and peripheral retina for BCs (c), HCs (d), ACs(e), RGCs (f). Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . Genes involved in these dystrophies were generally enriched in photoreceptors (e.g., PRPH2, PROM1), but others were also expressed in non-neural cells – e.g., EFEMP1 selectively in Müller Glia and TIMP3 selectively in vascular endothelium (Fig. 8e). TIMP3, which is mutated in Sorsby Macular Dystrophy, encodes a protein involved in matrix remode- ling and suppression of retinal angiogenesis. Although the primary site of pathogenesis in this disease is believed to be at the level of retinal pigment epithelium or Bruch’s membrane, our results suggest that it might also act within retinal vasculature. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 8. Expression of disease-related genes in retinal cell classes. (a-l) Heat maps show expression of genes implicated in each of 12 groups of blinding diseases described in the text. Color represents the scaled expression level of genes among all cell classes. Thus, the heat maps accurately reflect the order of expression among cell classes for each gene but not the absolute levels of expression. Asterisks mark genes that were included because of clinical interest but fell below the threshold noted to the text. Fi 8 E i f di l d i Figure 8. Expression of disease-related genes in retinal cell classes. (a-l) Heat maps show expression of genes implicated in each of 12 groups of blinding diseases described in the text. Color represents the scaled expression level of genes among all cell classes. Thus, the heat maps accurately reflect the order of expression among cell classes for each gene but not the absolute levels of expression. Asterisks mark genes that were included because of clinical interest but fell below the threshold noted to the text. Leber’s hereditary optic neuropathy (LHON) is the most common inherited mitochondrial disease with oph- thalmic manifestations. It is caused by mutations in genes primarily encoding respiratory complex chain 1 pro- teins (e.g., ND1, ND4, ND6), leading to defects in NADH-ubiquinone oxidoreductase chains that may impair glutamate transport and increase production of reactive oxygen species39. The result of these impairments is RGC dysfunction and, eventually, apoptosis, and atrophy of the retinal nerve fiber layer. Consistent with this pathogen- esis, all LHON genes were predominantly expressed in RGCs (Fig. 8f). g p y p g Autosomal Dominant Optic Atrophy, the most common hereditary optic neuropathy, is characterized by gradual loss of visual acuity that is generally bilateral and symmetric. www.nature.com/scientificreports/ Other genes, including FOXC1, CYP1B1, LMX1B and MYOC were not expressed substantially in any neural retina cell classes, consistent with their proposed role predomi- nantly in the anterior segment with mutations leading to high intraocular pressure, a major risk factor for glau- coma (Fig. S3). Indeed, in a parallel study, we have demonstrated expression of these genes in cells of the aqueous humor outflow pathways53. l p y In general, these patterns of expression match those we previously documented for macaque. For example, of the 94 genes shown in Fig. 8, 85 were profiled in macaque and of these, 78 (or 92%) were expressed at highest levels in the same cell class in both species. p We next compared foveal and peripheral cohorts of cell classes in which genes were highly expressed (Fig. 9a,b). Several patterns were consistent with clinical features of the associated conditions. For example, many genes with causative mutations leading to Retinitis Pigmentosa, including RHO, NRL, and NR2E3 demonstrated foveal rod enrichment (Figs. 9a and S3). RP1 was preferentially expressed in foveal rods and cones. In contrast, PDE6H, a cone-rod dystrophy gene, demonstrated preferential expression in peripheral rods. Genes implicated in macular dystrophies were generally enriched in the fovea compared to the peripheral retina. ABCA4, which har- bors causative mutations leading to Stargardt disease, was enriched in foveal photoreceptors (Fig. S3); EFEMP1, implicated in Doyne Honeycomb Dystrophy, was predominantly expressed in foveal Müller glia. OPTN and APOE, implicated in Open Angle Glaucoma, demonstrated class-specific foveal enrichment among RGCs and Müller glia, respectively. In contrast, VEGFA, polymorphisms of which have been linked to severity of Diabetic Retinopathy, was expressed at higher levels in the periphery compared to fovea.i y g y Finally, we assessed type-specific expression in RGCs for genes implicated in dominant optic atrophy, diabetic retinopathy, diabetic macular edema, Mac Tel 2 and primary open angle glaucoma, and type-specific expres- sion in bipolar cells for genes implicated in CSNB (Fig. 9b,c). Genes with type-specific RGC expression patterns included the glaucoma-associated genes SIX6, which was enriched in midget ganglion cells; CAV2 and POU6F2 enriched in ON parasol RGCs and AFAP1, enriched in peripheral ON parasol RGCs. (POU6F2 was expressed at highest levels in RGC types 5, 11 and 12, which include ipRGCs.) Several but not all genes implicated in Mac Tel 2 (e.g. PHGDH, PSPH, LINC00461 and GBAS) were enriched in foveal RGCs. www.nature.com/scientificreports/ action. Alleles in the CFH gene and other complement pathway genes have also emerged as risk factors for AMD; we noted expression of multiple complement genes (CFH, CFI, C2, C3) in different cell classes (Fig. 8i). gf g Diabetic retinopathy (DR) and Diabetic Macular Edema (DME) together represent the leading cause of blind- ness and visual disability among working-age adults of all races living in industrialized nations44. While many genes implicated in diabetic retinopathy – classically considered a predominantly microvascular complication of diabetes mellitus – were expressed in non-neuronal retinal cell classes (particularly, vascular endothelial cells), a large proportion, such as HS6ST345, DPP1046, and VEGFB, were almost exclusively expressed in RGCs (Fig. 8j). g p p y p g j Macular Telangiectasia type 2 (Mac Tel 2) is a rare retinal neurodegenerative condition that leads to late-onset progressive central vision loss47,48. Early clinical findings, including retinal discoloration and capillary telangiec- tasis, are limited to the perifoveal region. Photoreceptor loss and foveal atrophy occur as the disease progresses. Mac Tel 2 is currently considered to be a primary neurodegenerative condition of the retina with secondary vas- cular involvement rather than (as previously hypothesized) a primary vasculopathy. Several risk alleles and genes in proximity to SNPs identified by GWAS studies implicate Müller cell dysfunction and dysregulation of serine metabolism in pathogenesis49,50. We found expression patterns compatible with these hypotheses. For example, PHGDH and PSPH, encoding enzymes involved in L-serine synthesis, were selectively expressed in RGCs with PHGDH also expressed in Müller glia. Finally, to gain insight into the protective effects of CNTF, which is cur- rently in phase 2 clinical trials for Mac Tel 2, we investigated the expression of CNTFR. We found expression in RGCs and Müller glia as well as amacrine cells and astrocytes (Fig. 8k). g y ( g ) Open Angle Glaucoma, an optic neuropathy characterized by loss of RGCs, is one of the most common causes of vision loss world-wide and the leading cause of irreversible blindness among African Americans51,52. Among genes implicated in glaucoma, either by GWAS or as rare Mendelian alleles, most were expressed predominantly within RGCs (e.g., OPTN, TMCO1, TBK1) (Fig. 8l). www.nature.com/scientificreports/ Patterns of expression differed, however, with PHGDH expressed primarily in foveal midget RGCS, LINC00461 primarily in foveal parasol RGCs, and PSPG and GBAS in both. MRPL19, one of the few genes implicated specifically in DME, which affects the fovea by clinical definition, was expressed preferentially in foveal RGCs54. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 Cholecystokinin (CCK) in C15. Thus, human amacrines appear to use a variety of neurotransmitters and neuro- modulators, as has been demonstrated for amacrines in other mammalian and non-mammalian retinas22 . RGC degeneration, particularly in the pap- illomacular bundle, has been implicated as the primary mechanism of disease40. Consistent with this pattern of expression, causative genes such as OPA1 and OPA3 were predominantly expressed in RGCs (Fig. 8g). p g p y p g g Inherited Vitreoretinopathies include Familial Exudative Vitreoretinopathy (FEVR), Norrie Disease and Coats Disease. Most genes mutated in these disorders were expressed in retinal vascular endothelium (Fig. 8h). In addition, however, NDP and LRP5 were also expressed in Müller glia41 and in astrocytes, a finding compatible with observations of NDP expression in a subset of cortical astrocytes42.i p y Age Related Macular Degeneration (AMD) is broadly classified into non-exudative (“dry”) and exudative or neovascular (“wet”) types. In dry AMD, mild forms are characterized by drusen accumulation between reti- nal pigment epithelium and Bruch’s membrane, which can progress to late forms with large patches of atrophic outer retina. In wet AMD, aberrant angiogenesis originating either within the choroid or retina leads to often catastrophic sub- or intraretinal hemorrhage43. HTRA1, a major susceptibility gene for neovascular AMD5, was expressed at high levels in HCs and Müller glia (Fig. 8i). While the disease-related effects of this gene are thought be exerted in the retinal pigment epithelium, its expression in HCs and Müller glia suggests additional sites of Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 10 www.nature.com/scientificreports/ Discussion We used high-throughput single-cell RNA-seq to generate a cell atlas of the adult human retina. From 55,736 foveal and 29,246 peripheral retinal cells, we identified 58 cell types. We then used the cell atlas to compare fovea with peripheral retina, and human with macaque retinal cell types. Finally, we probed region-, cell class-, and cell type-specific expression of genes associated with blinding retinal diseases. Together, our atlas provides a roadmap for human retinal research and paves the way for further research on the pathology of ocular diseases. Human cell atlas. Non-diseased retina is seldom excised during ocular surgery, so tissue must be obtained postmortem. Given that cell viability declines and transcriptomic profiles change after death, with dramatic alter- ations after 10 hours post-mortem, data quality hinges in large part on the time between death and tissue process- ing. For example, Lukowski et al.55 showed that rods began to degenerate and their expression of MALAT1—a long non-coding RNA—decreased at this point. In our dataset, retinas from 6 of the 7 donors were obtained within 6.5 hr post-mortem, all rod photoreceptors clustered together, and MALAT1 levels were high. These results affirm the high quality of the cells from which the atlas was generated. fi We and others have recently reported results of scRNA-seq studies on human retina,12,55–61 (see Table S2). However, our initial study was focused on bipolar cells, and some groups used fetal rather than adult cells, and/or did not distinguish foveal from peripheral cells. Four of these groups did, however, use adult retina and separated fovea from peripheral retina. These studies generated valuable data, but were disadvantaged in that rods comprise Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Figure 9. Differential expression of disease-related genes by region and cell type. (a) Violin and superimposed box plots showing differential expression of select disease genes between foveal and peripheral cell classes. (b) Heat maps showing expressions of select disease genes in four major RGC types. Cell types are segregated by their regions: fovea versus periphery. (c) Heat map showing expression of GRM6 and TRPM1, two previously reported congenital stationary night blindness genes in ON bipolar cells (blue bar top) but not in OFF bipolar f Figure 9. Differential expression of disease-related genes by region and cell type. (a) Violin and superimposed box plots showing differential expression of select disease genes between foveal and peripheral cell classes. Discussion Moreover, the fovea, and the macula within which it is embedded, are the principal site of pathology among diseases such as age-related macular degeneration, diabetic macular edema, hereditary macular dystrophy, and macular telangiectasia. Lacking fovea and macula, it is unsurprising that rodent models of these diseases have severe limitations. Our results address this issue in two ways. First, by generating a human cell atlas, and a comprehensive data- base on expression of disease-related genes, we provide a foundation for both translational and basic studies. Second, by documenting close similarities between human retinal cell types and the macaque types we described recently12, we both validate the use of this non-human primate model and point out some important differences that will need to be considered in interpreting studies of non-human primates. Although some differences could result from imperfections in gene and transcript annotation, it is likely that the vast majority are genuine.hf The structural and functional difference between the fovea and peripheral retina could result from the exist- ence of specialized foveal cell types. We show however, that nearly all retinal cell types are shared between fovea and periphery in human retina. Instead, there are substantial regional differences in gene expression and abun- dance between foveal and peripheral cohorts of shared types. Limitations to the comparison include low cell number in periphery for some cell types, and potential bias introduced by the methods we used to deplete rods and enrich RGCs from peripheral samples. Nonetheless, many of the differences in abundance we observed were consistent with those reported by others based on morphological analysis, and we reported histological validation of some of the DE genes in a recent study12. Thus, in humans as in macaques12, the fovea and peripheral retina are composed of similar cell types, with the structural and functional differences between them likely arising from differences in abundance of shared types and specific aspects of gene expression. Mapping disease genes to cell classes and types. We analyzed expression of 636 genes associated with retinal diseases, chosen from an initial list of 1,756. Although long, the list is incomplete, because retinal pigment epithelium, which plays a major role in the pathogenesis of many retinal diseases such as retinitis pigmentosa and age-related macular degeneration, was not included in our dataset. Discussion (b) Heat maps showing expressions of select disease genes in four major RGC types. Cell types are segregated by their regions: fovea versus periphery. (c) Heat map showing expression of GRM6 and TRPM1, two previously reported congenital stationary night blindness genes, in ON bipolar cells (blue bar, top), but not in OFF bipolar cells (red bar, top). a large fraction of all cells (>70%), reducing power to distinguish cell types among less abundant classes. This problem is most severe for RGCs, which comprise <2% of retinal cells. We circumvented these limitations by depleting rods in some samples (using anti-CD73) to enrich other neuronal classes, and by selecting RGCs in other samples (using anti-CD90). These strategies allowed us to distinguish more types within classes than in previous studies. For example, we were able to characterize vGlut3 excitatory ACs (0.7% of total retinal cells), ipRGCs (0.02% of total retinal cells), S cones (0.07% of total retinal cells), and the primate-specific OFFx bipolar type12. Thus, our cell atlas represents the most complete classification to date of cell types in adult human retina. a large fraction of all cells (>70%), reducing power to distinguish cell types among less abundant classes. This problem is most severe for RGCs, which comprise <2% of retinal cells. We circumvented these limitations by depleting rods in some samples (using anti-CD73) to enrich other neuronal classes, and by selecting RGCs in other samples (using anti-CD90). These strategies allowed us to distinguish more types within classes than in previous studies. For example, we were able to characterize vGlut3 excitatory ACs (0.7% of total retinal cells), ipRGCs (0.02% of total retinal cells), S cones (0.07% of total retinal cells), and the primate-specific OFFx bipolar type12. Thus, our cell atlas represents the most complete classification to date of cell types in adult human retina. Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Comparison of fovea and peripheral retina. A major hurdle for studying human retinal biology and diseases is that retinas of accessible animal models differ from human retina in critical respects. Perhaps most important is that among mammals, only primates have a fovea or macula. The fovea comprises only ~1% of reti- nal area in humans, but accounts for most of our high-acuity vision, much of our chromatic vision, and supplies ~50% of the visual input to the cortex62,63. Discussion On the other hand, this confound would not affect the intriguing result that we documented RGC type-selectivity in expression of many disease-associated genes. Of particular interest are selective expression patterns of genes implicated in POAG, given speculation that specific RGC types may exhibit selective vulnerability in this disease64.f h In contrast, several disease-associated genes were expressed by cells not definitively linked to previously reported phenotypes or pathogenic mechanisms. For example, many RP-associated genes were expressed in RGCs. While loss of RGCs could explain waxy pallor of the optic disc—a common clinical finding in RP—the molecular underpinnings leading to this finding remain incompletely understood. Our observations that some RP-associated genes such as IDH3B, ACBD5 and ASRGL1 were highly expressed in RGCs motivate further investigation. Similarly, several genes implicated in diabetic retinopathy – classically considered a retinal vascu- lopathy– were differentially expressed by RGCs (e.g AKR1B1, HS6ST3 and MPRIP), suggesting that a primary neuropathic process may also be involved. This conclusion is complicated by the fact that we detect more genes in RGCs than in other classes, likely because of their large size. On the other hand, this confound would not affect the intriguing result that we documented RGC type-selectivity in expression of many disease-associated genes. Of particular interest are selective expression patterns of genes implicated in POAG, given speculation that specific RGC types may exhibit selective vulnerability in this disease64.f yp y y Together, our results offer new insights into many rare and common retinal diseases, and may contribute to a more comprehensive understanding of their pathogenesis and the discovery of novel therapeutic targets. Discussion Moreover, our criterion for inclusion was expression in more than 20% of cells of at least one class in either fovea or peripheral retina, so genes expressed at slightly lower levels or in only a few minor types within a class would have been excluded. We added 12 genes that fell below threshold to the 624 that met the criterion based on their known clinical relevance, but others remain to be analyzed.i y We investigated cell-class and cell-type specific expression patterns for disease-associated genes meeting the above criteria; expression patterns for many of them supported prior reports of pathogenetic mechanisms (detailed in Results). For example, many genes harboring mutations implicated in retinal degenerations charac- terized predominantly by night blindness–such as RHO, SLC24A1, and GNAT1–were confirmed to be selectively expressed in rods. Similarly, genes characterized by optic nerve degeneration or atrophy – such as OPA1, OPTN and WFS1 – were selectively expressed in RGCs. These patterns are expected and many have been documented33.i We investigated cell-class and cell-type specific expression patterns for disease-associated genes meeting the above criteria; expression patterns for many of them supported prior reports of pathogenetic mechanisms (detailed in Results). For example, many genes harboring mutations implicated in retinal degenerations charac- terized predominantly by night blindness–such as RHO, SLC24A1, and GNAT1–were confirmed to be selectively expressed in rods. Similarly, genes characterized by optic nerve degeneration or atrophy – such as OPA1, OPTN and WFS1 – were selectively expressed in RGCs. These patterns are expected and many have been documented33. In contrast, several disease-associated genes were expressed by cells not definitively linked to previously reported phenotypes or pathogenic mechanisms. For example, many RP-associated genes were expressed in RGCs. While loss of RGCs could explain waxy pallor of the optic disc—a common clinical finding in RP—the molecular underpinnings leading to this finding remain incompletely understood. Our observations that some RP-associated genes such as IDH3B, ACBD5 and ASRGL1 were highly expressed in RGCs motivate further investigation. Similarly, several genes implicated in diabetic retinopathy – classically considered a retinal vascu- lopathy– were differentially expressed by RGCs (e.g AKR1B1, HS6ST3 and MPRIP), suggesting that a primary neuropathic process may also be involved. This conclusion is complicated by the fact that we detect more genes in RGCs than in other classes, likely because of their large size. Materials and Methods 2500 (Paired end reads: Read 1, 26 bp, Read 2, 98 bp). Single cell isolation, library preparation and sequencing. Single cell libraries were generated by minor modifications of methods developed for macaque retina12. Briefly, a ~1.5 mm diameter circular region centered on the foveal pit was dissected from the retina, and peripheral retinal pieces were pooled from all retinal quadrants. Dissected tissues were digested with papain (Worthington, LS003126) for 30 min at 37 °C. Following digestion, samples were dissociated and triturated into single cell suspensions with 0.04% bovine serum albu- min (BSA) in Ames. Dissociated cells from digested peripheral retinas were incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253; 1 ml per 107 cells) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2; 5 ml per 107 cells) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102; 10 ml per 107 cells) to deplete rods. Incubations were at room temperature for 10 min. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Foveal samples were used without further processing. Single cell suspensions were diluted to 500–1800 cells/µL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell v2 or v3 Chips. Following collection, cDNA libraries were prepared following the manufacturer’s protocol, and sequenced on the Illumina HiSeq. 2500 (Paired end reads: Read 1, 26 bp, Read 2, 98 bp). Bioinformatics analysis. Clustering. Sequencing reads were demultiplexed and aligned to a human tran- scriptomic reference (GRCh38) with the Cell Ranger software (version 2.1.0, 10X Genomics for the v2 sam- ples, and version 3.0.2 for the v3 samples) for each 10X channel separately. The resulting digital gene expression (DGE) matrices representing the transcript counts for each gene (rows) in each cell (columns) were combined for all samples (foveal and peripheral), and analyzed further using the R statistical language following methods described by Peng et al.12, with minor modifications, as follows.i y gi A threshold of 600 detected genes per cell was applied to filter out low quality cells or debris from the com- bined DGE matrix. DGE matrix was normalized, log-transformed where expression values Ei,j for gene i in cell j were calculated following65. Highly variable genes (HVGs) were identified as in66, and used for dimensionality reduction. Batch correction was performed using the linear regression approach adapted from the R package ‘Seurat’. Materials and Methods Principal component analysis (PCA) was performed and statistically significant PCs were estimated using Random Matrix Theory67. Cell clustering was performed using Louvain algorithm with Jaccard correction65. The work flow was first performed to group cells into major cell classes. In addition to the nine cell classes described in the main text, we detected small numbers of epithelial cells and melanocytes (<100 cells); they were not con- sidered further, because of the likelihood that they arise from non-retinal contaminants. For each of the neuronal classes, a second round of clustering was performed to assign cells into molecularly distinct types within that class. We noted a significant higher number of genes/transcripts detected in RGCs than other cell classes (the averaged number in RGC is 2.4~5 fold of other classes), so the gene expression matrix was re-normalized when analyzing RGCs.ti y g Some low quality cells or doublets became apparent only after clustering. We identified and eliminated them before re-running the analysis. Doublets were detected in two ways: (1) During initial analysis, clusters exhibiting high expression levels of canonical markers from more than cell class were further investigated. If no uniquely expressed marker genes were found (as assessed by statistical testing with R package ‘MAST’), and the average number of genes in these clusters was higher than observed in related clusters, the cluster was classified as com- prising doublets. (2) When analyzing types within one class, no pre-filtering was applied. Clusters were identified as comprising doublets if its cells lacked uniquely expressed marker gene and had on average higher numbers of transcripts/genes per cell than related types. Clusters of low quality cells were identified by low numbers of tran- scripts detected and high proportion of reads mapped to mitochondrial genes. The rationale is that damaged cells “leak” cytoplasmic RNAs but retain organelle-bound mitochondrial RNAs. These quality indices (average genes/ transcripts per cell and proportion of mitochondrial gene transcripts) are shown for each collection in Table S3, along with the number of cells in which >600 gene/cell were detected. On average, 17% of the cells that passed the initial filter of 600 genes/cell were later identified as low quality cells or doublets. The median number of genes and transcripts detected per cell was 1314 and 2577, respectively. Materials and Methods Human tissue. Acquisition and use of post-mortem human tissue samples was approved by the Human Study Subject Committees of Harvard Medical School (DFCI Protocol Number: 13–416 and MEE - NHSR Protocol Number 18–034 H) and in compliance with the National Human Genome Research Institute (NHGRI) policies. Informed consent was obtained from participants if they were enrolled antemortem or their legal guard- ians if post-mortem. All human eyes used for sequencing and histological studies were collected 3–14 hours post mortem through the Rapid Autopsy Program, Massachusetts General Hospital, with all but one collected ≤6.5 hours post mortem (Table S1). The globe was immediately transported back to the lab in a humid chamber. Hemisection was performed to remove the anterior chamber, and the posterior pole was immersed in Ames equilibrated with 95% O2/5% CO2 before further dissection and dissociation. All donors were confirmed to have no history or clinical evidence of ocular disease or intraocular surgery.i y g y For histological studies, dissected retinal tissues were fixed in ice-cold 4% PFA for 2hrs. For transcriptomic analysis, retinal cells were dissociated as described in the next section. 13 Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ Single cell isolation, library preparation and sequencing. Single cell libraries were generated by minor modifications of methods developed for macaque retina12. Briefly, a ~1.5 mm diameter circular region centered on the foveal pit was dissected from the retina, and peripheral retinal pieces were pooled from all retinal quadrants. Dissected tissues were digested with papain (Worthington, LS003126) for 30 min at 37 °C. Following digestion, samples were dissociated and triturated into single cell suspensions with 0.04% bovine serum albu- min (BSA) in Ames. Dissociated cells from digested peripheral retinas were incubated with CD90 microbeads (Miltenyi Biotec, 130-096-253; 1 ml per 107 cells) to enrich RGCs or with anti-CD73 (BD Biosciences, clone AD2; 5 ml per 107 cells) followed by anti-mouse IgG1 microbeads (Miltenyi Biotec, 130-047-102; 10 ml per 107 cells) to deplete rods. Incubations were at room temperature for 10 min. CD90 positive cells or CD73 negative cells were selected via large cell columns through a MiniMACS Separator (Miltenyi Biotec). Foveal samples were used without further processing. Single cell suspensions were diluted to 500–1800 cells/µL in 0.04% BSA/Ames for loading into 10X Chromium Single Cell v2 or v3 Chips. Following collection, cDNA libraries were prepared following the manufacturer’s protocol, and sequenced on the Illumina HiSeq. Immunohistochemistry and fluorescence in situ hybridization. Proce Immunohistochemistry and fluorescence in situ hybridization. Procedures for tissue preparation, immunohistochemistry and in situ hybridization have been described in12,65,70. Briefly, eyes were fixed in ice-cold 4% paraformaldehyde, rinsed with PBS, immersed in 30% sucrose overnight at 4 °C, embedded in Tissue Freezing Medium (EMS) and cryosectioned at 20 μm. For immunohistochemistry, antibodies were diluted in 3% donkey serum (Jackson, 017-000-121), and 0.3% Triton-X in PBS. Antibodies used for immunostaining were as fol- lows: goat anti anti-CHX10 (1:300, Santa Cruz); rabbit anti-TFAP2A (1:500, DSHB); rabbit anti-Secretagogin (1:10,000; BioVendor). For in situ hybridization, sections were mounted on Superfrost slides (Thermo Scientific), treated with 1.5 mg/mL of proteinase K (NEB, P8107S), and then post-fixed and treated with acetic anhydride for deacetylation. Probe detection was performed with anti-DIG HRP (1:1000) and anti-DNP HRP (1:500), followed by tyramide amplification. Image acquisition and processing. Images were acquired on Zeiss LSM 710 confocal microscopes with 405, 488–515, 568, and 647 lasers, processed using Zeiss ZEN software suites, and analyzed using ImageJ (NIH). Images were acquired with 16×, 40× or 63× oil lens at the resolution of 1,024 × 1,024 pixels, a step size of 0.5–1.5 µm, and 90 µm pinhole size. ImageJ (NIH) software was used to generate maximum intensity projections. Adobe Photoshop CC was used for adjustments to brightness and contrast. Mapping disease genes. SNP-trait associations (N = 980) were downloaded on 09/03/2019 from the NHGRI-EBI GWAS Catalog71 for the traits, “open-angle glaucoma” (n = 108), “intraocular pressure measure- ment” (n = 504),“diabetic retinopathy” (n = 138), “age-related macular degeneration” (n = 230) and Macular Telangiectasia Type 2 (n = 5). A list of genes and loci associated with retinal diseases was downloaded on 09/03/2019 from the Retinal Information Network, last updated on 07/01/2019 (RetNet, https://sph.uth.edu/ retnet/). References References 1. Khandhadia, S., Cherry, J. & Lotery, A. J. Age-related macular degeneration. Adv. Exp. Med. Biol. 724, 15–36, https://doi org/10.1007/978-1-4614-0653-2_2 (2012).h 1. Khandhadia, S., Cherry, J. & Lotery, A. J. Age-related macular degeneration. Adv. Exp. Med. Biol. 724, 15–36, https://doi. org/10.1007/978-1-4614-0653-2_2 (2012).h g ( ) 2. Weinreb, R. 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A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat. Genet. 48, 134–143, https://doi.org/10.1038/ng.3448 (2016).i g g 5. Fritsche, L. G. et al. A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat. Genet. 48, 134–143, https://doi.org/10.1038/ng.3448 (2016).i g g 6. Choquet, H. et al. A multiethnic genome-wide association study of primary open-angle glaucoma identifies novel risk loci. Nat Commun. 9, 2278, https://doi.org/10.1038/s41467-018-04555-4 (2018). p g 7. Ferris, F. L. III & Patz, A. Macular edema. A complication of diabetic retinopathy. Surv. Ophthalmol. 28(Suppl), 452–461, https://doi org/10.1016/0039-6257(84)90227-3 (1984). 7. Ferris, F. L. III & Patz, A. Macular edema. A complication of diabetic retinopathy. Surv. Ophthalmol. 28(Suppl), 452–461, https://doi. org/10.1016/0039-6257(84)90227-3 (1984). g 8. O’Brien, K. M. B. in Visual Transduction and Non-Visual Light Perception (eds Joyce Tombran-Tink & Colin J. Barnstable) 17-33 (Humana Press (2008). 9. Crawford, M. L., Harwerth, R. S., Smith, E. L. III, Shen, F. & Carter-Dawson, L. Glaucoma in primates: cytochrome oxidase reactivity in parvo- and magnocellular pathways. Invest. Ophthalmol. Vis. Sci. 41, 1791–1802 (2000).f p g p y p ( ) 10. Umeda, S. et al. Molecular composition of drusen and possible involvement of anti-retinal autoimmunity in two different forms of macular degeneration in cynomolgus monkey (Macaca fascicularis). FASEB J. Data availability Th d d y The raw and processed single cell RNAseq data reported in this study can be accessed at GEO: GSE148077. Data can be visualized at the Broad Institute’s Single Cell Portal: https://singlecell.broadinstitute.org/single_cell/study/ SCP839. Received: 11 February 2020; Accepted: 12 May 2020; Published: xx xx xxxx where # means number of. Comparing foveal and peripheral cells. To evaluate the extent of similarity between foveal and peripheral cell types in human retina (Fig. 7a), we measured the number of DE genes identified between foveal and peripheral cells for each human type. Only types with at least 20 cells in both regions were considered. DE genes in this com- parison were those with differences in expression ≥1*log fold change and adjusted p-value < 0.001. www.nature.com/scientificreports/ i this resuled in two DE lists, one each for human (hDEi) and macaque (mDEi) respectively. The proportion of type-specific DE genes that were shared across species (pDEi) was computed for each type i as, i this resuled in two DE lists, one each for human (hDEi) and macaque (mDEi) respectively. The proportion of type-specific DE genes that were shared across species (pDEi) was computed for each type i as, = ∗ pDE intersect hDE mDE hDE # ( , ) # 100, i i i i Materials and Methods To visualize the data in 2D space, we applied t-distributed stochastic neighbor embedding (t-SNE) to the nor- malized cell factors using the functions quantileAlignSNF and runTSNE (with default setting of perplexity =30) from the R package ‘liger’68, which uses an integrative non-negative matrix factorization framework. Averaged expression matrix of HVGs for each cluster were calculated to build dendrogram (Fig. 3b upper panel, hierarchi- cal agglomerative clustering of Euclidean distance metric with complete linkage), which revealed transcriptomic relationships among types. Neighboring clusters on this dendrogram were iteratively merged if no more than five DE gene was found showing ≥1.1 log fold change with adjusted p value < 0.001 using the R package ‘MAST’69. Comparing human and macaque cell types. We sought correspondence between human and macaque cell types using the multi-class classification approach described earlier12. Briefly, for each cell class in macaque, we trained a multi-class classifier using the R package ‘xgboost’14 to map cells to discrete type labels based on their transcrip- tional signatures. This classifier was then applied to each human cell of the same class to assign it a macaque type label based on its expression of 1:1 gene orthologs, but in a manner completely agnostic to its cluster identity. Confusion matrices (e.g. Fig. 2b,e,h) were used to identify correspondences between macaque and human types within each class. Shared types were defined as those exhibiting a near 1:1 correspondence using this classification approach. Each pair of shared human and macaque types were assessed for DE genes compared to other types of the same class (Fig. 5g). 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Genome-wide association studies for diabetic macular edema and proliferative diabetic retinopathy. BMC Med Genet. 19, 71, https://doi.org/10.1186/s12881-018-0587-8 (2018). 16 Scientific Reports \| (2020) 10:9802 \| https://doi.org/10.1038/s41598-020-66092-9 www.nature.com/scientificreports/ 5. Lukowski, S. W. et al. A single-cell transcriptome atlas of the adult human retina. EMBO J. 38, e100811, https://doi.org/10.15252 embj.2018100811 (2019). j ( ) 56. Hu, Y. et al. Author contributions Author contributions W.Y., Y.-R.P., T.v.Z., and J.R.S. conceived and designed experiments, analyzed data and wrote the paper. Y.-R.P. and T.v.Z., performed experiments. W.Y. performed bioinformatic analyses. K.S. and A.R. advised on computational approaches. D.J. provided human tissue. Acknowledgements g We thank the donors and their families for their generous and essential role in this research. This work was supported by National Institutes of Health grants K12 EY016335-13, R21 EY028633, K99 EY028625 and U01 MH105960 and a Chan Zuckerberg Initiative Human Cell Atlas grant CZF2019-002459. g We thank the donors and their families for their generous and essential role in this research. This work was supported by National Institutes of Health grants K12 EY016335-13, R21 EY028633, K99 EY028625 and U01 MH105960 and a Chan Zuckerberg Initiative Human Cell Atlas grant CZF2019-002459. g We thank the donors and their families for their generous and essential role in this research. This work was supported by National Institutes of Health grants K12 EY016335-13, R21 EY028633, K99 EY028625 and U01 MH105960 and a Chan Zuckerberg Initiative Human Cell Atlas grant CZF2019-002459. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66092-9. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66092-9. Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-66092-9. Correspondence and requests for materials should be addressed to J.R.S. Correspondence and requests for materials should be addressed to J.R.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W2291223100	https://europepmc.org/articles/pmc4776391?pdf=render	English	null	Knockdown of SALL4 inhibits the proliferation and reverses the resistance of MCF-7/ADR cells to doxorubicin hydrochloride	BMC molecular biology	2,016	cc-by	8,603	© 2016 Chen et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Breast cancer is the most frequent malignancy in women and drug resistance is the major obstacle for its successful chemotherapy. In the present study, we analyzed the involvement of an oncofetal gene, sal-like 4 (SALL4), in the tumor proliferation and drug resistance of human breast cancer. Results: Our study showed that SALL4 was up-regulated in the drug resistant breast cancer cell line, MCF-7/ADR, compared to the other five cell lines. We established the lentiviral system expressing short hairpin RNA to knockdown SALL4 in MCF-7/ADR cells. Down-regulation of SALL4 inhibited the proliferation of MCF-7/ADR cells and induced the G1 phase arrest in cell cycle, accompanied by an obvious reduction of the expression of cyclinD1 and CDK4. Besides, down-regulating SALL4 can re-sensitize MCF-7/ADR to doxorubicin hydrochloride (ADMh) and had potent synergy with ADMh in MCF-7/ADR cells. Depletion of SALL4 led to a decrease in IC50 for ADMh and an inhibitory effect on the ability to form colonies in MCF-7/ADR cells. With SALL4 knockdown, ADMh accumulation rate of MCF-7/ADR cells was increased, while the expression of BCRP and c-myc was significantly decreased. Furthermore, silencing SALL4 also suppressed the growth of the xenograft tumors and reversed their resistance to ADMh in vivo. Conclusion: SALL4 knockdown inhibits the growth of the drug resistant breast cancer due to cell cycle arrest and reverses tumor chemo-resistance through down-regulating the membrane transporter, BCPR. Thus, SALL4 has poten‑ tial as a novel target for the treatment of breast cancer. words: SALL4, Proliferation, Chemo-resistance, Breast cancer, Doxorubicin Keywords: SALL4, Proliferation, Chemo-resistance, Breast cancer, Doxorubicin Knockdown of SALL4 inhibits the proliferation and reverses the resistance of MCF‑7/ADR cells to doxorubicin hydrochloride Yuan‑Yuan Chen1,2†, Zhi‑Zhen Li1,2†, Yuan‑Yuan Ye1,2†, Feng Xu1,2, Rui‑Jie Niu1,2, Hong‑Chen Zhang1,2, Yi‑Jian Zhang1,2, Ying‑Bin Liu1,2 and Bao‑San Han1,2* Chen et al. BMC Molecular Biol (2016) 17:6 DOI 10.1186/s12867-016-0055-y Chen et al. BMC Molecular Biol (2016) 17:6 DOI 10.1186/s12867-016-0055-y BMC Molecular Biology BMC Molecular Biology Background Thereafter, SALLL4 aberrant expression in hematopoietic stem cells, in the leukemic cells of AML patients, and in some solid tumors has been reported by various groups [19–22]. Up-regulating SALL4 in cancer cells can promote their proliferative and invasive abilities. The overexpression of SALL4 in tumor patients often correlates with adverse progression and poor prognosis. Besides, the expression of SALL4 is higher in drug resistant primary acute mye- loid leukemic patients than those from drug-responsive cases. SALL4 characterizes a feature of drug resistance through the maintenance of side population cancer stem cells and affects the side population cells by regulation of ABC drug transport genes ABCG2 and ABCA3 in leu- kemic cells [23]. In liver cancer, SALL4 also enhances chemo-resistance by up-regulating ABCG2 [24]. In endo- metrial cancer, however, SALL4 promotes tumor chemo- resistance by activating c-myc to regulate MDR1/P-gp [25]. Furthermore, silencing SALL4 can enhance the chemo-sensitivity of tumor cells to anticancer drugs in liver cancer, endometrial cancer and lung cancer [26, 27]. Nevertheless, studies on the relation between SALL4 and drug resistance and chemotherapeutic sensitivity in breast cancer are still lacking. linked to chemotherapy resistance. In addition, overex- pression c-myc can up-regulate the expression of BCRP in human mammary epithelial cells [31]. Therefore, we are interested in determining whether P-gp, BCRP and c-Myc are related with SALL4 involved in chemotherapy resistance in breast cancer. causes of chemo-resistance [5–8].Interestingly, recent studies have shown that sal-like 4 (SALL4) gene, a mem- ber of the mammal homologs of Drosophila homeotic gene spalt, is shared by stem cells with tumor cells pro- moting their proliferation and conferring drug resist- ance on them. SALL4 gene maps to chromosome 20q13 in human and encodes zinc-finger transcription factor maintaining self-renewal and pluripotency of embry- onic stem cells through a variety of signaling pathways, transcription factors, and epigenetic modulators [9–16]. And SALL4 gene mutations are known to cause Oki- hiro syndrome, an autosomal dominant disease involv- ing dysplasia of multiple organs [17]. In mice, SALL4 homozygous knockout is embryonic lethal, and Ma et al. [18] reported that SALL4 can function as an oncogene in leukemogenesis by using a transgenic mouse model with SALL4 overexpression in most organs. Thereafter, SALLL4 aberrant expression in hematopoietic stem cells, in the leukemic cells of AML patients, and in some solid tumors has been reported by various groups [19–22]. Up-regulating SALL4 in cancer cells can promote their proliferative and invasive abilities. Cell lines and cultureh The human breast cancer cell lines, MCF-7, MDA- MB-231, SK-BR-3, ZR-75-1 and the human mammary epithelial cell line, HBL-100, were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China). Human multidrug resistant breast cancer cell line, MCF-7/ADR, was obtained from the Cancer Institute of Zhejiang University. HBL-100 was cultured in RPMI-1640 medium (Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco) as well as 1 % penicillin–streptomycin (Gibco). The other cells were all cultured in Dulbecco modified Eagle medium (DMEM, Gibco), supplemented with 10 % FBS and 1 % penicillin–streptomycin. Cells were all pas- saged by trypsinization every 2–3 days and maintained at 37 °C in 5 % CO2. And all cells were cultured following the guidelines of Laboratory of Xinhua Hospital, affiliated to Shanghai JiaoTong University, School of Medicine, China. Background The overexpression of SALL4 in tumor patients often correlates with adverse progression and poor prognosis. Besides, the expression of SALL4 is higher in drug resistant primary acute mye- loid leukemic patients than those from drug-responsive cases. SALL4 characterizes a feature of drug resistance through the maintenance of side population cancer stem cells and affects the side population cells by regulation of ABC drug transport genes ABCG2 and ABCA3 in leu- kemic cells [23]. In liver cancer, SALL4 also enhances chemo-resistance by up-regulating ABCG2 [24]. In endo- metrial cancer, however, SALL4 promotes tumor chemo- resistance by activating c-myc to regulate MDR1/P-gp [25]. Furthermore, silencing SALL4 can enhance the chemo-sensitivity of tumor cells to anticancer drugs in liver cancer, endometrial cancer and lung cancer [26, 27]. Nevertheless, studies on the relation between SALL4 and drug resistance and chemotherapeutic sensitivity in breast cancer are still lacking. In this study, we confirmed that SALL4 was overex- pressed in the drug resistant cell line, MCF-7/ADR, and we down-regulated SALL4 gene in MCF-7/ADR through the lentiviral-mediated short hairpin RNA (shRNA) interference system. Then we examined the effects of SALL4 in MCF-7/ADR cells on proliferation and sensi- tivity to doxorubicin hydrochloride (ADMh) in vitro and in vivo, and explored the possible molecular mechanisms underlying these effects, to provide experimental evi- dence for the potential application of SALL4 gene as a therapeutic target in breast cancer. Background advanced and recurrent breast cancer still has very poor prognosis due to the primary or secondary resistance to chemotherapy drugs. Chemo-resistance that limits the therapeutic potential of anticancer drugs, result- ing in failure of cancer treatment, still remains the main obstacle in terms of successful treatment of breast can- cer [3, 4]. Therefore, novel effective attempts to over- come chemo-resistance are urgently needed in order to improve the outcome of patients with advanced and recurrent breast cancer. Breast cancer, the most common neoplasm, is the lead- ing cause of cancer-induced death in women worldwide [1, 2]. Surgery combined with adjuvant therapy, such as chemotherapy and radiotherapy, may have increased the patient survival rate in some way. Unfortunately, Correspondence: phdhanbaosan@126.com †Yuan‑Yuan Chen, Zhi‑Zhen Li and Yuan‑Yuan Ye contributed equally to this work 1 Department of General Surgery and Laboratory of General Surgery, Xinhua Hospital, Affiliated with Shanghai Jiao Tong University, School of Medicine, No. 1665 Kong Jiang Road, 200092 Shanghai, China Full list of author information is available at the end of the article The mechanism of chemo-resistance is very complex, but the overexpression of some ATP-binding cassette (ABC) transporters and cancer stem cells, are two main 1 Department of General Surgery and Laboratory of General Surgery, Xinhua Hospital, Affiliated with Shanghai Jiao Tong University, School of Medicine, No. 1665 Kong Jiang Road, 200092 Shanghai, China Full list of author information is available at the end of the article Chen et al. BMC Molecular Biol (2016) 17:6 Page 2 of 11 causes of chemo-resistance [5–8].Interestingly, recent studies have shown that sal-like 4 (SALL4) gene, a mem- ber of the mammal homologs of Drosophila homeotic gene spalt, is shared by stem cells with tumor cells pro- moting their proliferation and conferring drug resist- ance on them. SALL4 gene maps to chromosome 20q13 in human and encodes zinc-finger transcription factor maintaining self-renewal and pluripotency of embry- onic stem cells through a variety of signaling pathways, transcription factors, and epigenetic modulators [9–16]. And SALL4 gene mutations are known to cause Oki- hiro syndrome, an autosomal dominant disease involv- ing dysplasia of multiple organs [17]. In mice, SALL4 homozygous knockout is embryonic lethal, and Ma et al. [18] reported that SALL4 can function as an oncogene in leukemogenesis by using a transgenic mouse model with SALL4 overexpression in most organs. Drugs and antibodies Doxorubicin hydrochloride (ADMh) and Verapamil were purchased from Sigma Chemical Co. (St. Louis, MO, USA). For in vitro studies, they were dissolved in Stroke- physiological saline solution (NS, NJCTT Pharmaceuti- cal Co., Ltd, Nangjing, China) to create a stock solution (10 mmol/L), which was stored at −20 °C. To prepare working solutions, the stock solution was further diluted with culture media to yield the desired ADMh concentra- tion. Primary antibodies against P-gp (goat-anti-rabbit) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies against c-myc, SALL4, BCRP, GAPDH and secondary antibodies (goat-anti- rabbit) were purchased from Cell Signaling Technology (Danvers, MA, USA). The human breast cancer resistance protein (BCRP) is encoded by ABCG2 gene and it is a member of the G sub- family of the large ABC transporter superfamily [28, 29]. MDR1, also named ABCB1, encodes P-glycoprotein(P- gp) and it is one of the earliest ABC transporters to be identified. The high expression of MDR1 was found in the majority of breast cancer tissues and human breast cancer drug-resistant cells [30]. P-gp and BCRP were considered to be responsible for the majority of drug efflux in human cancer. A rise in their expression had something to do with a poor prognosis in many types of cancer. C-Myc gene encodes an evolutionarily conserved basic transcription factor and its overexpression is closely Page 3 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 buffer (Beyotime, Shanghai, China) from the treated cells. Protein concentrations were quantified by the BCA protein assay reagent kit (Beyotime) according to the manufacturer’s instructions. Equal quantities of cellular proteins were resolved by SDS-PAGE and then electro- phoretically transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). Each membrane was blocked with 5 % skim milk and immuneblotted with a primary antibody. After incubation with a secondary antibody, the bands were visualized by enhanced chemi- luminescence (Millipore, Billerica, MA). GAPDH was used as the loading control. Lentivirus‑mediated RNA interference and transfection According to the gene data of SALL4 (NM‑020436) in GenBank, the designed sequence of the short hairpin RNA (shRNA) used to target SALL4 was 5′-GCCT TGAAACAAGCCAAGCTA-3′. And the sequence of the negative control shRNA was 5′-TTCTCCGAACGTG TCACGT-3′.The shRNAs were synthesized and inserted into the pGMLV-SC5 lentivirus core vector containing a cytomegalovirus-driven enhanced green fluorescent protein (GFP) reporter gene. Cell cycle analysis MCF-7/ADR cells infected by the recombinant lentivi- ruses or not were inoculated into 6 well plates for 48 h. Then, cells were harvested by trypsinization, washed twice in cold PBS, and fixed in 70 % ethanol at 4 °C over- night. Next, the cells were washed and resuspended in cold PBS and incubated in a solution of 10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma-Aldrich) at 37 °C in the dark for 30 min. Finally, the samples were analyzed by flow cytometry (BD Biosciences). The percentage of cells in the G1, S, and G2/M phases was determined using Cell Quest acquisition software (BD Biosciences). Quantitative real‑time PCR (qRT‑PCR) Total RNA was extracted from the control and transfected cells using Trizol reagent (Takara, Shiga, Japan) and reverse transcribed into cDNA with the M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA). RNA expres- sion was measured by qRT-PCR using the SYBR-Green method (Takara) according to the manufacturer’s instruc- tions. The relative expression level of the target gene was calculated by 2−ΔΔCT (ΔCT = CTtarget − CTGADPH, ΔΔCT = ΔCTtarget − ΔCTcon) and normalized to the relative expression detected in the corresponding control cells, which was defined as 1.0 [32]. GAPDH was used as the referral gene. And HBL-100 cells was used as the control cells in calculating the rela- tive expression of SALL4 among the six breast cell lines. Primer sequences were as follows: GAPDH for- ward, 5′-GAGAGACCCTCACTGCTG-3′; and reverse, 5′-GACTGGTAGATGACAAGGTGC-3′; SALL4 for- ward, 5′-CCAATAGTCAAGAAAGC-3′; and reverse, 5′-ATCGCTCCGACCTTCCATC-3′; ABCG2 forward, 5′-TTATCCGTGGTGTGTCTGGA-3′; and reverse, 5′-GATGATTGTTCGTCCCTGCT-3′; c-myc forward, 5′-GCTGCTTAGACGCTGGATTT-3′; and reverse, 5′-CGAGGTCATAGTTCCTGTTGG-3′; MDR1 for- ward, 5′-CCGCTGGTTTCCTTTAG-3′; and reverse, 5′-CTTCTTTGCTCCTCCATTGC-3′. Cell viability assayh The cells were inoculated into 96-well plates and treated according to the experimental grouping requirements. The 10 ul of CCK-8 (Dojindo, Kumamoto, Japan) was added to each well of the plate, and the plates were main- tained at 37 °C and 5 % CO2 for 3 h, and the absorb- ance at 450 nm (A450) was measured with a microplate reader (Bio-Tek, Winooski, VT, USA). The results were expressed as the average of three independent experiments. The absorbance values were used to draw growth curve and calculate the relative proliferation rate (RPR). The inhibition ratios (IR), resistance indi- ces (RI) and the changes in IC50 values (the concentra- tion of drug inhibiting 50 % of the cells) at 24 and 48 h were also calculated based on A450. RPR is the ratio of the A450 value of the tested day and that of the first day in a group cells. And RI is the ratio of IC50 values of resistant cells and that of parental cells. IR, PRP and RI were calculated according to the following formu- las: PRP = A450 (the tested day)/A450 (the first day), IR = [1 − (A450treatment − A450blank)/(A450con- trol − A450blank)] × 100 %, and RI = IC50(MCF-7/ADR cells)/IC50(MCF-7 cells). Drugs and antibodies Recombinant lentiviruses expressing SALL4-shRNA (Lv-shSALL4) or negative con- trol shRNA (Lv-shNC) were produced by Genomeditech Co.Ltd (Shanghai, China). The experiment of MCF-7/ ADR cells set up an experimental group (Lv-shSALL4), the negative control group (Lv-shNC) and blank control group (CON). MCF-7/ADR cells were infected with con- centrated virus in serum-free medium. The supernatant was replaced with complete culture medium after 24 h. After being transfected for 96 h, mRNA and protein expression of SALL4 in the infected cells was validated by quantitative real-time PCR (qRT-PCR) analysis and west- ern blot assays. Colony formation assay Cells in the logarithmic growth phase were aliquoted as single cell suspension and 400 cells were placed into Western blot analysis was carried out as described before [33]. Briefly, cellular proteins were extracted using lysis Chen et al. BMC Molecular Biol (2016) 17:6 Page 4 of 11 Lv-shNC were also randomly divided into two groups (5 mice/group) and subsequently treated as the Lv-shRNA group. On Day 20, all nude mice were anesthetized with CO2, and the tumor tissue was excised from mice and weighed. each well of 6-well plates (Corning, Corning, NY, USA). After adherence, cells were treated with ADMh (5 and 10 umol/L) for 12 h. Then, all plates were incubated for 14 days to allow colony formation. Next, cells were fixed with 4 % paraformaldehyde and stained with 0.1 % crys- tal violet (Sigma-Aldrich) for 20 min. After washing, the plates were air-dried, and stained colonies were photo- graphed with a microscope (Leica, Wetzlar, Germany). The total number of colonies (>50 cells/colony) was counted. each well of 6-well plates (Corning, Corning, NY, USA). After adherence, cells were treated with ADMh (5 and 10 umol/L) for 12 h. Then, all plates were incubated for 14 days to allow colony formation. Next, cells were fixed with 4 % paraformaldehyde and stained with 0.1 % crys- tal violet (Sigma-Aldrich) for 20 min. After washing, the plates were air-dried, and stained colonies were photo- graphed with a microscope (Leica, Wetzlar, Germany). The total number of colonies (>50 cells/colony) was counted. SALL4 is overexpressed in chemo‑resistant breast cancer cell line MCF‑7/ADR To assess the role of SALL4 in the drug resistant breast cancer cells, we detected the endogenous expression of SALL4 in the normal mammary epithelial cell line HBL-100 and five breast cancer cell lines including MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and MCF-7/ ADR by qRT-PCR and Western blot. MCF-7, MDA- MB-231, SK-BR-3 and ZR-75-1 cell lines are sensitive to chemotherapy drugs such as anthracycline, taxane and so on. But MCF-7/ADR cells are resistant to many drugs, despite the diversity in their chemical structures and mechanisms of action. And it was established from MCF-7cell line by exposing to adriamycin with step- wise increasing concentration [35]. The relative expres- sion level of SALL4 was significantly higher in MCF-7/ ADR cells compared with that in the other five cell lines (P < 0.05, Fig. 1a). And the results of western blot of SALL4 were consistent with the results of mRNA (Fig. 1b). Previously, gain- and loss-of-function studies have revealed that overexpression of SALL4 was cor- related with chemo-resistance in myeloid leukemia, endometrial cancer, lung cancer and liver cancer. Taken together, these results illustrate that SALL4 may also play an important role in regulating the resistance to chemo- therapeutics in breast cancer. Statistical analysis Statistical analyses were carried out using GraphPad Prism 5.0 software (La Jolla, CA, USA). All assays were performed independently three times and data were expressed as the mean ± SD. The independent Student’s t test was used to compare the means of two groups. The analysis of variance (ANOVA) test was performed in 2 × 2 factorial design to test a synergistic effect of shRNA-driven knockdown of SALL4 and drug treatment on tumor growth. The difference was considered statisti- cally significant when P < 0.05. Experimental animalsh This study was approved by the ethics committee of Xin- hua Hospital of Shanghai Jiaotong University,School of Medicine, China. Female BALB/c nude mice, 4–6 weeks old, were purchased from Shanghai SLAC Labora- tory Animal Co., Ltd. (Shanghai, China). The mice were housed in groups of five in specific pathogen-free con- ditions following the guidelines of Laboratory Animals and the Ethics Committee of Xinhua Hospital, affiliated to Shanghai JiaoTong University. To explore the effects of SALL4 on tumor growth and drug resistance in vivo, MCF-7/ADR cells (3 × 106) stably expressing Lv-shRNA suspended in 150 ul PBS were subcutaneously injected into the right axilla of the mice. On Day 7, these mice carrying xenograft tumor of MCF7/ADR cells infected with Lv-shSALL4 were randomly divided into two groups (5 mice/group). The first group received an intraperito- neally (i.p.) injection of NS every 3 days and the other group were administered an i.p. injection of ADMh (3 mg/kg) every 3 days. Simultaneously, these mice car- rying xenograft tumor of MCF7/ADR cells infected with Analysis of intracellular accumulation of ADMhh The assay of intracellular accumulation of ADMh were conducted as previously described [34]. In short, the intracellular accumulation of ADMh in MCF-7/ADR cells and parental MCF-7 cells was measured by flow cytometry (BD Biosciences). The excitation and emis- sion wavelengths of ADMh were 488 and 566 nm, respec- tively. The cells were incubated with ADMh (5 umol/L) for 2 h under normal cell culture conditions. Next, the cells were removed from the culture dishes by trypsini- zation and centrifuged and suspended in ice-cold PBS. Then, the samples were analyzed by flow cytometry (BD Biosciences). In another experiment, to show the effect of P-gp and BCRP inhibitor on the anthracycline drugs accumulation, cells were preincubated with Verapamil (5 umol/L) for 30 min and then incubated with ADMh (5 umol/L) for 2 h. Cellular drug fluorescence was meas- ured using an argon laser at FL2 channel. For all samples 20,000 cells were counted and the analysis was performed using flow cytometry. Knockdown of SALL4 inhibits cell proliferationf To explore the effects of SALL4 on the chemo-resistant breast cancer, we established a lentiviral system express- ing shRNA to transfect MCF-7/ADR cells. The trans- fection efficiency was confirmed by qRT-PCR (Fig. 2a) and western blot (Fig. 2f).SALL4 mRNA detection in the cells showed the shRNA sequence targeting SALL4 Page 5 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 Fig. 1 Expression of the transcription factor SALL4 (sal-like 4) in breast cell lines. a MRNA levels of SALL4 expressed in the indicated cell lines were evaluated by quantitative real-time PCR (qRT-PCR). Data are expressed as mean ± standard deviation (SD) of at least three independent experiments. P < 0.01, when compared to MCF-7/ADR cells; and b protein levels of SALL4 were evaluated by western blot in the indicated cell lines knockdown may exert a significant influence on MCF-7/ ADR cell viability and proliferation. y p Arresting cell cycle progression can inhibit the prolif- eration of cells [36]. To investigate the potential molec- ular mechanisms of SALL4 on the cell proliferation, we examined the cell-cycle analyses in MCF-7/ADR using flow cytometry, and the results showed that knockdown of SALL4 significantly changed cell cycle distributions in MCF-7/ADR cells by increasing the percentage of cells in the G1 phase accompanied with reducing the percentage of cells in the S and G2/M phase (P < 0.05, Fig. 2d), dem- onstrating typical cell cycle arresting at the G1 phase. In terms of all of the detected indices, there were no differ- ence between the Lv-shNC group and the CON group (P > 0.05). These findings are consistent with the previous reports that down-regulation of SALL4 suppresses the growth of cancer cells and induces G1 phase arrest [37, 38]. We also evaluated the levels of cycle-related proteins cyclinD1 and cyclin-dependent kinase 4 (CDK4) by qRT- PCR and western blot analysis. CyclinD1 is one of the main factors in cell cycle regulation [39, 40], which bind- ing with cyclin-dependent kinase, then leading G1 phase cells into S phase and promoting cell proliferation. A sig- nificant decrease of mRNAs and proteins (cyclinD1 and CDK4) was observed in MCF-7/ADR cells in the target gene knockdown group (Fig. 2e, f), indicating that knock- down SALL4 induced G1 phase arrest in these cells. These results suggest that SALL4 has an impact on the proliferative activity of the resistant breast cancer cells through cell cycle arrest. Fig. Knockdown of SALL4 inhibits cell proliferationf 1 Expression of the transcription factor SALL4 (sal-like 4) in breast cell lines. a MRNA levels of SALL4 expressed in the indicated cell lines were evaluated by quantitative real-time PCR (qRT-PCR). Data are expressed as mean ± standard deviation (SD) of at least three independent experiments. P < 0.01, when compared to MCF-7/ADR cells; and b protein levels of SALL4 were evaluated by western blot in the indicated cell lines Fig. 1 Expression of the transcription factor SALL4 (sal-like 4) in breast cell lines. a MRNA levels of SALL4 expressed in the indicated cell lines were evaluated by quantitative real-time PCR (qRT-PCR). Data are expressed as mean ± standard deviation (SD) of at least three independent experiments. P < 0.01, when compared to MCF-7/ADR cells; and b protein levels of SALL4 were evaluated by western blot in the indicated cell lines Silencing SALL4 re‑sensitizes MCF‑7/ADR cells to ADMh in vitro significantly inhibited SALL4 expression compared with the CON group (P < 0.001). On the contrary, the nega- tive control sequence (Lv-shNC) did not show statisti- cally effect on the target gene (P > 0.05). The results of western blot of SALL4 also coincided exactly with the results of mRNA. These data suggest that we have suc- cessfully down-regulated SALL4 in MCF-7/ADR cells by the approach lentivirus-mediated shRNA interference. Silencing SALL4 in MCF-7/ADR cells evidently enhanced its sensitivity to ADMh. As shown in the Table 1, the IC50 values of ADMh at 24 and 48 h in the Lv-shSALL4 group decreased from (152.57 ± 2.33) and (29.66 ± 1.14) umol/L to (42.09 ± 3.42) and (6.48 ± 0.42) umol/L, respectively. And RI also became apparently lower than that in the CON group (P < 0.01). Besides this, a marked inhibition of colony forma- tion ability was observed in the colony formation assay. The colony count and area were smaller in Lv-shSALL4 group (P < 0.01, Fig. 3). And the ANOVA test indicated there was a synergistic effect of SALL4 knockdown and ADMh treatment on inhibition of colony formation abil- ity (P < 0.05). The results mentioned above suggest that knockdown SALL4 can re-sensitize MCF-7/ADR cells to ADMh and reduce it growth. pp By comparing the growth curves of MCF-7/ADR cells with or without SALL4 knockdown, SALL4 knock- down seemed to significantly inhibit the cell viability. The cell viability in SALL4 knockdown group was sig- nificantly lower than that in the CON group at the third day (P < 0.05), and the inhibitory effect on cell viabil- ity became more obvious at the fourth and fifth day (P < 0.001, Fig. 2b). The relative proliferation rate also indicated cell proliferation was markedly reduced in Lv- shSALL4 group since the third day when compared to the CON group (P < 0.05, Fig. 2c).But there was no observ- able difference between the CON group and the Lv- shNC group (P > 0.05). According to those data, SALL4 The cytotoxic effect of anticancer drugs is associated with the dose which can be accumulated in the cells [41]. In order to investigate the potential mechanisms of SALL4 and drug resistance of MCF-7/ADR cells, we Page 6 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 detected intracellular accumulation rate of ADMh in compared with the CON group (P < 0 01) Moreover we Fig. Silencing SALL4 re‑sensitizes MCF‑7/ADR cells to ADMh in vitro CON group * P < 0.01 Intervention time/h IC50 (uM) Resistance index (RI) MCF-7 MCF-7/ADR MCF-7/ADR CON Lv-shNC Lv-shSALL4 CON Lv-shNC Lv-shSALL4 24 1.06 ± 0.09 152.57 ± 2.33 149.03 ± 3.28 42.09 ± 3.42 143.93 ± 2.20 140.60 ± 3.60 39.70 ± 33.23 48 0.22 ± 0.01 29.66 ± 1.14 30.01 ± 1.88 6.48 ± 0.42 134.82 ± 5.17 136.41 ± 5.40 29.63 ± 1.59 Fig. 3 Synergistic cytotoxic effect of SALL4 knockdown and ADMh on MCF-7/ADR cells. a The photo-micrographic differences, and b, c influence of colonies (mean ± SD, n = 3) on colony formation are shown. All data were from three independent experiments. MCF-7/ADR cells were treated with various concentration of ADMh (0, 5 and 10 μmol/L) for 12 h and were allowed to form colonies in fresh medium for 14 days, P < 0.01, P < 0.001, when compared to the CON group Table 1 The analysis of the IC50 and RI of MCF-7 and MCF-7/ADR cells towards ADMh Lv-shSALL4 group vs. CON group * P < 0.01 Intervention time/h IC50 (uM) Resistance index (RI) MCF-7 MCF-7/ADR MCF-7/ADR CON Lv-shNC Lv-shSALL4 CON Lv-shNC Lv-shSALL4 24 1.06 ± 0.09 152.57 ± 2.33 149.03 ± 3.28 42.09 ± 3.42 143.93 ± 2.20 140.60 ± 3.60 39.70 ± 33.23 48 0.22 ± 0.01 29.66 ± 1.14 30.01 ± 1.88 6.48 ± 0.42 134.82 ± 5.17 136.41 ± 5.40 29.63 ± 1.59 Table 1 The analysis of the IC50 and RI of MCF-7 and MCF-7/ADR cells towards ADMh Table 1 The analysis of the IC50 and RI of MCF-7 and MCF-7/ADR Fig. 3 Synergistic cytotoxic effect of SALL4 knockdown and ADMh on MCF-7/ADR cells. a The photo-micrographic differences, and b, c influence of colonies (mean ± SD, n = 3) on colony formation are shown. All data were from three independent experiments. MCF-7/ADR cells were treated with various concentration of ADMh (0, 5 and 10 μmol/L) for 12 h and were allowed to form colonies in fresh medium for 14 days, P < 0.01, P < 0.001, when compared to the CON group Fig. 3 Synergistic cytotoxic effect of SALL4 knockdown and ADMh on MCF-7/ADR cells. a The photo-micrographic differences, and b, c influence of colonies (mean ± SD, n = 3) on colony formation are shown. All data were from three independent experiments. Silencing SALL4 re‑sensitizes MCF‑7/ADR cells to ADMh in vitro 2 Down-regulation of SALL4 inhibits proliferation and changes cell cycle distributions in MCF-7/ADR cells. a MRNA levels of SALL4 in the indi‑ cated cells were assessed by qRT-PCR (*P < 0.001); and b growth curves of MCF-7/ADR cells and c the relative proliferation rate of the cells with or without SALL4 knockdown (P < 0.05 and **P < 0.001); and d cell cycle distribution in percentages of different groups (P < 0.05 and P < 0.01); and e effects of SALL4 on the mRNA levels of cyclinD1 and CDK4 genes. GAPDH was used as the referral gene. (P < 0.01); and f the levels of indi‑ cated proteins, GAPDH was used as the loading control, and the experiments were performed in triplicate Fig. 2 Down-regulation of SALL4 inhibits proliferation and changes cell cycle distributions in MCF-7/ADR cells. a MRNA levels of SALL4 in the indi‑ cated cells were assessed by qRT-PCR (**P < 0.001); and b growth curves of MCF-7/ADR cells and c the relative proliferation rate of the cells with or without SALL4 knockdown (P < 0.05 and **P < 0.001); and d cell cycle distribution in percentages of different groups (P < 0.05 and P < 0.01); and e effects of SALL4 on the mRNA levels of cyclinD1 and CDK4 genes. GAPDH was used as the referral gene. (P < 0.01); and f the levels of indi‑ cated proteins, GAPDH was used as the loading control, and the experiments were performed in triplicate detected intracellular accumulation rate of ADMh in MCF-7/ADR and parental MCF-7 cells. As shown in Fig. 4a, c, a markedly higher intracellular accumulation of ADMh was observed in the cells of Lv-shSALL4 group compared with the CON group (P < 0.01). Moreover, we also found that the intracellular accumulation of ADMh in the Verapamil pretreated cells was higher in MCF-7/ ADR cells. But the ADMh accumulation rate was similar Page 7 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 in parental MCF-7 cells with Verapamil pretreated or not (P > 0 05 Fig 4b d) Furthermore the ANOVA c-myc in SALL4-knocked-down MCF-7/ADR cells was effectively inhibited while the function of MDR1in Table 1 The analysis of the IC50 and RI of MCF-7 and MCF-7/ADR cells towards ADMh Lv-shSALL4 group vs. Silencing SALL4 re‑sensitizes MCF‑7/ADR cells to ADMh in vitro MCF-7/ADR cells were treated with various concentration of ADMh (0, 5 and 10 μmol/L) for 12 h and were allowed to form colonies in fresh medium for 14 days, P < 0.01, P < 0.001, when compared to the CON group c-myc in SALL4-knocked-down MCF-7/ADR cells was effectively inhibited, while the function of MDR1in SALL4-knocked-down MCF-7/ADR cells was not signifi- cantly inhibited. Verapamil is a competitive inhibitor of both P-gp and BCRP, and they are ABC trans-membrane efflux proteins sharing the ability to actively extrude drugs and toxins out of the cells, which may conduce to increasing the drug resistance in cancer cells. ADMh is the substrate of both P-gp and BCRP. The intracellular accumulation rate of ADMh will increase when anyone of the trans-membrane proteins is inhibited. In view of this, we assumed that the accumulation rate of SALL4- knocked-down MCF-7/ADR cells was higher than the in parental MCF-7 cells with Verapamil pretreated or not (P > 0.05, Fig. 4b, d). Furthermore, the ANOVA test indicated that knockdown of SALL4 had synergy with Verapamil to increase ADMh accumulation rate in MCF-7/ADR cells (P < 0.05).We also investigated the effect of SALL4 on membrane transporters, BCRP and P-gp, as well as c-myc by qRT-PCR and western blot. With SALL4 knockdown, the expression of both c-myc and ABCG2/BCRP in MCF-7/ADR cells decreased sig- nificantly (P < 0.01). But with SALL4 knockdown, the expression of MDR1/P-gp only had a slight declining tendency, when compared to the CON group (P > 0.05, Fig. 4e, f). In other words, the function of ABCG2 and Page 8 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 Fig. 4 ADMh accumulation rates of MCF-7/ADR and MCF-7 cells and effects of SALL4 on ABCG2, MDR1 and c-myc. a, b The intracellular accumu‑ lation of ADMh in the tested cells was analyzed by flow cytometry, and c, d the intracellular accumulation of ADMh in percentages of different groups. All data were from three independent experiments. P < 0.01, when compared to the CON group; and e effects of SALL4 on the mRNA levels of indicated genes. GAPDH was used as the referral gene. All data were from 3 independent experiments. P < 0.01, when compared with the CON group; and f effects of SALL4 on the levels of indicated proteins. Results were representative of 3 independent experiments. GAPDH was used as the loading control Fig. Silencing SALL4 re‑sensitizes MCF‑7/ADR cells to ADMh in vitro 4 ADMh accumulation rates of MCF-7/ADR and MCF-7 cells and effects of SALL4 on ABCG2, MDR1 and c-myc. a, b The intracellular accumu‑ lation of ADMh in the tested cells was analyzed by flow cytometry, and c, d the intracellular accumulation of ADMh in percentages of different groups. All data were from three independent experiments. P < 0.01, when compared to the CON group; and e effects of SALL4 on the mRNA levels of indicated genes. GAPDH was used as the referral gene. All data were from 3 independent experiments. P < 0.01, when compared with the CON group; and f effects of SALL4 on the levels of indicated proteins. Results were representative of 3 independent experiments. GAPDH was used as the loading control Chen et al. BMC Molecular Biol (2016) 17:6 Page 9 of 11 Fig. 5 Knockdown of SALL4 re-sensitizes the in vivo tumor to chemotherapy. a Tumors were excised from the animals and photos of them were presented to show the sizes of the resulting tumors; and b tumors were weighed. P < 0.001, when compared to the Lv-shNC group cells without SALL4 knockdown due to ABCG2/BCRP down-regulation. And the accumulation rate of SALL4- knocked-down MCF-7/ADR cells was increased again by Verapamil treatment caused by MDR1/P-gp inhibition. This was consistent with Yhee et al. [42], who reported drug accumulation rate increased in MCF-7/ADR cells by P-gp inhibition. However, the results of MCF-7 cells were not in agreement with Rogalska et al. [43], who reported that doxorubicin accumulation rate in MCF-7 cells pre- incubated with Verapamil increased higher. The reason for this difference probably was that the concentration, 5-umol/L of ADMh, was a supersaturated concentration for MCF-7 and the membrane transport proteins can- not continue extruding ADMh out of cells in our experi- ment. According to these data, we speculated SALL4 conferred breast cancer cells on drug resistance, at least partly through manipulating ABCG2 and c-myc genes. Knockdown SALL4 of MCF-7/ADR cells can enhance intracellular ADMh accumulation, leading to a higher therapeutic effect of ADMh. SALL4 down‑regulation re‑sensitizes drug‑resistant tumor to chemotherapy in vivof SALL4 in MCF-7/ADR decreased its proliferation and induced G1 phase arrest accompanied by down-regula- tion of cell cycle related proteins (cyclinD1 and CDK4). Analyses of the IC50 values and resistance indices indi- cated knockdown of SALL4 enhanced the chemo-sen- sitivity of MCF-7/ADR cells to ADMh. The observed difference in cytotoxicity and intracellular accumulation of ADMh might be related to the inhibition of SALL4 affecting the function of BCRP, c-myc and P-gp. The strongest suppression of tumor growth in vivo observed in SALL4 knockdown combined with ADMh group sug- gested knockdown of SALL4 and ADMh had synergistic inhibition of tumor growth. In summary, our study pro- vided solid evidence that knockdown of SALL4 can sig- nificantly inhibit the proliferative captivity and reverse the drug resistance of breast cancer in vitro and in vivo; and these results suggest that SALL4 interference may have a profound effect on the chemotherapy of breast cancer. In order to increase the efficacy of breast cancer therapy, it is of great importance to identify candidate genes that are essential in cancer cell proliferation and drug resistance. SALL4, a stem cell factor plays vital roles in development and tumorigenesis. Its expression has been previously linked with tumor treatment status and drug resistance. SALL4 knockdown affects tumor growth in various malignancies including breast can- cer. However, whether up-regulation of SALL4 is related with chemo-resistance of breast cancer and the under- ling mechanisms remained unclear. In this study, using multidrug resistant breast cancer cells MCF-7/ADR and its parental MCF-7 cells, we demonstrated that SALL4 was overexpressed in MCF-7/ADR cells. Knockdown of SALL4 down‑regulation re‑sensitizes drug‑resistant tumor to chemotherapy in vivof To investigate the effects of SALL4 down-regulation on tumor growth and drug resistance in vivo, stroke-phys- iological saline solution (NS) or ADMh (3 mg/kg) were intraperitoneally injected into nude mice carrying sub- cutaneous MCF-7/ADR tumor every 3 days for up to 20 days. The tumors removed from these animals were shown in Fig. 5a, and their mean weights were provided in Fig. 5b. The results showed an obvious inhibition of tumor growth in mice with SALL4 knockdown compared with the Lv-shNC group (P < 0.001). And the ANOVA test indicated SALL4 knockdown had potent syn- ergy with ADMh to suppress the tumor growth in vivo (P < 0.05). Those were consistent well with the results of colony formation assay in vitro that knockdown of SALL4 can reduce the chemo-resistance and prolifera- tion of MCF-7/ADR cells.fi Fig. 5 Knockdown of SALL4 re-sensitizes the in vivo tumor to chemotherapy. a Tumors were excised from the animals and photos of them were presented to show the sizes of the resulting tumors; and b tumors were weighed. P < 0.001, when compared to the Lv-shNC group Fig. 5 Knockdown of SALL4 re-sensitizes the in vivo tumor to chemotherapy. a Tumors were excised from the animals and photos of them were presented to show the sizes of the resulting tumors; and b tumors were weighed. **P < 0.001, when compared to the Lv-shNC group SALL4 in MCF-7/ADR decreased its proliferation and induced G1 phase arrest accompanied by down-regula- tion of cell cycle related proteins (cyclinD1 and CDK4). Analyses of the IC50 values and resistance indices indi- cated knockdown of SALL4 enhanced the chemo-sen- sitivity of MCF-7/ADR cells to ADMh. The observed difference in cytotoxicity and intracellular accumulation of ADMh might be related to the inhibition of SALL4 affecting the function of BCRP, c-myc and P-gp. The strongest suppression of tumor growth in vivo observed in SALL4 knockdown combined with ADMh group sug- gested knockdown of SALL4 and ADMh had synergistic inhibition of tumor growth. 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Author details 1 1 Department of General Surgery and Laboratory of General Surgery, Xinhua Hospital, Affiliated with Shanghai Jiao Tong University, School of Medicine, No. 1665 Kong Jiang Road, 200092 Shanghai, China. 2 Institute of Biliary Tract Disease, Shanghai Jiao Tong University, School of Medicine, 200092 Shanghai, China. 17. Kohlhase J, Chitayat D, Kotzot D, Ceylaner S, Froster UG, Fuchs S, Mont‑ gomery T, Rosler B. SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders. Hum Mutat. 2005;26(3):176–83. 18. Ma Y, Cui W, Yang J, Qu J, Di C, Amin HM, Lai R, Ritz J, Krause DS, Chai L. SALL4, a novel oncogene, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Blood. 2006;108(8):2726–35. Abbreviations l l k 10. Rao S, Zhen S, Roumiantsev S, McDonald LT, Yuan GC, Orkin SH. Differen‑ tial roles of Sall4 isoforms in embryonic stem cell pluripotency. Mol Cell Biol. 2010;30(22):5364–80. SALL4: sal-like 4; shRNA: short hairpin RNA; ADMh: doxorubicin hydrochloride; NS: stroke-physiological saline solution; FBS: fetal bovine serum; Ver: verapamil; P-gp: P-glycoprotein; BCRP: breast cancer resistance protein; Lv-shSALL4: the experimental group; Lv-shNC: the negative control group; CON: the blank control group; h: hour; RPR: the relative proliferation rate; IR: the inhibition ratios; RI: resistance index; IC50: the concentration of drug inhibiting 50 % of the cells. 11. Yang J, Gao C, Chai L, Ma Y. A novel SALL4/OCT4 transcriptional feedback network for pluripotency of embryonic stem cells. PLoS One. 2010;5(5):e10766. 12. Bard JD, Gelebart P, Amin HM, Young LC, Ma Y, Lai R. Signal transducer and activator of transcription 3 is a transcriptional factor regulating the gene expression of SALL4. FASEB Journal: official publication of the Federation of American Societies for Experimental Biology. 2009;23(5):1405–14. Authors’ contributions YYC, ZZL, YYY, FX, RJN, HCZ, YJZ, YBL and BSH designed and conducted the study, analyzed the data. YYC, ZZL and YYY wrote the manuscript. BSH is the principal investigators, and revised and edited the manuscript. All authors read and approved the final manuscript. 13. Yang F, Yao Y, Jiang Y, Lu L, Ma Y, Dai W. Sumoylation is important for stability, subcellular localization, and transcriptional activity of SALL4, an essential stem cell transcription factor. J Biol Chem. 2012;287(46):38600–8. Conclusions In conclusion, this study suggests that knockdown of SALL4 inhibits the proliferation of MCF-7/ADR cells through arresting the cell cycle in the G1 phase Page 10 of 11 Chen et al. BMC Molecular Biol (2016) 17:6 7. Dong Y, Li L, Wang L, Zhou T, Liu JW, Gao YJ. Preliminary study of the effects of beta-elemene on MCF-7/ADM breast cancer stem cells. Genet Mol Res. 2015;14(1):2347–55. and that down-regulation of SALL4 reverses the drug resistance of breast cancer by reducing the expres- sion of ABCG2 and c-myc. However, further mech- anisms of SALL4 affecting tumor growth and drug sensitivity of tumor to chemotherapy are required to investigate. 8. Chun SY, Kwon YS, Nam KS, Kim S. Lapatinib enhances the cytotoxic effects of doxorubicin in MCF-7 tumorspheres by inhibiting the drug efflux function of ABC transporters. Biomed Pharmacother. 2015;72:37–43. 9. Yang J, Chai L, Fowles TC, Alipio Z, Xu D, Fink LM, Ward DC, Ma Y. Genome-wide analysis reveals Sall4 to be a major regulator of pluri‑ potency in murine-embryonic stem cells. Proc Natl Acad Sci USA. 2008;105(50):19756–61.f Authors’ information 14. Zhang L, Luo YB, Bou G, Kong QR, Huan YJ, Zhu J, Wang JY, Li H, Wang F, Shi YQ et al: Overexpression Nanog activates pluripotent genes in porcine fetal fibroblasts and nuclear transfer embryos. Anatomical record (Hoboken, NJ: 2007) 2011, 294(11):1809–17. Yuan-Yuan Chen, Zhi-Zhen Li, Yuan-Yuan Ye, Feng Xu, Rui-Jie Niu and Hong- Chen Zhang are surgery graduate students. Bao-San Han, Yi-Jian Zhang and Ying-Bin Liu are doctors with Ph.D. Bao-San Han is the deputy director of general surgery, and Ying-Bin Liu is the director of general surgery and Cheung Kong Scholars. And all authors are greatly interested in the treatment of tumors and surgery, especially breast cancer and gallbladder cancer. 15. Shuai X, Zhou D, Shen T, Wu Y, Zhang J, Wang X, Li Q. Overexpression of the novel oncogene SALL4 and activation of the Wnt/beta-catenin pathway in myelodysplastic syndromes. Cancer Genet Cytogenet. 2009;194(2):119–24. 16. Yang J, Aguila JR, Alipio Z, Lai R, Fink LM, Ma Y. Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4. J Hematol Oncol. 2011;4:38. Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 81172078) and the Medical Engineering Cross Foundation of Shanghai Jiaotong University (No. YG2013MS16). 19. Forghanifard MM, Moghbeli M, Raeisossadati R, Tavassoli A, Mallak AJ, Boroumand-Noughabi S, Abbaszadegan MR. Role of SALL4 in the pro‑ gression and metastasis of colorectal cancer. J Biomed Sci. 2013;20:6. Competing interests The authors declare that they have no competing interests. 20. Aguila JR, Liao W, Yang J, Avila C, Hagag N, Senzel L, Ma Y. SALL4 is a robust stimulator for the expansion of hematopoietic stem cells. Blood. 2011;118(3):576–85. Received: 1 October 2015 Accepted: 25 January 2016 21. Liao W, Aguila JR, Yao Y, Yang J, Zieve G, Jiang Y, Avila C, Senzel L, Lai R, Xu D, et al. Enhancing bone marrow regeneration by SALL4 protein. J Hematol Oncol. 2013;6:84. 22. Zhang L, Yan Y, Jiang Y, Cui Y, Zou Y, Qian J, Luo C, Lu Y, Wu X. 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Am J Cancer Res. 2015;5(7):2127–38. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: 36. Park SJ, Kong HK, Kim YS, Lee YS, Park JH. 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https://openalex.org/W4293239265	https://www.researchsquare.com/article/rs-1592437/latest.pdf	English	null	The risk factors and outcomes for radiological abnormalities in early convalescence of COVID-19 patients caused by the SARS-CoV-2 Omicron variant in Tianjin, China: a retrospective, multicenter follow-up study	Research Square (Research Square)	2,022	cc-by	9,461	The risk factors and outcomes for radiological abnormalities in early convalescence of COVID-19 patients caused by the SARS-CoV-2 Omicron variant in Tianjin, China: a retrospective, multicenter follow- up study The risk factors and outcomes for radiological abnormalities in early convalescence of COVID-19 patients caused by the SARS-CoV-2 Omicron variant in Tianjin, China: a retrospective, multicenter follow- up study j p up study Hong Wang Tianjin Institute of imaging medicine, Tianjin First Central Hospital Qingyuan Yang Haihe Hospital, Tianjin University, Tianjin Institute of Respiratory Diseases Fangfei Li Tianjin Institute of imaging medicine, Tianjin First Central Hospital Huiying Wang Nankai University Jing Yu Tianjin Institute of imaging medicine, Tianjin First Central Hospital Xihong Ge Tianjin Institute of imaging medicine, Tianjin First Central Hospital Guangfeng Gao Tianjin Institute of imaging medicine, Tianjin First Central Hospital Shuang Xia Tianjin Institute of imaging medicine, Tianjin First Central Hospital Zhiheng Xing Haihe Hospital, Tianjin University, Tianjin Institute of Respiratory Diseases Wen Shen (  shenwen66happy@163.com ) Tianjin Institute of imaging medicine, Tianjin First Central Hospital Research Article Keywords: COVID-19, convalescence, CT abnormalities, follow-up, Omicron, residual pulmonary abnormalities, risk factors, SARS-CoV-2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research Article Posted Date: April 28th, 2022 se:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read cense License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 2/25 Abstract Background: The emergence of the SARS-CoV-2 Omicron variant has been triggering the new wave of COVID- 19 globally. However, the risk factors and outcomes for radiological abnormalities in the early convalescent stage (1 month after onset) of Omicron infected patients are still unknown. Methods: Patients were retrospectively enrolled if they were admitted to the hospital due to COVID-19 and underwent chest CT scans during hospitalization between 8 January and 24 February 2022. The chest CTs and clinical data obtained at admission and 1 month after onset were longitudinally analyzed. Spearman rank correlation analysis was used to investigate the correlation between laboratory markers and CT scores. Uni-/multi-variable logistic regression tests were performed to explore independent risk factors for radiological abnormalities at admission and residual pulmonary abnormalities after 1 month. Results: We assessed 316 COVID-19 patients, including 47% with radiological abnormalities at admission and 23% with residual pulmonary abnormalities at 1-month follow-up. In a multivariate regression analysis, age ≥ 50 years, body mass index ≥ 23.87, days after vaccination ≥ 81 days, lymphocyte count ≤ 1.21×10- 9/L, Interleukin-6 ≥ 10.05 pg/mL and IgG ≤ 14.140 S/CO were independent risk factors for CT abnormalities at admission (odds ratio = 2.870, 4.171, 2.360, 2.826, 3.886 and 2.828, respectively; P < 0.05 for each comparison). The presence of interlobular septal thickening and IL-6 ≥ 5.85 pg/mL were the independent risk factors for residual pulmonary abnormalities at 1-month follow-up (odds ratio = 3.619 and 3.197, respectively; P < 0.05 for each comparison). There were no significant changes in the number of involved lung lobes (P = 0.308) and total CT score (P = 0.068) during the early convalescent stage. And the level of IL- 6 showed positive correlation with CT score at admission (spearman r = 0.202, P = 0.030). Conclusions: The higher IL-6 level was a common independent risk factor for CT abnormalities at admission and residual pulmonary abnormalities at 1-month follow-up. And there were no obvious radiographic changes during the early convalescent stage in patients with residual pulmonary abnormalities. Introduction Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to global public health, and has caused more than 5 million deaths by February 25, 2022.1 On 25 November 2021, a new variant was reported to World Health Organization from South Africa, which was designated as a variant of concern and named it Omicron variant (B.1.1.529) of SARS-CoV‐2.2–4 Compared with Delta (B.1.617.2) variant, Omicron harbors a large number of the mutations that could be more infectious, more transmissible and more capable of evading from neutralizing antibodies. But it causes mild disease in a majority of patients with a favorable prognosis due to vaccination or natural infection.4,5 Unfortunately, Tianjin became the first city to face the challenges of the first wave of the Omicron pandemic in China on January 8, 2022. Although the nucleic acid amplification test is the only method confirmed by WHO for diagnosing SARS-CoV- 2 infections, chest CT remains an essential tool for disease evaluation.6–8 Numbers of studies have described the typical chest CT manifestations of COVID-19 pneumonia, including ground-glass opacity Page 3/25 (GGO) and consolidation with bilateral peripheral distribution, mainly in the lower lung lobes.9,10 Recently, the efforts have focused on the dynamic radiographic changes from the initial diagnosis to the convalescent period at 3, 6, 9 and 12 months.11–16 COVID-19 patients, especially the severe group, often sustain significant residual lung abnormalities on chest CT comprising inflammatory sequelae in 50% patients and fibrotic sequelae in 29% patients at the time of discharge or at follow-up.13–16 But little is known about the radiological features at admission and outcomes in early convalescence (1 month after onset) of the Omicron infected patients. According to “Diagnosis and Treatment Protocol for Coronavirus Disease 2019 (trial version 8)” issued by the National Health Commission of the People’s Republic of China, COVID-19 patients were required to follow up at 2 and 4 weeks after discharge with or without imaging surveillance.17 But imaging follow-up is recommended for COVID-19 patients 3 months after discharge according to the guidelines published by the British Thoracic Society.18 However, without enough follow-up studies in early convalescence, it is unknown who need follow-up CT examinations and when to do the first follow-up CT examination for the Omicron infected patients. In this study, we hypothesized some risk factors and short-term changes for radiological abnormalities in Omicron infected patients. Study Design and Population We retrospectively evaluated patients diagnosed with COVID-19 and admitted to Tianjin First Central Hospital for convalescence from January 22 to February 24, 2022 after leaving Haihe Hospital (Fig. 1). As shown in Fig. 1, patients were excluded if they were less than 14 years old, or infected with Delta variant, or with bacterial pneumonia, or without two serial chest CT examinations at admission and one month after onset. Enrolled patients were divided into two groups: patients with CT abnormalities and patients without CT abnormalities at admission in Haihe Hospital. Patients with CT abnormalities were further divided into two subgroups: complete resolution (CR) and residual abnormalities (RA) at 1-month follow-up in Tianjin First Central Hospital. Introduction We systematically reviewed the clinical data of enrolled patients and assessed the impact of potential risk factors, including demographic characteristics, general condition, laboratory markers and radiological features. Secondly, we sought to determine whether the changes of radiological abnormalities detected in patients at admission and 1 month after onset were significantly different, which may provide evidence to make optimal follow-up plans for different patients. Chest CT Scans and Image Analysis Non-contrast chest CT examinations were performed using two Canon 64-slice spiral CT scanners (Siemens Go-Top and Toshiba Canon). Images were obtained during breath-holding at full inspiration from the apex to the base of the lungs. The acquisition parameters were as follows: patient in the supine position; tube voltage, 120kVp with automatic tube current modulation; scan speed, 0.5 s/r; collimation width, 64 × 0.5 mm; matrix size, 512 × 512 and section thickness after reconstruction, 1.0mm and1.25 mm. All acquired chest CT images were independently read on a picture archiving and communication system by three chest radiologists (Li FF, Yang QY, and Yu J) with 5-, 8-, and 10-years’ experience in thoracic radiology, respectively. Afterward, any inconsistency in interpretation was resolved by discussion and consensus. All readers were blinded to the clinical progress of all patients. The CT abnormalities were described with the standard terminology defined in the Fleischner Society glossary19 and previous works of literature on COVID- 199,11,13−15. All imaging manifestations of pulmonary lesions were recorded as listed below: (1) distribution: subpleural, peribronchovascular, or both; (2) number of involved pulmonary lobes; (3) CT abnormalities: GGO, consolidations, parenchymal bands, interlobular septal thickening, or pleural involvement (including pleural thickening and/or effusion) (Fig. 2). To further quantify the extent of pulmonary lesions, a semiquantitative visual scoring method was used9. Each single lung lobe was scored according to the area percentages of the lesions in the single lung lobe, and the criteria were as followed: 0, 0%; 1, < 25%; 2, 25–49%; 3, 50–74%; and 4, > 75%. The summation scores of the five lobes were calculated as the total CT score, ranging from 0 to 20. Demographic and Clinical Data Patient demographic characteristics, clinical information, and laboratory parameters were retrieved from the electronic medical records. The following patient characteristics during hospitalization were recorded: age, gender, body mass index (BMI), initial symptom (including fever, pharyngodynia, cough, rhinorrhea, hyposmia/hypogeusia, myalgia, and fatigue), prior comorbidities [including hypertension, type 2 diabetes Page 4/25 Page 4/25 mellitus (type 2 DM), cardio-cerebral vascular diseases (CCVD) and prior lung disease], liver dysfunction during hospitalization, length of hospital stay (LHS), vaccination [including fully vaccinated (three doses of vaccine), partially vaccinated (one or two doses of vaccine) and not vaccinated], days after vaccination (DAV), and vaccine type (including inactivated, adenovirus vector, and recombinant vaccine). The laboratory data were recorded as follows: blood routine [including white cell blood (WBC) count, neutrophil (NEUT) count, lymphocyte (LY) count, and platelet (PLT) count), inflammatory markers (C-reactive protein (CRP) and interleukin-6 (IL-6)], and SARS-CoV-2 test (IgM and IgG). mellitus (type 2 DM), cardio-cerebral vascular diseases (CCVD) a during hospitalization, length of hospital stay (LHS), vaccination vaccine), partially vaccinated (one or two doses of vaccine) and n (DAV), and vaccine type (including inactivated, adenovirus vector data were recorded as follows: blood routine [including white cell count, lymphocyte (LY) count, and platelet (PLT) count), inflamm interleukin-6 (IL-6)], and SARS-CoV-2 test (IgM and IgG). Statistical Analysis Normally distributed continuous variables were expressed as mean ± standard deviation (x ± s), and between- group comparisons were subjected to independent sample t-tests. Non-normally distributed continuous variables were expressed as median (interquartile ranges [IQRs]), and compared using the Mann–Whitney U- test. The categorical variables were expressed as counts and percentages, and compared using chi-square test and Fisher’s test. To compare CT abnormalities between admission and 1-month follow-up, Wilcoxon signed-rank test or McNemar’s test were performed when appropriate. To explore the independent risk factors associated with residual pulmonary abnormalities, all the clinical, laboratory, and imaging features at admission with a P < 0.05 were further assessed in the multivariate logistic regression analysis (Enter method), and odds ratios with 95% CIs were calculated. Furthermore, Spearman rank correlation analysis was used to test for correlations between laboratory parameters and CT scores. Page 5/25 Page 5/25 All the statistical analyses were performed using SPSS statistics software (IBM Corp., Armonk, NY, USA, version 27.0). All significance tests were two-sided, and statistical significance was defined as P < 0.05. All correlation graphs were generated in R (version 3.5.2), OriginPro (version 2020), and GraphPad Prism (version 8.0). Characteristics of the Study Population A total of 316 patients were screened for our study, and their demographic and clinical characteristics for each group are presented in Table 1. The mean age of patients was 46 years (IQR, 34–58 years), ranging from 15 to 90 years, with a male-to-female ratio of 1:1.34. The mean BMI of patients was 25.08 (IQR, 22.31– 27.76). Pharyngodynia (53%), fever (29%), and cough (49%) were the most common initial symptoms. 42% of patients had prior comorbidities, including hypertension (22%), type 2 DM (11%), CCVD (9%), and chronic pulmonary disease (1%). There were 18% of patients with liver dysfunction during hospitalization. The mean length of hospital stay was 13 days (IQR, 11–15 days). More than 90% of patients were vaccinated, including 43% with complete vaccination and 50%. And more than 80% of patients were vaccinated with inactivated vaccine, while 17% of patients were vaccinated with adenovirus vector vaccine. The mean days after vaccination was 78 days (IQR, 42–194 days). Page 6/25 Table 1 Demographics and clinical characteristics for each group at admission Characteristic All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value Age, y 46 (34, 58) 40 (32, 53) 53 (38, 63) 0.000 41 (31, 58) 58 (47, 66) 0.000 Gender 0.523 0.434 Male 135 (42.7) 75 (44.4) 60 (40.8) 18 (36.0) 31 (43.1) Female 181 (57.3) 94 (55.6) 87 (59.2) 32 (64.0) 41 (56.9) BMI, kg/m2 25.08 (22.31,27.76) 23.85 (21.61, 27.23) 25.60 (23.79, 28.13) 0.001 25.39 (23.20, 27.93) 25.39 (23.87, 27.98) 0.958 Initial symptom Fever 93 (29.4) 52 (30.8) 41 (7.98) 0.576 14 (28.0) 20 (27.8) 0.979 Pharyngodynia 167 (52.8) 98 (58.0) 69 (77.7) 0.050 24 (48.0) 34 (47.2) 0.933 Cough 154 (48.7) 90 (53.3) 64 (43.5) 0.085 22 (44.0) 30 (41.7) 0.798 Rhinorrhea 45 (14.2) 23 (13.6) 22 (15.0) 0.731 10 (20.0) 10 (13.9) 0.370 Data are presented as No. (%), or median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. Characteristics of the Study Population BMI = Body Mass Index; DM = diabetes mellitus; CCVD = Cardio-Cerebral Vascular Diseases; LDDH = Liver Dysfunction During Hospitalization; LHS = Length of Hospital Stay; DAV = Days After Vaccination; CR = Complete Resolution; RA = Residual Abnormalities. The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. F i fl t k th di l l f CRP d IL 6 4 01 (IQR 1 60 8 74) /L d 6 90 Table 1 Table 1 The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. For inflammatory markers, the median levels of CRP and IL-6 were 4.01 (IQR, 1.60–8.74) mg/L and 6.90 (IQR, 4.30–10.50) pg/mL, respectively. For SARS-CoV-2 test, the median levels of IgM and IgG were 0.19 (IQR, 0.09–0.42) S/CO and 20.24 (IQR, 2.18–62.14) S/CO, respectively. Although the median values of almost all laboratory tests were within normal limits, increases in levels of IgG were observed. Characteristics of the Study Population Page 7/25 Characteristic All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value Hyposmia and /or Hypogeusia 6 (1.9) 3 (1.8) 3 (2.0) 1.000 2 (4.0) 1 (1.4) 0.567 Myalgia 23 (7.3) 9 (5.3) 14 (9.5) 0.152 8 (16.0) 4 (5.6) 0.110 Fatigue 25 (7.9) 12 (7.1) 13 (8.8) 0.567 5 (10.0) 7 (9.7) 1.000 Prior comorbidities Hypertension 68 (21.5) 25 (14.8) 43 (29.3) 0.002 14 (28.0) 21 (29.2) 0.889 Type 2 DM 35 (11.1) 7 (4.1) 28 (19.0) 0.000 7 (14.0) 17 (23.6) 0.189 CCVD 27 (8.5) 9 (5.3) 18 (12.2) 0.028 5 (10.0) 11 (15.3) 0.396 Prior lung diseases 4 (1.3) 3 (1.8) 1 (0.7) 0.626 0 (0.0) 1 (1.4) 0.590 LDDH 58 (18.4) 22 (13.0) 36 (24.5) 0.009 12 (24.0) 19 (26.4) 0.766 LHS, d 13 (11, 15) 12 (11, 14) 13 (12, 15) 0.000 14 (12, 16) 13 (12, 15) 0.176 Vaccination 0.015 0.214 Data are presented as No. (%), or median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. BMI = Body Mass Index; DM = diabetes mellitus; CCVD = Cardio-Cerebral Vascular Diseases; LDDH = Liver Dysfunction During Hospitalization; LHS = Length of Hospital Stay; DAV = Days After Vaccination; CR = Complete Resolution; RA = Residual Abnormalities. The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR 2 21 4 40) ×10−9/L 1 37 (IQR 0 97 1 90) ×10−9/L and 221 (IQR 184 255) ×10−9/L respectively The P values reflect comparisons between patients with CT abnormalities and or patients with complete resolution and residual CT abnormalities, respective The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. Characteristics of the Study Population For inflammatory markers, the median levels of CRP and IL-6 were 4.01 (IQR, 1.60–8.74) mg/L and 6.90 (IQR, 4.30–10.50) pg/mL, respectively. For SARS-CoV-2 test, the median levels of IgM and IgG were 0.19 (IQR, 0.09–0.42) S/CO and 20.24 (IQR, 2.18–62.14) S/CO, respectively. Although the median values of almost all laboratory tests were within normal limits, increases in levels of IgG were observed. Page 8/25 Characteristic All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value Complete vaccination 159 (50.3) 95 (56.2) 64 (43.5) < 0.05 24 (48.0) 24 (33.3) Partial vaccination 136 (43.0) 68 (40.2) 68 (46.5) ༞0.05 20 (40.0) 40 (55.6) Not vaccination 21 (6.6) 6 (3.6) 15 (10.2) < 0.05 6 (12.0) 8 (11.1) Vaccine type 0.209 0.718 Inactivated vaccine 244 (82.7) 133 (81.6) 111 (84.1) 37 (84.1) 52 (81.3) Adenovirus vector vaccine 49 (16.6) 30 (18.4) 19 (14.4) 7 (15.9) 10 (15.6) Recombinant vaccine 2 (0.7) 0 (0.0) 2 (1.5) 0 (0.0) 2 (3.1) DAV, d 78 (42, 194) 65 (40, 155) 108 (45, 201) 0.023 110 (48, 197) 143 (54, 202) 0.552 Data are presented as No. (%), or median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. BMI = Body Mass Index; DM = diabetes mellitus; CCVD = Cardio-Cerebral Vascular Diseases; LDDH = Liver Dysfunction During Hospitalization; LHS = Length of Hospital Stay; DAV = Days After Vaccination; CR = Complete Resolution; RA = Residual Abnormalities. The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. For inflammatory markers, the median levels of CRP and IL-6 were 4.01 (IQR, 1.60–8.74) mg/L and 6.90 (IQR, 4.30–10.50) pg/mL, respectively. For SARS-CoV-2 test, the median levels of IgM and IgG were 0.19 (IQR, 0.09–0.42) S/CO and 20.24 (IQR, 2.18–62.14) S/CO, respectively. Although the median values of almost all laboratory tests were within normal limits, increases in levels of IgG were observed. Characteristics of the Study Population The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. For inflammatory markers, the median levels of CRP and IL-6 were 4.01 (IQR, 1.60–8.74) mg/L and 6.90 (IQR, 4.30–10.50) pg/mL, respectively. For SARS-CoV-2 test, the median levels of IgM and IgG were 0.19 (IQR, 0.09–0.42) S/CO and 20.24 (IQR, 2.18–62.14) S/CO, respectively. Although the median values of almost all laboratory tests were within normal limits, increases in levels of IgG were observed. The laboratory test variables in all patients at admission are shown in Table 2 and Fig. 3. For routine blood tests, the median levels of WBC, NEUT, LY, and PLT count were 5.33 (IQR, 4.41–6.85) ×10− 9/L, 3.19 (IQR, 2.21–4.40) ×10− 9/L, 1.37 (IQR, 0.97–1.90) ×10− 9/L, and 221 (IQR, 184–255) ×10− 9/L, respectively. For inflammatory markers, the median levels of CRP and IL-6 were 4.01 (IQR, 1.60–8.74) mg/L and 6.90 (IQR, 4.30–10.50) pg/mL, respectively. For SARS-CoV-2 test, the median levels of IgM and IgG were 0.19 (IQR, 0.09–0.42) S/CO and 20.24 (IQR, 2.18–62.14) S/CO, respectively. Although the median values of almost all laboratory tests were within normal limits, increases in levels of IgG were observed. Characteristics of the Study Population Page 9/25 Table 2 Laboratory parameters for each group at admission Laboratory tests All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value Blood routine WBC count, ×10− 9/L 5.33 (4.41, 6.85) 5.64 (4.55, 7.21) 5.17 (4.23, 6.37) 0.014 5.36 (4.42, 6.69) 5.02 (4.02, 6.17) 0.116 NEUT count, ×10− 9/L 3.19 (2.21, 4.40) 3.16 (2.26, 4.59) 3.22 (2.14, 4.36) 0.462 3.23 (2.31, 4.45) 3.13 (2.10, 4.10) 0.583 LY count, ×10− 9/L 1.37 (0.97, 1.90) 1.51 (1.18, 1.97) 1.26 (0.85, 1.72) 0.000 1.32 (0.86, 1.96) 1.15 (0.74, 1.48) 0.086 PLT count, ×10− 9/L 221 (184, 255) 231 (201, 277) 200 (166, 243) 0.000 199 (181, 233) 195 (161, 239) 0.612 Inflammatory markers CRP, mg/L 4.01 (1.60, 8.74) 3.37 (1.30, 8.04) 4.63 (1.99, 9.29) 0.068 4.31 (1.96, 9.79) 4.82 (2.03, 8.78) 0.883 IL-6, pg/mL 6.90 (4.30, 10.50) 6.05 (3.85, 8.60) 7.80 (5.00, 12.30) 0.000 6.65 (3.38, 11.35) 8.90 (6.03, 15.25) 0.014 SARS-CoV-2 test IgM, S/CO 0.19 (0.09, 0.42) 0.23 (0.11, 0.54) 0.14 (0.07, 0.35) 0.002 0.19 (0.87, 0.40) 0.12 (0.68, 0.25) 0.072 Data are presented as median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. WBC White blood cell; NEUT Neutrophil; LY Lymphocyte; PLT Platelet; CRP C reactive protein; IL 6 Table 2 Laboratory parameters for each group at admission Page 10/25 Laboratory tests All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value IgG, S/CO 20.24 (2.18, 62.14) 36.50 (10.07, 89.16) 5.59 (1.14, 28.71) 0.000 8.90 (0.84,29.32) 2.85 (1.02, 15.98) 0.191 Data are presented as median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6; CR = Complete Resolution; RA = Residual Abnormalities. Characteristics of the Study Population Laboratory tests All patients (n = 316) At admission (n = 316) P value At 1-month follow-up (n = 122) Without CT abnormalities (n = 169) With CT abnormalities (n = 147) CR (n = 50) RA (n = 72) P value IgG, S/CO 20.24 36.50 5.59 0.000 8.90 (0.84,29.32) 2.85 0.191 (2.18, 62.14) (10.07, 89.16) (1.14, 28.71) (1.02, 15.98) Data are presented as median (interquartile range). Data are presented as median (interquartile range). The P values reflect comparisons between patients with CT abnormalities and without CT abnormalities, or patients with complete resolution and residual CT abnormalities, respectively. WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6; CR = Complete Resolution; RA = Residual Abnormalities. or patients with complete resolution and residual CT abnormalities, respectively. WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6; CR = Complete Resolution; RA = Residual Abnormalities. WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6; CR = Complete Resolution; RA = Residual Abnormalities. The CT features at admission are shown in Table 3. Regarding CT abnormalities, GGO (98%), consolidation (40%), and interlobular septal thickening (35%) were the predominant findings (Fig. 4 and Table 3). And most of the lesions (85%) were distributed in the subpleural area, and often involved lower lobes (71% for the left lower lobe and 73% for the right lower lobe, respectively). The median number of involved lung lobes is 3 (IQR, 1–5), and the median total CT score is 3 (IQR, 1–5). Table 3. Characteristics of the Study Population Comparison of CT features between CR and RA at admission and CT patterns of RA at 1 month after onset Page 11/25 Features All patients (n = 122) CR at admission (n = 50) RA at admission (n = 72) P value RA at 1 month (n = 72) Pa value Distribution 0.478 Subpleural 104 (85.2) 42 (84.0) 62 (86.1) 55 (76.4) 0.230 Peribronchovascular 14 (11.5) 5 (10.0) 9 (12.5) 4 (5.6) 0.227 Both 4 (3.3) 3 (6.0) 1 (1.4) 13 (18.1) 0.002 Pulmonary involvement Left upper lobe 63 (51.6) 23 (46.0) 40 (55.6) 0.299 40 (55.6) 1.000 Left lower lobe 86 (70.5) 34 (68.0) 52 (72.2) 0.615 58 (80.6) 0.238 Right upper lobe 60 (49.2) 22 (44.0) 38 (52.8) 0.340 44 (61.1) 0.337 Right middle lobe 52 (42.6) 17 (34.0) 35 (48.6) 0.109 34 (47.2) 1.000 Right lower lobe 89 (73.0) 36 (72.0) 53 (73.6) 0.844 61 (84.7) 0.115 Number of involved lung lobes 3 (1, 5) 2 (1, 4) 3 (1, 5) 0.228 4 (2, 5) 0.308 CT abnormalities GGO 119 (97.5) 48 (96.0) 71 (98.6) 0.567 72 (100) 1.000 Consolidation 49 (40.2) 19 (38.0) 30 (41.7) 0.685 14 (19.4) 0.010 Parenchymal bands 21 (17.2) 8 (16.0) 13 (18.1) 0.767 21 (29.2) 0.115 Interlobular septal thickening 43 (35.2) 10 (20.0) 33 (45.8) 0.003 16 (22.2) 0.004 Pleural involvement 31 (25.4) 9 (18.0) 22 (30.6) 0.117 5 (6.9) < 0.001 Data are presented as No. (%), or median (interquartile range). The P values reflect comparisons between CR and with RA at admission. Features All patients (n = 122) CR at admission (n = 50) RA at admission (n = 72) P value RA at 1 month (n = 72) Pa value CT score assessments Left upper lobe 0 (0, 1) 0 (0, 1) 1 (0, 1) 0.272 1 (0, 1) 0.274 Left lower lobe 1 (0, 1) 1 (0, 1) 1 (0, 1) 0.361 1 (1, 1) 0.664 Right upper lobe 0 (0, 1) 0 (0, 1) 1 (0, 1) 0.339 1 (0, 1) 0.238 Right middle lobe 0 (0, 1) 0 (0, 1) 0 (0, 1) 0.168 0 (0, 1) 0.974 Right lower lobe 1 (0, 1) 1 (0, 1) 1 (0, 1) 0.969 1 (1, 2) 0.011 Total CT Score 3 (1, 5) 2 (1, 4) 3 (1, 5) 0.250 4 (2, 5) 0.068 Data are presented as No. (%), or median (interquartile range). Characteristics of the Study Population The P values reflect comparisons between CR and with RA at admission. The Pa values reflect comparisons of RA between admission and 1 month after symptom onset. GGO = ground-glass opacity; CR = Complete Resolution; RA = Residual Abnormalities. Data are presented as No. (%), or median (interquartile range). The P values reflect comparisons between CR and with RA at admission. The P values reflect comparisons between CR and with RA at admission. Radiological Changes of RA from Admission to 1-Month F ll Over time, predominant GGOs (100%) were remain the most common CT abnormalities patients with residual CT abnormalities, without statistically significant changes at 1 month (P = 1.000). And consolidation, interlobular septal thickening and pleural involvement significantly decrease over time (19% vs. 42%, P = 0.010, 22% vs. 46%, P = 0.004 and 7% vs. 31%, P < 0.001, respectively) (Table 3, Fig. 4). The detection rate of both subpleural and peribronchovascular distribution was significantly higher after 1 month (18% vs. 1%, P = 0.002). But there were no significant changes in the number of involved lung lobes [3 (IQR, 1–5) vs. 4 (IQR, 2–5), P = 0.308] and total CT score [3 (IQR, 1–5) vs. 4 (IQR, 2–5), P = 0.068] from admission to 1 month (Table 3, Fig. 4). Comparisons between Patients with and without CT Abnormalities at Admission At admission, there were 47% patients with CT abnormalities and 53% patients without CT abnormalities (Fig. 1). For demographics and clinical characteristics, the median age [40 (IQR, 32–53) years vs. 53 (IQR, 38–63) years, P < 0.001] and BMI [23.85 (IQR, 21.61–27.23) vs. 25.74 (IQR, 23.86–28.26), P = 0.001] of patients with CT abnormalities was significantly larger than those without CT abnormalities (Table 1). The proportion of patients with hypertension, type 2 DM and CCVD at admission, liver dysfunction during hospitalization and not vaccination in patients with CT abnormalities was significantly higher than those without CT abnormalities (P < 0.05 for each comparison) (Table 1). The median LHS and DAV of patients with CT abnormalities was significantly longer than those without CT abnormalities [13 (IQR, 12–15) days vs. 12 (IQR, 11–14) days, P < 0.001) and 108 (IQR, 45–201) days vs. 65 (IQR, 40–155) days, P = 0.023), respectively] (Table 1). Regarding laboratory parameters, patients with CT abnormalities had significantly higher level of IL-6 [7.80 (IQR, 5.00-12.30) pg/mL vs. 6.05 (IQR, 3.80–8.60) pg/mL, P < 0.001], but had lower level of WBC count [5.17 (IQR, 4.23–6.37) ×10− 9/L vs. 5.64 (IQR, 4.55–7.21) ×10− 9/L, P = 0.014], LY count [1.26 (IQR, 0.85–1.72) ×10− 9/L vs. 1.51 (IQR, 1.18–1.97) ×10− 9/L, P < 0.001], PLT count [200 (IQR, 166–243) ×10− 9/L vs. 231 (IQR, 201–277) ×10− 9/L, P < 0.001], IgM [0.14 (IQR, 0.07–0.35) S/CO vs. 0.23 (IQR, 0.11– 0.54) S/CO, P = 0.002] and IgG [5.59 (IQR, 1.14–28.71) S/CO vs. 36.50 (IQR, 10.07–89.16) S/CO, P < 0.001] than those without CT abnormalities (Table 2). Page 13/25 Age ≥ 50 years, BMI ≥ 23.87, DAV ≥ 81 days, LY count ≤ 1.21×10− 9/L, IL-6 ≥ 10.05 pg/mL and IgG ≤ 14.140 S/CO were independent risk factors for CT abnormalities at admission (odds ratio = 2.870, 4.171, 2.360, 2.826, 3.886 and 2.828, respectively; P < 0.05 for each comparison) (Fig. 5, 6). And CT score of patients with CT abnormalities showed low correlations with the level of IL-6 (spearman r = 0.202, P = 0.030) (Fig. 7). Comparisons between CA and RA at admission For patients with CT abnormalities, there were 41% patients with CR and 59% patients with RA (Fig. 1). The median age of patients with CT abnormalities was significantly larger than those without CT abnormalities (58, (IQR, 47–66) years vs. 41, (IQR, 31–58) years, P < 0.001) (Table 1). Regarding laboratory findings, levels of IL-6 differed between the two groups; patients with RA had significantly higher level of [8.90 (IQR, 6.03– 15.25) pg/mL vs. 6.65 (IQR, 3.38–11.35) pg/mL, P = 0.014] than those with CA (Table 2). On the CT examinations at admission, the detection rates of interlobular septal thickening (46% vs. 20%, P = 0.003) were significantly higher in patients with CT RA (Table 3). The presence of interlobular septal thickening and IL-6 ≥ 5.85 pg/mL were the independent risk factors for residual pulmonary abnormalities at 1 month after symptom onset (odds ratio = 3.619 and 3.197, respectively; P < 0.05 for each comparison) (Fig. 5, 6). But the level of IL-6 showed no correlations with CT score of patients with RA (spearman r = 0.108, P = 0.374) (Fig. 7). Discussion The risk factors and short-term changes for radiological abnormalities in Omicron infected patients are unknown. In this multi-center retrospective study, we identified a group of patients with CT abnormalities at admission, including patients with CR and RA at 1-month follow-up, and a group without CT abnormalities. We demonstrated that age ≥ 50 years, BMI ≥ 23.87, DAV ≥ 81 days, LY count ≤ 1.21×10− 9 /L, IL-6 ≥ 10.05 pg/mL, and IgG ≤ 14.140 S/CO were independent risk factors for radiological abnormalities at admission. And compared with the patients with CR, patients with RA showed a statistically high Page 14/25 Page 14/25 incidence rate of interlobular septal thickening and IL-6 ≥ 5.85 pg/mL. Additionally, there were no significant differences in radiological abnormalities between admission and 1-month follow-up. In the present study, abnormal CT findings were identified in 46% Omicron infected patients at admission. Similar to previous studies, bilateral subpleural GGOs with partial consolidation were the most frequent CT findings at admission9,10,14. We found that longer DAV, lymphopenia, higher IL-6 level, and lower IgG level were independent risk factors for radiological abnormalities at admission. Dong S et.al20 have shown that the percentage GGO and consolidation were significantly correlated with lymphocyte count in severe patients, and lymphopenia has been identified as a prognostic factor for COVID-19. But we found no significant correlation between lymphocyte count and CT score, because a large number of mild patients were included in our study. Several mechanisms have been suggested to explain lymphopenia, including cytokine storm, the damage to the cytoplasmic component of the lymphocytes, and the direct attack on lymphatic organs by SARS-CoV-2 viral particles 9,20,21. And higher IL-6 level is the independent risk factor for both CT abnormalities and residual lesions. Discussion The present study finding was consistent with that of a previous report, in which CT score showed significant positive correlations with levels of IL-6.22,23 Recent data have indicated that the high concentrations of IL-6 in COVID-19 patients due to cytokine storm was related to pulmonary inflammation, extensive lung damage, and even multiple organ failure.22,24 Moreover, basic researches confirmed the increased serum levels of IL-6 may take part in the induction of lymphopenia through inducing apoptosis in T cells.21,25 According to the findings of the previous study, vaccine effectiveness was lower for the Omicron variant than for the delta variant, and the vaccine effectiveness reduced over time from 75.1% after 2 to 4 weeks to 14.9% after 25 or more weeks.26 Moreover, the IgG levels in the breakthrough infections group rapidly increased due to a quick immune response generated by IgG + memory B cell in response to vaccination.27,28 Therefore, it can explain why longer DAV and lower IgG levels increased the potential risk for radiological abnormalities at admission. In the present study, residual CT abnormalities were identified in 59% Omicron infected patients at a 1-month follow-up. We found that the presence of interlobular septal thickening was the other independent risk factor for residual radiological abnormalities. With the limited follow-up period for our study, we avoided radiological terms such as “reticular pattern” associated with lung fibrosis; instead, we used the terms “interlobular septal thickening”. Previous studies had found that consolidation may wane and interstitial septal thickening may emerge during the absorption stage of the first two weeks of infection. However, these abnormalities improved three months after cure and discharge without residual pulmonary interstitial fibrosis.9,11,14,29 But, other studies found that the interstitial septal thickening was more common in severe patients, and did not resolve fully in 24% of patients at 12 months after discharge, which was potentially consistent with evolving fibrosis.9,30,31 Compared to the time of admission, the detection rate of both subpleural and peribronchovascular distribution was significantly higher after 1 month in our study. This may be caused by some patients with disease progression. Our study found that consolidation, interlobular septal thickening, and pleural involvement significantly improved over time, whereas GGOs and parenchymal bands tended to increase after 1 month, but without significantly different. Discussion After 1 month, the total CT score and the number of Page 15/25 Page 15/25 Page 15/25 involved lung lobes showed no significant difference from those at admission. According to previous studies11,14, complete resolution mainly occurred in the first 3 months and more severe acute disease was linked with CT abnormalities at 3 months, but after 3 months, residual lesions became increasingly persistent with insignificant decrease in total CT score. Another meta-analysis literature also had described those inflammatory changes such as GGOs and consolidation reduced to 44% at a median follow-up of 3 months, with the timing of follow-up strongly associated with estimates of inflammatory sequelae16. More than that, the CT score has been shown to be correlated with pathologic specimens and identified as a marker for disease severity and prognosis.32 For these reasons, reducing follow-up chest CT scans during the first month may be considered for these patients at high risk for the residual CT abnormalities. This retrospective investigation has several limitations. First, our studies mainly predict the risk factors according to baseline (at admission) information without dynamic characteristics analysis, because the follow-up period was relatively short. Further long-term follow-up studies are required to determine the evolution of the identified pulmonary residual abnormalities. And future studies of the effects of continuous laboratory markers monitoring on radiological outcomes during convalescence would be welcome. Another limitation of our study is that patients were not divided according to severity because of the large proportion of mild patients in this pandemic. Strengths of our study include the follow-up study of Omicron infected patients during its first pandemic in China, the inclusion of relatively complete clinical and radiological information at admission, and the use of baseline characteristics to predict radiological outcomes at 1 month and the investigation of changes in radiological abnormalities in the early convalescence to provide evidence to make optimal first follow-up plans. Interpretation We demonstrated that age ≥ 50 years, BMI ≥ 23.87, DAV ≥ 81 days, LY count ≤ 1.21×10− 9/L, IL-6 ≥ 10.05 pg/mL, and IgG ≤ 14.140 S/CO were independent risk factors for radiological abnormalities at admission, and especially the presence of interlobular septal thickening and IL-6 ≥ 5.85 pg/mL were the independent risk factors for residual CT abnormalities at 1 month. For the patients with residual pulmonary abnormalities, we found that radiological changes were not statistically significant during the early convalescence. Therefore, a longer follow-up interval in the early convalescence should be considered for these patients who have risk factors for residual CT abnormalities. Further studies are needed to identify the exact follow-up interval for different patients. Ethics approval and consent to participate This multicenter retrospective study was approved by the Ethics Review Committee of Tianjin First Central Hospital (No. 2022N062KY) and Haihe Hospital Ethics Committee (No. 2020HHKT-008), and waived the patient’s informed consent. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Consent for publication Not applicable Abbreviations BMI Body Mass Index CCVD Cardio-Cerebral Vascular Diseases CR Complete Resolution CRP C-reactive Protein Page 16/25 DAV Days After Vaccination DM Diabetes Mellitus GGO Ground-Glass Opacity IL-6 Interleukin-6 LDDH Liver Dysfunction During Hospitalization LHS Length of Hospital Stay LY Lymphocyte NEUT Neutrophil PLT Platelet RA Residual Abnormalities WBC White Blood Cell DAV Days After Vaccination DM Diabetes Mellitus GGO Ground-Glass Opacity IL-6 Interleukin-6 LDDH Liver Dysfunction During Hospitalization LHS Length of Hospital Stay LY Lymphocyte NEUT Neutrophil PLT Platelet RA Residual Abnormalities WBC White Blood Cell Competing interests The authors declare that they have no competing interests. References 1. World Health Organization (WHO). Coronavirus Disease (COVID-19) Pandemic. Available online: https://www.who.int/emergencies/diseases/novel-coronavirus-2019 (accessed on 24 January 2022).. 2. Callaway E. 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Can J Infect Dis Med Microbiol. 2022;2022:1728446. 30. Wu X, Liu X, Zhou Y, et al. 3-month, 6-month, 9-month, and 12-month respiratory outcomes in patients following COVID-19-related hospitalisation: a prospective study. Lancet Respir Med. 2021;9(7):747–754. 30. Wu X, Liu X, Zhou Y, et al. 3-month, 6-month, 9-month, and 12-month respiratory outcomes in patients following COVID-19-related hospitalisation: a prospective study. Lancet Respir Med. 2021;9(7):747–754. 31. Zheng Y, Wang L, Ben S. Meta-analysis of chest CT features of patients with COVID-19 pneumonia. J Med Virol. 2021;93(1):241–249. 31. Zheng Y, Wang L, Ben S. Meta-analysis of chest CT features of patients with COVID-19 pneumonia. J Med Virol. 2021;93(1):241–249. 32. Francone M, Iafrate F, Masci GM, et al. Chest CT score in COVID-19 patients: correlation with disease severity and short-term prognosis. Eur Radiol. 2020;30(12):6808–6817. 32. Francone M, Iafrate F, Masci GM, et al. Chest CT score in COVID-19 patients: correlation with disease severity and short-term prognosis. Eur Radiol. 2020;30(12):6808–6817. Figure 1 Flowchart showing COVID-19 patients included in the study. Page 20/25 ure 2 e pattern of CT abnormalities observed in this study. A, CT scan shows subpleural GGO (red arrow). B, C n shows subpleural consolidation (red arrow). C, CT scan shows subpleural interlobular septal thicken d arrow). D, CT scan shows subpleural parenchymal bands (red arrow). GGO = Ground-Glass Opacity. Figure 2 The pattern of CT abnormalities observed in this study. A, CT scan shows subpleural GGO (red arrow). B, CT scan shows subpleural consolidation (red arrow). C, CT scan shows subpleural interlobular septal thickening (red arrow). D, CT scan shows subpleural parenchymal bands (red arrow). GGO = Ground-Glass Opacity. Figure 3 Figure 3 Page 21/25 Page 21/25 The correlation heat map showed the correlations between age, BMI, DAV, LHS, WBC, NEUT, LY, PLT, CRP, IL-6, IgM, and IgG for the patient with CT abnormalities (A) and the patient with RA (B). Correlations were computed by Spearman rank correlation. The color in each tile was coded by the strength of Spearman correlation coefficients, and intensity is proportional to the degree of correlation. * P value < 0.05. RA = Residual Abnormalities; BMI = Body Mass Index; LHS = Length of Hospital Stay; DAV = Days After Vaccination; WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6. The correlation heat map showed the correlations between age, BMI, DAV, LHS, WBC, NEUT, LY, PLT, CRP, IL-6, IgM, and IgG for the patient with CT abnormalities (A) and the patient with RA (B). Correlations were computed by Spearman rank correlation. The color in each tile was coded by the strength of Spearman correlation coefficients, and intensity is proportional to the degree of correlation. * P value < 0.05. RA = Residual Abnormalities; BMI = Body Mass Index; LHS = Length of Hospital Stay; DAV = Days After Vaccination; WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6. The correlation heat map showed the correlations between age, BMI, DAV, LHS, WBC, NEUT, LY, PLT, CRP, IL-6, IgM, and IgG for the patient with CT abnormalities (A) and the patient with RA (B). Correlations were computed by Spearman rank correlation. The color in each tile was coded by the strength of Spearman correlation coefficients, and intensity is proportional to the degree of correlation. * P value < 0.05. RA = Residual Abnormalities; BMI = Body Mass Index; LHS = Length of Hospital Stay; DAV = Days After Vaccination; WBC = White blood cell; NEUT = Neutrophil; LY = Lymphocyte; PLT = Platelet; CRP = C-reactive protein; IL-6 = Interleukin-6. Figure 4 Graphs show the changes in CT abnormalities over time. A, The bar graph shows main patterns of lung abnormalities on CT at admission and 1-month follow-up for different groups. B, The stacked bar graphs show the distribution of the percentage of involved lung lobes. Paired Wilcoxon signed-rank test comparing changes in CT scores (C) and the number of involved lung lobes (D) between admission and 1-month follow- up. CR = complete resolution, RA = residual abnormalities, GGO = Ground-Glass Opacity. * P value < 0.05, P value < 0.01, * P value < 0.001. Graphs show the changes in CT abnormalities over time. A, The bar graph shows main patterns of lung abnormalities on CT at admission and 1-month follow-up for different groups. B, The stacked bar graphs show the distribution of the percentage of involved lung lobes. Paired Wilcoxon signed-rank test comparing changes in CT scores (C) and the number of involved lung lobes (D) between admission and 1-month follow- up. CR = complete resolution, RA = residual abnormalities, GGO = Ground-Glass Opacity. * P value < 0.05, P value < 0.01, * P value < 0.001. Page 22/25 Figure 5 Risk factors associated with CT abnormalities at admission (A), and RA at 1-month follow-up. OR and corresponding 95% CI were obtained from multivariable logistic regression analysis (Enter method). CR = complete resolution, RA = residual abnormalities, OR = Odds Ratio, CI = Confidence Intervals. Figure 5 Risk factors associated with CT abnormalities at admission (A), and RA at 1-month follow-up. OR and corresponding 95% CI were obtained from multivariable logistic regression analysis (Enter method). CR = complete resolution, RA = residual abnormalities, OR = Odds Ratio, CI = Confidence Intervals. Figure 5 Figure 5 Risk factors associated with CT abnormalities at admission (A), and RA at 1-month follow-up. OR and corresponding 95% CI were obtained from multivariable logistic regression analysis (Enter method). CR = complete resolution, RA = residual abnormalities, OR = Odds Ratio, CI = Confidence Intervals. Page 23/25 Page 23/25 Figure 6 The Omicron infected patient whose IL-6 was 3.6 pg/mL at admission. A, Chest CT obtained at admission showed bilateral subpleural GGOs. B, Chest CT obtained at 1-week follow-up showed increased extent of bilateral subpleural consolidations with interlobular septal thickening or reticular lesion. C, Chest CT obtained 1-month follow-up showed residual consolidations with parenchymal bands. The Omicron infected patient whose IL-6 was 2.0 pg/mL at admission. D, Chest CT obtained at admission showed slight subpleural GGOs in the right low lobe. E, Chest CT obtained at 1-week follow-up showed increased extent of subpleural consolidations in the right low lobe. C, Chest CT obtained 1-month follow-up showed complete resolution of the lesion in the right low lobe. GGO = Ground-Glass Opacity. Figure 6 The Omicron infected patient whose IL-6 was 3.6 pg/mL at admission. A, Chest CT obtained at admission showed bilateral subpleural GGOs. B, Chest CT obtained at 1-week follow-up showed increased extent of bilateral subpleural consolidations with interlobular septal thickening or reticular lesion. C, Chest CT obtained 1-month follow-up showed residual consolidations with parenchymal bands. The Omicron infected patient whose IL-6 was 2.0 pg/mL at admission. D, Chest CT obtained at admission showed slight subpleural GGOs in the right low lobe. E, Chest CT obtained at 1-week follow-up showed increased extent of subpleural consolidations in the right low lobe. C, Chest CT obtained 1-month follow-up showed complete resolution of the lesion in the right low lobe. GGO = Ground-Glass Opacity. Page 24/25 Page 24/25 Figure 7 Scatterplots showing relationship between admission IL-6, LY count and IgG in those with, relative to those without CT changes (A-C). D, Scatterplots showing relationship between admission IL-6 in those with RA, relative to those with CR. Median and interquartile ranges shown (A-D). Spearman rank correlation of total CT score and IL-6, LY count and IgG in those with CT abnormalities at admission (E-G). H, Spearman rank correlation of total CT score and IL-6 in those with RA at admission. CR = complete resolution, RA = residual abnormalities. Figure 7 Scatterplots showing relationship between admission IL-6, LY count and IgG in those with, relative to those without CT changes (A-C). D, Scatterplots showing relationship between admission IL-6 in those with RA, relative to those with CR. Median and interquartile ranges shown (A-D). Spearman rank correlation of total CT score and IL-6, LY count and IgG in those with CT abnormalities at admission (E-G). H, Spearman rank correlation of total CT score and IL-6 in those with RA at admission. CR = complete resolution, RA = residual abnormalities. Scatterplots showing relationship between admission IL-6, LY count and IgG in those with, relative to those without CT changes (A-C). D, Scatterplots showing relationship between admission IL-6 in those with RA, relative to those with CR. Median and interquartile ranges shown (A-D). Spearman rank correlation of total CT score and IL-6, LY count and IgG in those with CT abnormalities at admission (E-G). H, Spearman rank correlation of total CT score and IL-6 in those with RA at admission. CR = complete resolution, RA = residual abnormalities. Page 25/25
https://openalex.org/W2944770736	https://www.nature.com/articles/s41598-019-43484-0.pdf	English	null	Activities of starch synthetic enzymes and contents of endogenous hormones in waxy maize grains subjected to post-silking water deficit	Scientific reports	2,019	cc-by	6,021	Activities of starch synthetic enzymes and contents of endogenous hormones in waxy maize grains subjected to post- silking water deficit Received: 26 November 2018 Accepted: 25 April 2019 Published: xx xx xxxx Huan Yang, Xiaotian Gu, Mengqiu Ding, Weiping Lu & Dalei Lu Rainfed maize in Southern China and frequently suffer water deficit at later plant growth periods. A pot trial in 2014–2015 was conducted to study the effects of drought stress (the relative soil moisture contents are 70–80% and 50–60% under control and water deficit conditions, respectively) after pollination on grain filling and starch accumulation, activities of starch synthetic enzymes, and contents of indole-3-acetic acid (IAA) and abscisic acid (ABA), with Suyunuo5 as test material. The grain fresh weight, volume, and dry weight were not affected by drought before 10 days after pollination but were restricted thereafter. The reduction at maturity was reduced by 33.3%, 40.0%, and 32.3% in 2014 and by 21.7%, 24.3%, and 18.3% in 2015. The grain filling rate was suppressed by water deficit, whereas grain moisture and starch content were slightly affected. The starch accumulation was decreased by 33.5% and 20.0% at maturity in 2014 and 2015, respectively. The activities of starch synthetic enzymes (sucrose phosphate synthase, sucrose synthase, ADP-glucose pyrophosphorylase, soluble starch synthase, and starch branching enzyme) were downregulated by post-silking drought. The ABA content was increased, whereas IAA content was decreased when plants suffered water deficit during grain filling. In conclusion, post-silking water deficit increased ABA content, decreased IAA content, and weakened the activities of starch synthetic enzymes, which suppressed grain development and ultimately reduced grain weight. Drought stress is a main environmental constraint that restrict crop productivity worldwide. Maize are prone to drought as they are mostly grown under rainfed conditions. The annual loss of maize yield caused by water deficit dominates all environmental stresses, and the incidence is acute with increasing intensity and frequency1.ii Grain filling duration and rate are crucial to final grain weight2. Studies have demonstrated that drought stress at early grain development disturb the cell division and differentiation processes required for organ establishment and starch biosynthesis3,4. Starch is the foremost grain component that determines grain weight, and subject to water deficit after flowering stage is, in most cases, associated with the reduced starch accumulation5. Water deficit occurring at initially grain development stage curtails the grain sink potential by reducing the number of endosperm cells and amyloplasts formed, thus reducing grain weight by decrease the endosperm cells to deposite starch, in terms of both duration and rate6,7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Activities of starch synthetic enzymes and contents of endogenous hormones in waxy maize grains subjected to post- silking water deficit Studies reported that postanthesis mild drought stress shortened the grain filling period and increased grain filling rate and starch accumulation rate by enhancing the activi- ties of starch synthetic enzymes (sucrose synthase (SuSy), ADP Glc pyrophosphorylase (AGPase), soluble starch synthase (SSS), and starch branching enzyme (SBE)), whereas the activities of those starch synthetic enzymes were weakened under severe drought stress8–10. The downregulation of AGPase activity at both the protein and transcript levels under water deficit conditions during grain filling is responsible for the significant decrease in Jiangsu Key Laboratory of Crop Genetics and Physiology/Jiangsu Key Laboratory of Crop Cultivation and Physiology, Agricultural College of Yangzhou University/Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou, 225009, China. Huan Yang and Xiaotian Gu contributed equally. Correspondence and requests for materials should be addressed to D.L. (email: dllu@yzu.edu.cn) Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 1 www.nature.com/scientificreports/ starch deposition11,12. The sorghum grain filling rate was reduced by water shortage at the flowering stage due to the reduced activities of SSS, SBE, and granule-bound starch synthase13.hi g y The grain endogenous hormones such as indole-3-acetic acid (IAA) and abscisic acid (ABA) significantly affect the rate and duration of grain filling14. Drought stress during grain filling increases the ABA content, which induces the increase of the rate and early suspension of duration15. Yang and Zhang2 found that high grain ABA content under moderately soil-dried condition increased the rate, whereas too high ABA content under severely soil-dried condition reduced the rate of grain filling. Application of exogenous ABA at middle grain filling stages could regulate sink activity, grain weight was noticeably reduced with high ABA spraying concentration and mildly increased with low concentration16. Guoth et al.17 found that drought-tolerant wheat genotypes only exhibited higher ABA content at early grain filling, and the sensitive cultivars maintained high ABA levels in the later grain filling stages, which led to a decreased grain yield. Lv et al.18 and Liu et al.19 observed that post-anthesis water stress decreased the wheat grain weight and grain filling rates due to the decrease of grain IAA and ABA contents at the early and middle stages of grain filling. Our early studies reported that waxy maize flour and starch physiochemical properties were changed by drought after pollination20,21. Results fi Grain filling. Water deficit did not affect the grain fresh weight, volume, and dry weight before 10 days after pollination (DAP), but they were restricted thereafter. The reduction gradually increased with grain development. At maturity (40 DAP), the grain fresh weight, volume, and dry weight were reduced by 33.3%, 40.0%, and 32.3% in 2014 and by 21.7%, 24.3%, and 18.3% in 2015, respectively (Fig. 1). The grain moisture content was slightly affected by drought, whereas the reduction was significantly affected at 10 and 25 DAP in 2014 and 30 DAP in 2015, and not affected at the other stages. The grain filling rate in 2014 was similar to control before 5 DAP, increased by drought at 6–10 DAP, and decreased thereafter. The value in 2015 was not affected by drought before 10 DAP, decreased at 11–15 DAP, not affected at 16–20 DAP, increased at 21–25 DAP, and decreased thereafter. Starch accumulation. The grain starch concentration (mg g−1) was slightly affected by water deficit throughout the grain filling in both years (Fig. 2). The starch accumulation (starch content × grain dry weight, mg grain−1) was not affected by drought before 15 DAP, and the value was reduced by water deficit thereafter, similar to the reduction of grain dry weight. At maturity, the reduction of starch accumulation under drought was 33.5% and 20.0% in 2014 and 2015, respectively. Activities of SPS and SuSy. The grain SPS activity was not affected by drought at 5–10 DAP and decreased at 15–25 DAP, and the difference disappeared at 30 DAP in both years (Fig. 3). The SuSy activity in 2014 was decreased by drought at 5–15 DAP and not affected thereafter, the value in 2015 was only reduced by drought at 15 DAP and was similar to drought at the other stages. In general, the average reductions of SPS and SuSy activi- ties throughout the grain filling were 16.1% and 19.8% in 2014 and 17.3% and 7.2% in 2015. Activities of AGPase, SSS, and SBE. The activities of starch synthetic enzymes (AGPase, SSS, and SBE) were significantly influenced by post-silking drought (Fig. 4). The AGPase activity in both years was decreased by drought, and the decrease was pronounced at 10–25 DAP in 2014 and 10–20 DAP in 2015. The SSS activity in 2014 was similar to control at 5 DAP, increased at 10 DAP, and decreased thereafter. Results fi The value in 2015 was not affected by drought at 5, 15, and 25–30 DAP; increased at 10 DAP; and reduced at 20 DAP. The SBE activity in 2014 was reduced by drought throughout the grain filling, and the value in 2015 was also reduced by drought at 5, 15, and 30 DAP and was not affected at 10 and 20–25 DAP. ABA and IAA content. The grain ABA content gradually increased with grain development, and it was increased by drought throughout the grain filling (Fig. 5). The IAA content in 2015 was decreased by drought, the value in 2014 was not affected at 15 DAP and reduced at the other stages, and the decrease was severe after 15 DAP in both years. Activities of starch synthetic enzymes and contents of endogenous hormones in waxy maize grains subjected to post- silking water deficit Starch formation and accumulation were co-adjusted by a series of starch synthetic enzymes and endogenous hormones, and clarfying the effects of post-silking water deficit on those enzymes and hormones could improve our understanding on starch develop- ment under drought conditions. In the present research, the grain filling and starch accumulation were clarified, the activities of starch synthetic enzymes and contents of endogenous hormones (ABA and IAA) in response to post-silking water deficit were studied. The results may offer a reference for drought-stressed starch formation of rainfed waxy maize that undergo water deficit after silking. Discussion In the present study, the grain weight (fresh or dry), volume, and average grain filling rate were decreased by post-silking drought stress. The severe reduction of in 2014 may be due to the high temperature and low sun- light duration (Post-silking day/night temperature and sunlight were 29.0/22.0 °C and 126 h in 2014 and were 28.2/21.5 °C and 145 h in 2015, respectively. Data available on http://www.tianqihoubao.com/lishi/yangzhou. html), as those heat and sunlight stresses also reduce grain weight1. The small volume and low weight of grains under water stress may be caused by the reduced endosperm cell numbers and few formed amyloplasts, which induced the decrease of grain sink potential6. The reduced rate of grain filling under drought condition induced the low grain weight, as it could serve as an indirect selection criterion for grain filling rate22. Grain moisture con- tent declines during the entire grain filling, and final grain weight is achieved at values closed to 35% and moder- ate stress conditions affecting plant development seem to have little impact over this value23,24. The similar grain moisture content under control (36.3% and 34.7% in 2014 and 2015) and drought (35.3% and 32.2% in 2014 and 2015) conditions indicated semblable grain filling progress, similar observations were also reported on fresh waxy maize21 and normal maize25, while extreme drought stress shortened this duration25. Studies on wheat and rice Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 2 www.nature.com/scientificreports/ Figure 1. Grain filling properties (fresh weight, fresh volume, dry weight, water content, and filling rate control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 not significant. Figure 1. Grain filling properties (fresh weight, fresh volume, dry weight, water content, and filling rate) under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. revealed that mild drought after anthesis shortened the duration but increased the rate of grain filling, whereas severe water shortage reduced both the rate and duration8–10. The possible explanation for this phenomenon may be due to the similar reduction of both source and sink potential in response to post-silking water deficit21, and the value mainly different among genotypes and little affected by moderate stresses23,24. Therefore, further study different water stress levels on maize grain filling may help clarify this different response. Discussion different water stress levels on maize grain filling may help clarify this different response. Grain development is dependent on many factors, including sucrose availability and the activities of enzymes involved in grain starch and sugar metabolism26. Drought during grain filling restricted the starch deposition were reported in many crops5. We observed that the grain starch concentration was not affected (only reduced by Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 3 www.nature.com/scientificreports/ Figure 2. Grain starch concentration and accumulation under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 2. Grain starch concentration and accumulation under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 3. Grain SPS and SuSy activities under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 3. Grain SPS and SuSy activities under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. 1.8% and 2.2% at maturity in 2014 and 2015, respectively), but the starch accumulation was severely depressed (reduction was 33.5% and 20.0% at maturity in 2014 and 2015, respectively) by post-silking water deficit, and the severe reduction of starch accumulation in 2014 may be due to the severe reduction of grain dry weight (32.3% and 18.3% in 2014 and 2015, respectively). Similar starch concentration between control and drought stress was observed on fresh waxy maize21 and normal maize27. A field study on normal maize28 and sorghum29 also observed that starch content was slightly affected by restrict irrigation. The starch accumulation was remark- ably reduced by drought, which may caused by the weakened activities of starch synthetic enzymes (SPS, SuSy, AGPase, SSS, and SBE), this opinion was also demonstrated at protein, transcript, and enzymology levels when plants suffered drought at late growth stages in wheat10–12 and sorghum13.if f ABA and IAA are two important endogenous hormones that significantly affect the grain endosperm cell division and expansion, grain size initiation, and grain filling rate and duration30. High IAA content can promote endosperm cell division and increase sink potential, which accelerate endosperm cell propagation and grain filling31,32. Materials and Methods Plant materials and growth conditions. Suyunuo5, a well-known waxy maize covering large plantation areas in Southern China, was analysed in farm at Yangzhou University (Yangzhou, China) in 2014 and 2015. Seeds were sown in March 15 and transplanted to plastic pots (two seedlings per pot, with one plant retained at the jointing stage) in March 28. Each plastic pot was 38 cm in height and 43 cm in diameter, and loaded with 30 kg of sieved sandy loam soil. The plants per pot were provided 10 g compound fertilizer (N/P2O5/K2O = 15%/15%/15%) at transplantation and 6.6 g urea (N = 46%) at the jointing stage. Plants were maintained up to silking stage under relative soil moisture content of approximately 70–80%. After manual pollination at same day, plants were imposed to water deficit treatment till maturity. The relative moisture contents of the soil subjected to the control and drought treatments were 70–80% and 50–60% by the weighing method. The evaporation of the plants was supplied at each morning by measuring the weight loss. The rainfall was excluded by a mobilizable transparent waterproof canopy. Each treatment has fifty pots. Samples preparation. Three ears were harvested at 5, 10, 15, 20, 25, 30 DAP and maturity (40 DAP). Approximately 50–60 grains at middle position were stripped from the ears and immediately frozen in liquid N2, and then stored at −75 °C until analysis. 100-grains randomly selected from the left grains on independent ear were weighed (fresh weight, mg grain−1) and the volume were measured by drainage (fresh grain volume, μl grain−1). After that, the grains were deactivated at 105 °C for 30 min and dried at 60 °C to consistent weight. Then, the grain dry weight (mg grain−1) and grain moisture content (%) was determined based on three independ- ent ears as triplicates. The grain filling rate (mg grain−1 d−1) was calculated based on the dry weight difference between two contiguous sampling periods. After drying and weighing, the grains were pulverized and passed through a filter screen (100-mesh, d = 0.149 mm) for starch content determination. Starch analysis. The grain starch content (mg g−1) was determined using the anthrone-sulfuric acid method38. Starch accumulation (mg grain−1) was determined using the following formula: grain dry weight × starch content. Starch analysis. The grain starch content (mg g−1) was determined using the anthrone-sulfuric acid method38. Discussion ABA is involved in the metabolic activity of key enzymes in sucrose decomposition and starch biosyn- thesis and regulates the process of grain weight formation16. Drought stress increases the amounts of ABA and Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 4 www.nature.com/scientificreports/ Figure 4. Grain AGPase, SSS, and SBE activities under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 4. Grain AGPase, SSS, and SBE activities under control and water deficit conditions. Bars are standar errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 5. Grain ABA and IAA contents under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. Figure 5. Grain ABA and IAA contents under control and water deficit conditions. Bars are standard errors of three replicates. Significant at p < 0.05 level; ns, not significant. affects the embryo mutation, grain development, and grain components33–35. The reduced IAA content under drought was also observed in rice and caused spikelet sterility36. In the present study, the increased ABA content and decreased IAA content under post-silking drought stress restricted grain filling, resulted the low grain weight, affects the embryo mutation, grain development, and grain components33–35. The reduced IAA content under drought was also observed in rice and caused spikelet sterility36. In the present study, the increased ABA content and decreased IAA content under post-silking drought stress restricted grain filling, resulted the low grain weight, Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 5 www.nature.com/scientificreports/ which may be due to drought restricted the endosperm cell division, grain sink size initiation, filling capacity, and grain weight16,30,35,37. which may be due to drought restricted the endosperm cell division, grain sink size initiation, filling capacity, and grain weight16,30,35,37. Conclusion lk Post-silking water deficit decreased the grain weight (dry or wet), volume, and filling rate, and the severe reduc- tion of those parameters in 2014 may be due to the high temperature and low sunlight duration at grain filling stage. The grain moisture content and starch concentration were slightly affected by drought stress, whereas the starch deposition was severe restricted due to the severe decrease of grain weight. The reduction of starch depo- sition in grains was caused by the weakened activities of starch synthetic enzymes (SPS, SuSy, AGPase, SSS, and SBE), reduced IAA content and increased ABA content. Scientific Reports \| (2019) 9:7059 \| https://doi.org/10.1038/s41598-019-43484-0 Materials and Methods Starch accumulation (mg grain−1) was determined using the following formula: grain dry weight × starch content. Assays of SPS and SuSy activities. All procedures for enzyme extraction were carried out in ice (0 °C–2 °C). The grain embryo and pericarp were discarded, and approximately 1 g fresh weight of the lyophilized samples were homogenized in the extraction buffer containing 50 mM HEPES-NaOH (pH 7.5) followed by cen- trifugation at 10,000 × g for 10 min39. The supernatant was used for enzyme assays. SuSy (EC 2.4.1.13) and SPS (EC 2.4.1.14) assay followed the method proposed earlier39 with modifications by Yang et al.40. Assays of SPS and SuSy activities. All procedures for enzyme extraction were carried out in ice (0 °C–2 °C). The grain embryo and pericarp were discarded, and approximately 1 g fresh weight of the lyophilized samples were homogenized in the extraction buffer containing 50 mM HEPES-NaOH (pH 7.5) followed by cen- trifugation at 10,000 × g for 10 min39. The supernatant was used for enzyme assays. SuSy (EC 2.4.1.13) and SPS (EC 2.4.1.14) assay followed the method proposed earlier39 with modifications by Yang et al.40. Assay of starch synthetic enzymes. The enzyme extracting solution was prepared following a procedure that detailed by Yang et al.40. The assays of the AGPase (EC 2.7.7.27), SSS (EC 2.4.1.21) and SBE (EC 2.4.1.18) followed the method proposed by Nakamura et al.41 with slight modifications40. Assay of starch synthetic enzymes. The enzyme extracting solution was prepared following a procedure that detailed by Yang et al.40. The assays of the AGPase (EC 2.7.7.27), SSS (EC 2.4.1.21) and SBE (EC 2.4.1.18) followed the method proposed by Nakamura et al.41 with slight modifications40. Endogenous hormone. The contents of endogenous hormones (IAA and ABA) in grains at different stages (5, 10, 15, 20, 25, and 30 DAP) were examined by enzyme-linked immunosorbent assay (ELISA). The IAA and ABA were extracted according to the method proposed by Lv et al.18. A plant hormone ELISA kit was purchased from Shanghai Jining Shiye Co., Ltd. (Shanghai, China), and IAA and ABA were measured according to the kit’s protocols. The recovery rates for IAA and ABA were 84.7% ± 3.2% and 91.2% ± 2.6%, respectively. Statistical analysis. The data are expressed as averages of triplicates. Data were subjected to ANOVA with the LSD test at p < 0.05 level using the Data Processing System (version 7.05). gi g g g g 2 g y ( ) 8. Yang, J. 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Acknowledgementsh This study was supported by the National Key Research and Development Program of China (2016YFD0300109, 2018YFD0200703), the National Natural Science Foundation of China (Grant No. 31771709, 31471436), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Qing Lan Project of Jiangsu. The data of grain dry weight, starch concentration, starch accumulation, and activities of SPS, SuSy, AGPase, SSS, and SBE in the present study were identical to the previous published paper (Yang et al., Scientific Reports, 2018, 8, 15665) and certified by all the authors. www.nature.com/scientificreports/ C. & Liu, Y. Effect of potassium foliage application post-anthesis on grain filling of wheat unde drought stress. Field Crop Res. 206, 95–105 (2017).fi g p 19. Liu, Y. et al. Effect of polyamines on the grain filling of wheat under drought stress. Plant Physiol. 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https://openalex.org/W4360980173	https://link.springer.com/content/pdf/10.1007/s40747-023-01016-4.pdf	English	null	RDQN: ensemble of deep neural network with reinforcement learning in classification based on rough set theory for digital transactional fraud detection	Complex & intelligent systems	2,023	cc-by	15,165	Complex & Intelligent Systems (2023) 9:5313–5332 https://doi.org/10.1007/s40747-023-01016-4 Complex & Intelligent Systems (2023) 9:5313–5332 https://doi.org/10.1007/s40747-023-01016-4 ORIGINAL ARTICLE Abstract All ﬁnancial sectors are facing the most common frauds, which are digital transactional frauds. Fraudsters have always engaged in illegal activities such as stealing personal information and logging in with unauthorised credentials. Many machine learning algorithms predict whether the transaction is factual or nonfactual but fail to decrease the processing time. Hybrid models are used in this case to identify the fraud in a quick and efﬁcient manner. This article demarcates to construct a novel model, RDQN, i.e., deep reinforcement learning, that combines with the rough set theory. This article has three steps, including data pre-processing to determine the quality of the data, which affects the learning ability of the model, determining the structural relationship and gaining useful features from the data set using rough set theory, and doing a hybridization of DNN (deep neural network) and Q learning, which is called DQN. It uses the MISH activation function and the ReLU activation function in different layers for training dynamics in the neural network. The proposed model classiﬁes and predicts that the transaction belongs to the category implemented by the agents by activating the reward function. The reinforcement-learning agent’s performance improves based on reward assessment. This reward function gives a more precise value for each transaction, and no fraudster can escape from the agent’s sight. This novel approach improves accuracy and reduces processing time by considering the best feature selection during the process. Keywords Digital transactions · Rough set theory · Deep neural network · Deep reinforcement learning (DQN) · Reward function · Agent RDQN: ensemble of deep neural network with reinforcement learning in classiﬁcation based on rough set theory for digital transactional fraud detection Chandana Gouri Tekkali1 · Karthika Natarajan1 Received: 27 September 2022 / Accepted: 17 February 2023 / Published online: 17 March 2023 © The Author(s) 2023 Received: 27 September 2022 / Accepted: 17 February 2023 / Published online: 17 March 2023 © The Author(s) 2023 B Karthika Natarajan karthika.n@vitap.ac.in Chandana Gouri Tekkali gouritekkali.20phd7181@vitap.ac.in 1 VIT-AP University, Amaravati, Andhra Pradesh, India Introduction Deep learning offers a promising answer to the problem of credit card fraud detection and digital fraud detection by allowing businesses or individuals to make the most use of both previous customer behaviour and real-time transaction details captured at the moment of the transac- tion. Various deep learning algorithms CNN (convolutional neural network), SOM (self-organizing maps), DNN (deep neural networks), LSTM (long short-term memory) are used for identiﬁcation of credit card frauds [9–12]. There are a few authors who work with reinforcement learning in the fraud detection era. The authors theoretically explained reinforcement learning but did not provide insight into its implementation. networks was trained using a mini-batch approach. For a higher classiﬁcation rate, we use Q learning to validate the results. Q-learning is an off-policy reinforcement learning method that uses current transaction data to determine the fraud transaction as a course of action. By calculating the reward function, we determine the rewards for the individual transaction, and that will lead to conclusions instead of a fraud prediction. So, that concluded the better classiﬁcation. The following points are made to highlight the contribu- tions of this study: – RST is a technique for extracting the most important features from a dataset and training different reduced minimal feature datasets. – createdanovelhybridapproachforclassiﬁcationbycom- bining DNN and reinforcement learning (Q). – Q learning is integrated into DNN. This DQN classiﬁer is new to this ﬁeld of digital transactional fraud detection application. – The statistical results also prove DQN outperforms other classiﬁers. This article contributes to the RDQN model, which con- sists of two modules: feature selection and classiﬁcation based on an expert reinforcement learning method that works with deep Q learning. As can be seen, the primary motiva- tion for using the feature selection algorithm is to improve the classiﬁcation rate. The best features always produce bet- ter results and reduce the computational time to obtain the decision value. Deep learning models yield higher accuracy as they train a network very well by updating the weights. However, the proposed DQN uses agents and an ensemble of agents’ actions in an environment to identify whether the transaction is fraud or not by voting majority. In spite of the single agent in reinforcement learning, we adopted a multi-agent system, which enhances the overall system per- formance. The rest of this paper is written in accordance with the rules. Introduction company may decrease, and that reﬂects on the economic sector. Online transactions that are high-risk and unlawful are always detected by fraud detection systems. Numerous systems continuously monitor real-time consumer behaviour and provide risk scores to detect potentially fraudulent trans- actions. E-commerce companies, digital banking systems, and other compliance departments employ solutions like Amazon, Flip Cart, and many others to continuously monitor for potential fraudulent actions that are taken by their users. Digital transactional fraud detection (DTFD) is the main success factor in the banking sector and business environ- ment for detecting fraud that is committed using different digital payment modes, like debit, credit, prepaid cards, or other electronic payment modes. The rise of digital payment systems in all modes has meant that losses are expected to increase as well. Mainly on online platforms and in online shops, e-commerce has grown to make purchases easier. As e-commerce has grown in popularity, so has the prevalence of fraud. With concerns related to this kind of mis- representation in various transactions, the reputation of the Financial institutions all across the world are attempting to improve their skills for preventing and detecting fraud in dig- ital transactions. Fraud detection is a proactive strategy that frequently has a cost to safeguard the system from damaging activity that is ongoing when conducting transactions. Fraud prevention is a challenging task. Researchers from all over the world are suggesting new strategies to increase deterrent effects [1]. B Karthika Natarajan karthika.n@vitap.ac.in Chandana Gouri Tekkali gouritekkali.20phd7181@vitap.ac.in 1 VIT-AP University, Amaravati, Andhra Pradesh, India Most of the credit card fraud issues are addressed by data mining and machine learning techniques that have been 123 12 3 Complex & Intelligent Systems (2023) 9:5313–5332 5314 commonly used to avoid fraud over the last few years and discussed by many authors [2–7]. The primary indicator of fraud location is the ability to detect unwanted behaviour by observing the activities of massive amounts of customer data. The methods utilised for fraud detection systems in machine learning and neural networks are KNN (K nearest neighbour), SVM (support vector machine), DT (decision trees), fuzzy logic, ANN (artiﬁcial neural networks), and many more [8]. Introduction The mechanisms for detecting digital transactional fraud are detailed in the next section of the article. The subsequent section provides an overview of the key terms that are relevant to deep neural networks and reinforce- ment learning followed by which all the system’s speciﬁcs are provided. Next, frameworks for experiments are listed. Findings are provided in the penultimate section. The paper concludes with ﬁndings and ideas that can be extended to further research. Review of related works Now, it is a major issue for customers if they are on the unsafe side while doing their transactions in digital mode. It has been adifﬁcultprocesstoidentifythefraudbeforesigniﬁcantdam- age has been done. However, many scientists and researchers are coming up with new ideologies and approaches to iden- tify fraud in digital transactions. Here, the survey section discusses several contributions available in terms of fraud detection and prevention. Feature selection is the major con- cern of the proposed model. Vijaya et al. [13] extracted vital features from RST for predicting customer churn in the tele- com sector. RST is one of the numerical practises to deal with vagueness or defective information. RST works based on the notion that links link information to each element in the universe (U) of discourse. The RST computes upper and lower approximations, followed by vagueness linked by the boundary region. RST was recommended by Minz et al. [14] to make the search for dominating attributes in information Feature selection: In view of the classiﬁcation problem, col- lecting data are an essential move. In this paper, Rough Set Theory (RST), a methodology that addresses ambiguity and uncertainty in data analysis, is utilised to access the dataset’s key features. This assists in obtaining reduced features as reducts and contributing reducts to our updated DQN archi- tecture. Classiﬁcation: The proposed renovated RDQN uses deep reinforcement learning to blend DNN with the reinforcement learning framework (i.e., Q-learning). It is further outlined by the agents in their approach to accomplishing their objectives here. The two activation functions—ReLU in a hidden layer of the network and a new MISH (non-monolithic) activation function—train a deep neural network. The neural networks can apply complex processes like data classiﬁcation to these activation functions. The backpropagation process of neural 123 Complex & Intelligent Systems (2023) 9:5313–5332 5315 systems easier. A rough set-based decision tree (RDT) pro- posal was designed to solve the issue of high computational time by combining RS tools with traditional DT capabilities. More accuracy was seen with the combination of RDTs than with the separate decision trees. A novel rough set approx- imation employing probabilistic values was introduced by Zhou [15], and it requires more decision classes than two to produce an information table. To create the information table, the author talked about Pawlak’s rough set-based the- ory and the rough set model. Review of related works Features generated by RST were based on existing training mod- els like machine learning (ML), deep learning (DL), and reinforcement learning (RL). Here are some of the best train- ing models created by various authors. Carrasco et al. [18] applied advanced fraud detection techniques based on com- plicated rules, statistical modelling, and ML. As is the case with this, he discussed several deep neural networks, such as MLP, CNN, and DAE, that are tested to quantify their poten- tial to detect the false-positive rate. Now, the most trending technologies are machine learning and deep learning. These techniques were used by different authors to solve binary or multi-classiﬁcation problems. For Example, Dighe et al. [19] investigated both machine learning algorithms (LR, NB, SVM, KNN, DT) and deep learning algorithms (Chebyshev Functional Link ANN and MLP) applied these algorithms to training and testing data to identify fraud in credit card trans- actions. MLP produced a better result in terms of accuracy. In the same way, Forough et al. [20] suggested a novel credit card fraud identiﬁcation model. He developed models based on both deep neural networks (LSTM) and probabilis- tic graphical models, i.e., conditional random ﬁelds (CRFs) that use sequence labeling. These methods were consid- ered for previous instances and also for predicted labels. They compared it with other HMMs, GRUs, and single LSTMs. But the model (LSTM-CRF) gave good results on the credit card data set. Mbunge et al. [21] implemented the deep learning method MLP in different perceptions and the HMM—hidden Markov model—to analyse the obser- vational data. It uses probability distributions to predict the likelihood of an event occurring, whether a transaction is fraudulent or legitimate. The probability of changing from The next module of our model is classiﬁcation. Features generated by RST were based on existing training mod- els like machine learning (ML), deep learning (DL), and reinforcement learning (RL). Here are some of the best train- ing models created by various authors. Carrasco et al. [18] applied advanced fraud detection techniques based on com- plicated rules, statistical modelling, and ML. As is the case with this, he discussed several deep neural networks, such as MLP, CNN, and DAE, that are tested to quantify their poten- tial to detect the false-positive rate. Now, the most trending technologies are machine learning and deep learning. These techniques were used by different authors to solve binary or multi-classiﬁcation problems. Review of related works The rough set model is a decision-theoretic rough set model that is created using a certain option and alpha and beta threshold values that are set to 0.5. To solve classiﬁcation difﬁculties, a crude set- based model with probabilistic values was employed, which results in a three-way decision-making process as an addi- tion to the often-used binary decision-making. For diverse goals, such as dimensionality reduction and feature extrac- tion, writers additionally created a large number of additional feature selection algorithms and heuristics [16,17]. Recog- nize that RST performs effectively, and the best feature is the selection, despite all of these issues. one state to another is represented in the matrix by transition probabilities. These probabilities are provided to a multilayer perceptron that is used to classify the transaction as suspi- cious or non-suspicious. Deep learning techniques have been thoroughly examined by Nguyen et al. [12], who have used them to solve chal- lenging issues like detecting credit card fraud. As can be seen, LSTM and CNN are 1DCNN (one-dimensional) and 2DCNN (two-dimensional), respectively, where the internal representation is learned from the input data via the feature mapping process. On various ﬁnancial data sets, he compared their performance with that of different machine learning models. To get the desired output, Pillai et al. [22] focused more on training the neural network for improved perfor- mance. In his proposed study, compared the performance of the neural network by varying nodes in the hidden layer and also with various types of activation functions Sigmoid, ReLU and Tanh of ANN. MLP with Hyperbolic Tangent (Tanh) gave the best result. They identiﬁed the Tanh activa- tion function which gives the minimum sensitivity value due to its operation and nature. They [23] conducted thorough monitoring of the activation function, which is important for both training and performance analysis of neural networks. An enactment is used to demonstrate the neural network non-linearity concept. Many activation functions, includ- ing sigmoid, hyperbolic tangent, ReLU, Leaky ReLU, and Swish, have been utilised in the past. Here, a brand-new, modiﬁed activation function called MISH is suggested, pro- viding performance robustness. Through extensive testing and experimenting, MISH produced results that were pre- ferred to those of Swish and ReLU by Diganta Misra. p The next module of our model is classiﬁcation. Review of related works For Example, Dighe et al. [19] investigated both machine learning algorithms (LR, NB, SVM, KNN, DT) and deep learning algorithms (Chebyshev Functional Link ANN and MLP) applied these algorithms to training and testing data to identify fraud in credit card trans- actions. MLP produced a better result in terms of accuracy. Many authors explored the deep Q learning algorithm and exploited it in a wide variety of complex applications, like collaborative business processes with cloud services, man- ufacturing assembly programs, many robotic applications, automated trading in equity stock markets, and many more [24–28]. Chatterjee et al. [29] discussed deep reinforcement learning for the application where the most phishing activ- ities are taking place on websites and detecting malicious URLs. By calculating the reward function, they calculated dynamic behaviour on websites to identify phishing activities and learned the attributes using Q learning. For the purpose of detecting credit card fraud, Zhinin-Vera et al. [30] addressed theQ-CCFDmodel, whichdivides transactions intovalidand fraudulent ones. They combined deep learning, an autoen- coder, and AI agents to build a model using AI approaches. With the aid of reinforcement learning, this framework cate- gorises issues by rewarding AI agents based on the expected variable. It assigns a reward as “positive” if it determines that a transaction is authentic; otherwise, it assigns a reward as “negative.” Similar to how a Q-learning algorithm was put into practise by Gopchandani et al. [32] for the purpose of detecting credit card fraud, they talked about the learning In the same way, Forough et al. [20] suggested a novel credit card fraud identiﬁcation model. He developed models based on both deep neural networks (LSTM) and probabilis- tic graphical models, i.e., conditional random ﬁelds (CRFs) that use sequence labeling. These methods were consid- ered for previous instances and also for predicted labels. They compared it with other HMMs, GRUs, and single LSTMs. But the model (LSTM-CRF) gave good results on the credit card data set. Mbunge et al. [21] implemented the deep learning method MLP in different perceptions and the HMM—hidden Markov model—to analyse the obser- vational data. It uses probability distributions to predict the likelihood of an event occurring, whether a transaction is fraudulent or legitimate. The probability of changing from 12 3 Complex & Intelligent Systems (2023) 9:5313–5332 5316 Rough set theory Rough sets constitute a sound basis for KDD (knowledge discovery in databases). Generally, the classical rough sets are used as a framework to design data mining and machine learning algorithms. Rough sets offer a mathematical tool to discover invisible patterns in data, and they give a tool for the induction of (learning) approximations of sets that would be used for hard computations. The classical rough set theory is based on equivalence relations, which can be thought of as a partition of the universe, and the partition is viewed as a type of knowledge composed of deﬁnable sets. In this theory, the uncertain sets are approximated by the deﬁnable ones. RST handles the certainty of the data. Basically, rough sets are used for several purposes, such as feature selection (FS), fea- ture extraction (FE), data reduction (DR), core identiﬁcation (CI), rule generation on decisions from data, straight-forward interpretation of pattern extraction, and obtaining results. So, it is called a predictive modelling approach. In our approach, we used the RST algorithm to reduce the number of attributes to ‘N’. But beforehand, we have to discuss the information table or decision system table, which is constructed in the form of tables with categorical data. Canese et al. [33] used both actor-critic algorithms and the taxonomy of reinforcement learning algorithms. They talked about non-stationarity, changing learning rates, and scalabil- ity as some of the drawbacks of multiagent reinforcement learning methods. This paper discusses many techniques for agents to operate in the environment and is well versed in the exposure of deep reinforcement algorithms. Methodologies such as partial observability, centralised learning of decen- tralised policies, COMA (counterfactual multi-agent policy gradient), which addresses the issue of multi-agent credit assignment, and agent-to-agent communication are exposed. Existing relevant works function of Q (quality). The Q learning model learns rules from a set of actions. If an action is different from the learn- ing rules, the model takes a precise step in choosing a random action. Tortolero et al. [31] discussed and explained various things in deep reinforcement learning. They proposed the Q- CCFD model, which employs three components for efﬁcient fraud detection: a deep autoencoder, a mediator network, and an agent. They have shown results from the training phase with 1000 episodes and predicted the class label in a testing phase. Mead et al. [32] extended their work on the reinforce- ment learning paradigm with the Markov decision process (MDP). In their experiments, an agent was always focused on predicting the optimal set of e-transactions, like steal- ing money as much as possible by committing the bank’s fraud. For each transaction, rewards are allocated based on the authorization activity of the cardholder. The card was charged −50 for the ﬁrst transaction. + 5 denotes a suc- cessful low-amount transaction, + 50 denotes a successful higher amount transaction, and 0 denotes a declined trans- action. This technique has shown better results in increasing the true positive rate in credit card fraud detection systems. Information system (I.S.) Informationsystemsarealsocalled“decisiontables.”Knowl- edge is a rough collection of facts expressed as domain values of attributes describing an object. All facts are expressed in the form of an information or data table. Every row in the data table is treated as an object. An information table is expressed with four tuples, as follows: To increase the robustness of the models, Huang et al. [34] presented work on quantitative and qualitative trading by predicting price change methods in the ﬁnancial industry market. By keeping an eye on the learning environment in a speciﬁc location, they developed a novel sampling strategy for determining which data are worth learning. The experi- ments’ ﬁndings showed that the adaptive sampling approach outperformed the random learning approach. Additionally, it increases the computational effectiveness of the model. A deep Q learning agent’s behaviour was taken advantage of by Carta et al. [35] with relevant training data from the real-world ﬁnancial market sector, an agent was taught repeatedly. The experimental results of this intraday trading show that it outperforms traditional methods such as the buy- and-hold strategy. The four tuples state, action, probability, and rewards used by the authors to express the MDP and DQN models that are suggested as a method for learning the Q-table. Here, it is demonstrated that the DQN is a clever approach for handling classiﬁcation-related issues. I.S. =< U, A, D, f > . (1) (1) In the preceding Eq. (1), I.S. represents an information system or database, whereas U represents a closed universe with an ‘n’ number of instances {x1, x2, x3, . . . xn}, which is essentially a non-empty set consisting of objects. An attribute or feature set is a collection of n attributes represented as A = {a1, a2, . . . an} is the domain value of that attribute A. Let us consider A = S ∪Q then the tuple or record DES = (U, S ∪Q, D, f ) is said to be a decision table or information table, where S is the conditional attribute and Q is the decision attribute. Obviously, every object and attribute will have some domain range that could be speciﬁed by the set D. Reducts (4) The concepts that are primarily used for rule discovery are reducibility and core attributes. As given in I.S, some attributes are redundant when classifying in terms of any sub- set insisted on in X’s attribute set. Then, redundant attributes were removed without affecting the classiﬁcation power of the diminished information system. The reduct (RED) is a minimal set of attribute lists from A that preserves similar divisions of objects in the universe U (considering the whole set of attributes). The concepts that are primarily used for rule discovery are reducibility and core attributes. As given in I.S, some attributes are redundant when classifying in terms of any sub- set insisted on in X’s attribute set. Then, redundant attributes were removed without affecting the classiﬁcation power of the diminished information system. The reduct (RED) is a minimal set of attribute lists from A that preserves similar divisions of objects in the universe U (considering the whole set of attributes). where f (x j) and f (yk) corresponds to the attribute value a of the object x j and yk, respectively. It should be noted that here IND(P) has been deﬁned as an equivalence relation. The clusterofallequivalenceclassesinIND(P)arerepresentedby the universe quotient set U, accredit as U/IND(P), in shortest form, U/P; and it is denoted by U/P = {[x]P∥x ∈U}. In case that (x j, yk) ∈IND(P), here x and y are forenamed as indiscernible (or identical) w.r.t P, and it is written as P- indiscernible. P-indiscernibility equivalence class relations are represented by [x]P. Accordingly, the elements in [x]P are indiscernible by attributes from P. By this elaboration the IND(P) equivalence classes of the relation recalled as P-elementary sets. IND(P) = IND(P −{b}). (8) (8) Thus, it extract partition. In decisive, Let T , P ⊆A and b ∈H.The following presumptions are assumed to be true: – IND(P) = IND(P −b),then b is redundant in P; otherwise P is indispensable. Indiscernibility For a subset P ⊆A of attributes in I.S., a relation is said to be indiscernibility relation, and this relation is denoted by IND on the universe U of any subset P with an objects {y1, y2, y3, . . . , yn} and denoted by IND(P), is deﬁned in the following Eq. (4). IND(P) = (x j, yk) : (x j, yk) ∈U 2, ∀a∈P( f (x j) = f (yk)) where 1 ≤j, k ≤n, (4) Information system (I.S.) So, if D = Ua∈ADa where Da means represents the set of values associated with the domain of the attribute a, Function to Write Information as f : U X A −> D, (2) (2) f : U X A −> D, f : U X A −> D, 12 123 plex & Intelligent Systems (2023) 9:5313–53 ere as in Eq. (2), ‘ f ’ is the total dec , a) ∈Da for every x ∈U, a ∈A is, in Eq. (3), function should satisf niverse U towards respective attribut given set of examples. So, it does no ues. So, we have to deﬁne these kind bute and the objects. The decision t es that are fully or partially reliable. scernibility Complex & Intelligent Systems (2023) 9:5313–5332 5317 where as in Eq. (2), ‘ f ’ is the total decision function. Such that where as in Eq. (2), ‘ f ’ is the total decision function. Such that Equation (6), indicates P-upper approximation of an element X. The objects absolutely or in sometimes may be related to the concept of X. Equation (6), indicates P-upper approximation of an element X. The objects absolutely or in sometimes may be related to the concept of X. (3) f (x, a) ∈Da for every x ∈U, a ∈A, INDP(X) = {x ∈U\|[x]P ∩Z ̸= NU LL}. (6) (6) The positive, negative, and boundary regions of a set X generated by dividing theU universe. Here, described bound- ary region (B) or the doubtful region of IND(X) by Eq. (7). BIND(X) = INDP(X) −INDP(X). (7) (7) From the equations it has been clearly said that if an object xi ∈PIND(X) means positive region [PIND(X) = INDP(X)], then xi corresponding to related target set X. Suppose, an object xi ∈NIND(X), i.e, negative region [NIND(X) = UINDP(X)], then it does not belongs to X which implies a state of uncertainty. If BIND(X) = φ, the set X is called to be crisp set w.r.t P; Otherwise, the set X is designated as rough w.r.t P.The pair of {INDP, INDP} is known as the Pawlak’s rough set of X w.r.t A attribute set. INDP(X) = {x ∈U\|[x]P ⊆Z}. (5) Deep Q networks (DQN) Learning methodology based on reinforcement to achieve proﬁciency for optimal behaviour, this RL approach has been used. This adaptive learning process is characterised as the issue of an agent performing an action based on “trial and error” via communications with an anonymous environment that gives feedback in the form of numerical values, i.e., “rewards.” The employed agent works based on reinforce- ment learning (RL). Its activation function is the learning ReLU (hidden) and MISH at the output layer, and the algo- rithm is a novel version of deep learning. This particular framework solves classiﬁcation problems using reinforce- ment learning (RL) with an agent who receives rewards depending on the state and action. If it marks a transaction as true, a positive reward is assigned; otherwise, a negative reward is assigned. CORE(P) = ∩RED(P). (9) Core Core is the attribute that is shared by all of the indispensable attribute’s IND(P) in the set A. Core is a common attribute in all reduct sets. If removing an attribute introduces inconsis- tency, then that attribute is used as a core; otherwise, it is not used as a core. The list of all essential features of P is referred to as the core of P. The relation between the reduct and the core is speciﬁed by the following Eq. (9) where RED(P) represents the list of all reducts in P. The list of all indispens- able features of P are called core of P. The relation between reduct and the core is speciﬁed by the following Eq. (9) where RED(P) represents the list of all reducts in P. f (x) = x · tanh(softplus(x)) = x · tanh(ln(1 + ex)). f (x) = x · tanh(softplus(x)) = x · tanh(ln(1 + ex)). (12) Multilayer perceptron (MLP) MLP is a class of ANN. The models of MLPs are the most basic deep neural networks. It is composed of a series of fully connected layers [36] in between the input, hidden, and out- put layers. Mainly, MLP works as an agent. Here, the inputs are the state and action that take place in the reinforcement environment. The user inputs will be multiplied by weights and then added with the bias that is shared by all the nodes of the hidden unit, as shown in Eq. (10). After the work is done by the activation function, the result will be given to the next layer. For every bit of a hidden layer, an activation function is applied, and that produces results. This process repeats, and the weights of the model are adjusted to minimise the error by the back propagation method until the desired output is obtained, either 0 for a non-fraud transaction or 1 for a fraud transaction. Figure1 represented terminologies used in the reinforce- ment learning (RL): – Agent: An agent learns the model state St by taking an input Xt, where t denotes the state transactions at t time. An agent performs the task in three ways (i) execute, (2) observe state and (3) receive reward. In the pro- posed model, the feature vector representation will be the agent’s input of given transactional data. Through actions Ut, an agent cooperates with the learning framework and provides rewards R(t +1), which can be used to enhance the function policy. The rewards are received from each transaction that is to be processed, and according to that, the Q-table is updated. Actually, the Q table is a refer- ence table that is in the format of a matrix, and the matrix stores the q-values as state-action (State S and Action U) pairs. In the initial stage, it allocates 0s to all states and is updated after completing each and every episode of the learning, and then an agent learns to execute the neces- sary action for a given state. By observing the rewards, the agent is able to provide the best action regardless of whether the transaction is true or false. N i=1 (Wi xi) + bias. (10) (10) i 1 (Wi xi) + bias. – ReLU activation function: ReLU is one kind of activation function. Approximation of sets – Set P is said to be independent if all of its attributes are required or indispensable. Lets consider P ⊆A as a subset of attributes and X ⊆ U as a subset of the universe. We can proximate X with the information was insisted on in P by constructing the P lower and P upper approximations on X. The description, i.e., X, deserved, which can be used to actuate the membership status of each object x in U w.r.t X. The rough membership function speciﬁes the rate of associated overlap between set X and [x]P. Equation (5), indicates P-lower approximation of a concept X. – Given T H. If G is independent and IND(T) = IND(P), then the set T is the Reduct of P. Equation (8) deﬁned that all these deﬁned attributes preserve the indiscernibility relation and, thereupon, set an approxi- mation. Indiscernibility is basically the state of things where there is ambiguity. After completing the entire process on the data, we can get several such attribute subsets of s, and those minimal subsets are called “reduced sets.” The reduc- tion occurs when you can make a decision based solely on (5) INDP(X) = {x ∈U\|[x]P ⊆Z}. 12 3 Complex & Intelligent Systems (2023) 9:5313–5332 5318 the attribute X. As a result, we will obviously try to ﬁnd the one with the fewest attributes. Genetic algorithms are also applied to the simultaneous computation of many reductases [1,8,10]. the attribute X. As a result, we will obviously try to ﬁnd the one with the fewest attributes. Genetic algorithms are also applied to the simultaneous computation of many reductases [1,8,10]. – MISH activation function: In a neural network, the con- cept of non-linearity is offered by an activation function that plays an important part in the network’s training and performance evaluation. The Mish, a unique neu- ral activation function, is introduced in this study. When compared to Swish, Mish is a smooth and non-monotonic activation function, which can be deﬁned as in Eq. (12). – MISH activation function: In a neural network, the con- cept of non-linearity is offered by an activation function that plays an important part in the network’s training and performance evaluation. The Mish, a unique neu- ral activation function, is introduced in this study. When compared to Swish, Mish is a smooth and non-monotonic activation function, which can be deﬁned as in Eq. (12). y(x) = max(0, x) Multilayer perceptron (MLP) Our dataset is applied to rough set theory (RST). The RST is dividing our data set into ‘M’ number of reducts RD1, RD2,…,RDM which produce a minimal set of attributes that amuse the target class based on the indiscernibility of the class label. A deep reinforcement learning network, i.e. DQN is an intelligent system divided into ‘N’ number of agents like DQN1, DQN2,…, DQNN. This ﬁgure exposes the overall performance in terms of identifying fraud in digital transactions. Phases in this the proposed model is explained in subsections below. Our dataset is applied to rough set theory (RST). The RST is dividing our data set into ‘M’ number of reducts RD1, RD2,…,RDM which produce a minimal set of attributes that amuse the target class based on the indiscernibility of the class label. A deep reinforcement learning network, i.e. DQN is an intelligent system divided into ‘N’ number of agents like DQN1, DQN2,…, DQNN. – Probability of state transition (Prob): Which calculates the conditional probability (Prob (st+1 \| st, ut)) to move from state st to state st+1. – Episodes:Thenumberofiterationsthattheagentrequired to ﬁnd the better optimal Q-values for all the state and action (S,U) pairs. Then, apply the renovated DQN architecture for each and every reduced set as RDQN, and that will cause better accu- racy in performance. Every DQN that produces an output with the predicted label indicates whether or not fraud exists. After combining the RD1 × DQN1, RD2 × DQN2 …RDM × DQNN results with a weighted average or with majority voting. Here, each DQN is formed with the MLP neural net- Multilayer perceptron (MLP) 2 Intelligent agent system for DTFD This ﬁgure exposes the overall performance in terms of identifying fraud in digital transactions. Phases in this the proposed model is explained in subsections below. Our dataset is applied to rough set theory (RST). The RST is dividing our data set into ‘M’ number of reducts RD1, RD2,…,RDM which produce a minimal set of attributes that amuse the target class based on the indiscernibility of the class label. A deep reinforcement learning network, i.e. DQN is an intelligent system divided into ‘N’ number of agents like DQN1, DQN2,…, DQNN. Then, apply the renovated DQN architecture for each and every reduced set as RDQN and that will cause better accu Fig. 1 Renovated DQN architecture Fig. 2 Intelligent agent system for DTFD Fig. 1 Renovated DQN architecture Fig. 1 Renovated DQN architecture Fig. 2 Intelligent agent system for DTFD neural network and how many feature vectors there were, different numbers of actions were altered. The collection of moves an agent is capable of doing in the environment. – State (S): The state of the environment at each time stamp ’t’ is what the agent is attempting to interact with or per- ceive, with some modiﬁcations affecting the action that an agent handles. The input transaction data xt deter- mines the state ’S’ in this model. – Policy(π(S)): The policy π describes the mapping between states and the best action to take for that state in the current environment. That means an action refer- ring to that state can maximize the value of a reward. The policy set is important to the RL algorithm because it speciﬁes the best decision to make. – Strategy: The policy should choose an action that max- imises the feature reward. – Reward (R): A reward (R) is immediate feedback from the environment that measures the fraud or non-fraud of the agent’s action. So, this is treated as the optimum action for that concerned state. Fig. 2 Intelligent agent system for DTFD – Discount factor (γ ): is described as balancing the agent’s performance in such a way that the agent may make the best decisions for both immediate and long-term beneﬁt. The discount factor has a value between 0 and 1. This ﬁgure exposes the overall performance in terms of identifying fraud in digital transactions. Phases in this the proposed model is explained in subsections below. Multilayer perceptron (MLP) The activation function calculates a weighted total and also uses bias to decide if a neuron ought to be actuated or not. The main goal of ReLU is to introduce the non-linearity concept into a neuron’s output. This takes the maximum value, and this is not fully interval- derivable, but we can take a subvariant. The value of the ReLU activation function is either 0 or 1. ReLU is deﬁned by the formulae in Eq. (11). – Action (U): The environment is updated as a result of the actions. Depending on how many layers there were in the – Action (U): The environment is updated as a result of the actions. Depending on how many layers there were in the y(x) = max(0, x) (11) (11) 3 Complex & Intelligent Systems (2023) 9:5313–5332 5319 Fig. 1 Renovated DQN architecture neural network and how many feature vectors there were, different numbers of actions were altered. The collection of moves an agent is capable of doing in the environment. – State (S): The state of the environment at each time stamp ’t’ is what the agent is attempting to interact with or per- ceive, with some modiﬁcations affecting the action that an agent handles. The input transaction data xt deter- mines the state ’S’ in this model. – Policy(π(S)): The policy π describes the mapping between states and the best action to take for that state in the current environment. That means an action refer- ring to that state can maximize the value of a reward. The policy set is important to the RL algorithm because it speciﬁes the best decision to make. – Strategy: The policy should choose an action that max- imises the feature reward. – Reward (R): A reward (R) is immediate feedback from the environment that measures the fraud or non-fraud of the agent’s action. So, this is treated as the optimum action for that concerned state. – Discount factor (γ ): is described as balancing the agent’s performance in such a way that the agent may make the best decisions for both immediate and long-term beneﬁt. The discount factor has a value between 0 and 1. – Probability of state transition (Prob): Which calculates the conditional probability (Prob (st+1 \| st, ut)) to move from state st to state st+1. – Episodes:Thenumberofiterationsthattheagentrequired to ﬁnd the better optimal Q-values for all the state and action (S,U) pairs. Fig. The proposed RDQN model for digital transaction fraud detection (DTFD) The overall framework of the proposed architecture, named intelligent agent system for DTFD, is illustrated in Fig.2. 123 12 3 5320 Complex & Intelligent Systems (2023) 9:5313–5332 work to train the agent to predict fraud cases in the digital transactional reinforcement environment. At each various time step t, the state xt ∈Xdeﬁnes an environment. And then agent look into the state and that yield an action ut which belongs to set of agent actions (ut ∈ U). Subsequently, an agent banking environment conversion of its state to some other state, xt+1 ∈X in keeping with the moving possibilities or probabilities that are given by the function f: the ﬁnal probability in xt+1 after ut actions performed and executed in xt is f (xt, ut, xt+1). So, an agent obtains scalar reward (rt+1 ∈R), by the value given in the reward function i.e., {ρ : rt+1} = ρ(xt, ut, xt+1). A reward value updated with an immediate matter of fact an action ut, i.e., the moving or transition from xt to xt+1. Moreover, it saysnothingwithregardtothelongstandingconsequencesof this policy. It is assumed that the reward function is bounded. work to train the agent to predict fraud cases in the digital transactional reinforcement environment. Dataset pre-processing Toassess theperformanceof themodel, weusedareal-world, unique data set and conducted different experiments. The credit card fraud data was accessed from the UCI machine learning repository. This is a highly imbalanced dataset. That means it does not provide much accuracy in classiﬁcation. For that, using the oversampling technique SMOTE to bal- ance the data. And then, to develop a model, multiple types of data are required to be processed, like numerical data and categorical data. To run the proposed model, we required categorical data, which is fed as input to the RST. It will automatically increase the efﬁciency of our model because dealing with large variables is difﬁcult and it is preferable to reduce the number of variables handled. In deterministic environment, the next state of the envi- ronment is purely determined by the present state and their action that is carried out by the agent in an environment rt+1 = ρ(xt,ut). It observes rewards in each episode based on determination (ρ : X × U →R). Each episode in which action is taken will have its own learning process. And the learning process does not work with the single feature vec- tor. In MDP, it has some terminal states. So, we can receive rewards from these terminal states. Here, the function of tran- sition probability “f” is updated with the transition function f : X × U →X. Renovated DQN architecture Inourproposedarchitecture,weintroducedarenovatedDQN architecture. This DQN architecture is made up of deep neural network multi-layer perceptron’s and reinforcement learning Q-learning. The renovated DQN architecture is built with different activation functions. In the hidden layer, ReLU activation is used, and MISH activation is used in the output layer, both to speed up the process and obtain accurate results in the identiﬁcation of fraud cases. The policy deﬁnes the agent’s behaviour, which explains how the agent accepts its actions in the present. Generally, a policy or rule may be either deterministic or stochastic. The derived equation for stochastic is h : X × U →[0, 1] and for deterministic is h : X →U. If a policy does not alter over time, it is called stationary. The agent’s main goal is to ﬁnd a better policy that maximizes with an expectation of discounted return γ in Eq. (13) for each state x. DQN as classifier There are processes done in two steps, such as analysing Q∗and the greedy policy in Q∗. Renovated DQN Algorithm 1. Initialize replay memory RDi to Capacity M 2. for each i = 1 to N 3. Assign function action-value Q with RDi features and random weights 4. for episode 1 to M do 5. Initialize sequences Si = {} 6. for t to T do 7. with prob ϵ select at 8. π( a s ) = {ϵ, Random Action (1 −ϵ), a∗= argmaxQ(S, A)wherea ∈A 9. Implement action at in Emulator 10. Examine Reward value rt and image value xt+1 11. Set st+1 = st, at, xt+1 and preprocess Qt+1 = φ(st+1) 12. Store Transition (φt, at,rt, φt+1) in D 13. Sample random method of minibatch on transactions (φ j, a j,r j, φ j+1) from D 14. Activation function {ReLU y(x) = max(0, x) Mish f (x) = x.tanh(sof tplus(x)) = x.tanh(ln(1 + ex)) 15. Set yi {1 If transaction in fraud 0 Non-Fraud 16. Perform Gradient Descent Step w.r.t Mean Squared Error yi −Q(φ j; a j; φ)2 17. End for RDi 18. Ensemble of DQN1, DQN2 . . . DQNN 19. Each entrance of DQNi is predicted based on Average weightage of DQN Agent. 1. Initialize replay memory RDi to Capacity M Q-learning said to be procedure for an iterative approxi- mation. At every time step, Q starts learning from arbitrary of Q-function, and it look into the transitions i.e, represented as (xt, ut, xt+1,rt+1), and the Q function is updated after each transition observable. Qt+1(xt,Ut) = Qt(xt,Ut) + αtrt+1 +γ maxU ′ Qt(xt+1, u ′) −Qt(xt, ut). (16) 12. Store Transition (φt, at,rt, φt+1) in D Equation (16) denotes the difference between the cur- rent estimate (xt, ut) and the best Q-value of (xt, ut) and for which the value updated or estimated rt+1 + γ max ′ u Qt(xt+1, u ′). Here, the new estimation is generated according to the bellman Eq. (14), i.e., applied to Qt in the state and action pair (xt, ut). The value of learning rate γ ∈ (0, 1] can be varied over time in discrete mode, and usually, it decreases over time. 18. Ensemble of DQN1, DQN2 . . . DQNN If an agent continues to try all activities with nonzero probabilities in all the states, then it can be called exploration and then polished. DQN as classifier In this paper, we presented a novel renovated deep Q-network model, as well as a custom open AI gym environment for deep RL agents that stores experience replay and an approximation value function. The DQN agent employs an epsilon-greedy policy to perform classiﬁcation action based on batches of input. The open AI environment prospects an agent’s action and then evaluates the rewards for the agent accordingly. The agent’s memory stores this entire experi- ence. At the end of batch completion, the DQN agent samples a batch of memory from its experience replay buffer, edits the Q-value by Q-network, evaluates the loss, and performs back-propagation to revise the weights. Our updated model successfully classiﬁed fraudulent and non-fraudulent digital transactions, achieving state-of-the-art performance. Rπ(X) = E ∞ t=0 γ krt+1\|x0 = x, π , (13) (13) where the discount factor γ lies between (0 < γ < 1], the number of possible transition states under the policy π, the expectation is taken into account. The discount factor γ can be described as capturing and increasing uncertainty in future rewards. A long-term beneﬁt gained by an agent is expressed by the return R. The different ways of representing the return of R are discussed [43]. The deﬁnition of Q (an optimal) Q-function as Q∗(x, u) = maxh Qh(x, u). The Bellman optimality satisﬁes this Eq. (14). The classic model of the MDP consisted of single-agent RL. A ﬁnite MDP is deﬁned as a record < X,U, f , p >, the deﬁnition contains X as a ﬁxed set of states in an envi- ronment, a ﬁnite set of agent actions U, the state probability function f : X × U × X →[0, 1] and the reward function ρ : X × U × X →R [37]. Q∗(State x, action U) = x ′ ∈f (x, u, x ′)ρ(x, u, x ′) (14) (14) 123 123 5321 Complex & Intelligent Systems (2023) 9:5313–5332 According to Eq. (15) an optimal action reward is obtained by taking u of x, which is a proven or expected prompt reward summation, and multiplying it by the discounted and most advantageous value that is reachable from the next sequen- tial state. Once Q∗is available, the precise policy, which improves the return, can be evaluated. It chooses an action in every state that maximizes the subsequent future reward. The optimal Q-value is expressed in Eq. DQN as classifier (15) as follows: Algorithm for Calculating Reducts 1. RDi be a set of all reducts 2. C be a set of all attributes 3. D be a decision attribute 4. Initialize (b1, b2, ....bN) =τ(a1, a2, ...aN) 5. where is permutation 6. RDi = {} 7. for i=1 to N 8. Di(Ci) ̸= Di(bi) 9. RDi < −bi 10. else 11. RDi < −RDi −bi 12. end for 13. End Renovated DQN Algorithm 1. Initialize replay memory RDi to Capacity M 2. for each i = 1 to N 3. Assign function action-value Q with RDi features and random weights 4. for episode 1 to M do 5. Initialize sequences Si = {} 6. for t to T do 7. with prob ϵ select at 8. π( a s ) = {ϵ, Random Action (1 −ϵ), a∗= argmaxQ(S, A)wherea ∈A 9. Implement action at in Emulator 10. Examine Reward value rt and image value xt+1 11. Set st+1 = st, at, xt+1 and preprocess Qt+1 = φ(st+1) 12. Store Transition (φt, at,rt, φt+1) in D 13. Sample random method of minibatch on transactions (φ j, a j,r j, φ j+1) from D 14. Activation function {ReLU y(x) = max(0, x) Mish f (x) = x.tanh(sof tplus(x)) = x.tanh(ln(1 + ex)) 15. Set yi {1 If transaction in fraud 0 Non-Fraud 16. Perform Gradient Descent Step w.r.t Mean Squared Error yi −Q(φ j; a j; φ)2 17. End for RDi 18. Ensemble of DQN1, DQN2 . . . DQNN 19. Each entrance of DQNi is predicted based on Average weightage of DQN Agent. Algorithm for Calculating Reducts Algorithm for Calculating Reducts 1. RDi be a set of all reducts 2. C be a set of all attributes 3. D be a decision attribute 4. Initialize (b1, b2, ....bN) =τ(a1, a2, ...aN) 5. where is permutation 6. RDi = {} 7. for i=1 to N 8. Di(Ci) ̸= Di(bi) 9. RDi < −bi 10. else 11. RDi < −RDi −bi 12. end for 13. End (15) π∗(x) = argmaxu Q∗(x, u). (15) When various activities achieve the highest Q-value, any of them can be chosen and the approach will remain optimal. In that case, the argmax operator was interpreted as a parameter for opting for good clariﬁcation. As shown in Eq. (16), a policy that improves or maximises a Q-function is said to be greedy in that Q-function. DQN as classifier Typically, the probability decreases over time. For instance, the selection of probability random action at each stage ϵ ∈(0, 1), and probability (1−ϵ) with a greedy action. So that, we can obtain this ϵ -greedy exploration. to the base classiﬁers. Among all feature selection tech- niques, RST provides essential features. The ﬁnal frame up 3 adapted RST in the DQN algorithm (i.e., RDQN), tested all of the reducts produced by RST with DQN, calculated aver- age weighted accuracy, and compared this average weighted accuracy to other existing fraud detection techniques. Frame-up: 1 It can be exposed in three different frame-ups. The over- all performance of the proposed model is determined by the operation of all frame-ups. Frame 1 compares the per- formances of different classiﬁers with a European data set (including all the features). In Frame 2, some wrapper and selection-based feature selection algorithms were applied Many machine learning algorithms, such as LR, SVM, NB, and ANN, are used for classiﬁcation. Classiﬁcation is done on the dataset by dividing it into training and test data. All models are trained based on the training data and then predict the target variable, whether the transaction is fraud or not, in the test data. Some of the base classiﬁers that are consid- 12 3 Complex & Intelligent Systems (2023) 9:5313–5332 5322 Table 1 Important features from RST Reducts from RST RD1 {Time, V4, V13, V14, V15, V17, V18, V21, V22, V24 V25, V26, Amount} RD2 {V4, V9, V11, V12, V14, V15, V22, V24, V26, Amount} RD3 {V4, V14, V15, V17, V19, V21, V23, V26, Amount} RD4 {Time, V1, V3, V28, V24, V11, V25, V12, V26, V4} RD5 {Time, V1, V3, V28, V24, V11, V12, V25, V26, V4} Table 1 Important features from RST Reducts from RST RD1 {Time, V4, V13, V14, V15, V17, V18, V21, V22, V24 V25, V26, Amount} RD2 {V4, V9, V11, V12, V14, V15, V22, V24, V26, Amount} RD3 {V4, V14, V15, V17, V19, V21, V23, V26, Amount} RD4 {Time, V1, V3, V28, V24, V11, V25, V12, V26, V4} RD5 {Time, V1, V3, V28, V24, V11, V12, V25, V26, V4} ered for proposed model consideration and comparison are explained here. Logistic regression (LR) It is a basic common classiﬁcation technique that shows the relationship between dependent and independent variables. When the dependent variable is cat- egorical or binary and the predictors are continuous, then categorical linear regression is easily described [38,39]. LR uses a nonlinear sigmoid function to ﬁnd the best-ﬁt param- eters. In the equation, the sigmoid function is denoted by the symbol σ, and the input variable x to the sigmoid function is shown in Eq. (17). label [39]. Here, the dataset is oftentimes partitioned using either a breadth-ﬁrst greedy (BFG) or a depth-ﬁrst greedy (DFG) strategy, and the process is stopped after all of the features have been assigned to a given class label. Frame-up: 2 P(A\|B) = (P(B\|A) ∗P(A)) P(B) . (18) (18) The feature selection process is one of the necessary steps in data pre-processing. It improves the model’s performance and also reﬂects the computational cost of the model. Hence, the proposed model takes feature selection into considera- tion and applies some ﬁlter- and wrap-based feature selection techniques. KNN The KNN classiﬁer examines the pattern space among its nearest neighbours. A new instance that should be near when it assigns an unknown sample to the space [39]. To examine the closeness among neighbouring instances, an algorithm uses the Euclidean distance function in Eq. (19). KNN The KNN classiﬁer examines the pattern space among its nearest neighbours. A new instance that should be near when it assigns an unknown sample to the space [39]. To examine the closeness among neighbouring instances, an algorithm uses the Euclidean distance function in Eq. (19). Chi-square feature selection (CSFS) This is the easiest method for feature selection that inﬂuences the target vari- able in supervised learning. Basically, this method focuses on categorical features in a dataset. Select the best number of features by calculating the chi-square score, which can be calculated between each variable or feature and assesses whether the sample’s association between two categorical variables reﬂects their true association in the population. A Chi-squared score is denoted by Eq. (22). k i=1 (xi −yi)2. (19) (19) SVM Which is one of the best in a deep classiﬁcation tasks. SVM Which is one of the best in a deep classiﬁcation tasks. Generally, SVM works in high-dimensional feature space. There are two factors that SVM prepare them strong— marginal space and kernel function in Eq. (20) depiction [38]. With the use of a kernel function, SVMs map into a high-dimensional feature space and then learn the model for classiﬁcation purposes without any added computational dif- ﬁculties. SVM Which is one of the best in a deep classiﬁcation tasks. Generally, SVM works in high-dimensional feature space. There are two factors that SVM prepare them strong— marginal space and kernel function in Eq. (20) depiction [38]. With the use of a kernel function, SVMs map into a high-dimensional feature space and then learn the model for classiﬁcation purposes without any added computational dif- ﬁculties. X2 = (Ofrq −Efrq)/(Efrq), (22) (22) where Ofrq is number if observations of the class and Efrq number of expected observations of class. Frame-up: 1 The best split is one that prevents subgroups from iterating, i.e., one that keeps them as distinct as possible. Formulae in Eq. (21) provide information. Y = 1/(1 + e−z). (17) (17) Y = 1/(1 + e−z). Naïve Bayes This is a bayesian statistical strategy that deter- mines the best decision based on the highest probabilities. From known values, bayesian probability estimates unknown probabilities. It also enables the application of prior knowl- edge and logic to questionable assertions. This technique makes the assumption that the features in the data are condi- tionally independent [39]. The NB classiﬁer performs based on the conditional probabilities, as shown in Eq. (18), of the targeted classes, i.e., fraud and non-fraud. KNN is one of the most popular supervised classiﬁcation algorithms. Naïve Bayes This is a bayesian statistical strategy that deter- mines the best decision based on the highest probabilities. Gain(IS, A) = Entropy(IS) − v∈values(A) \|ISv\| \|IS\| Entropy(ISv). (21 (21) Entropy(ISv). Frame-up: 2 W T x + b = 0. (20) (20) W T x + b = 0. DT Another extensively used method for classiﬁcation pur- poses is the DT. A tree has some interior knobs that reﬂect tests on a characteristic; each branch indicates the occur- rence of that test, a concurrent terminal node grasps a target Pearson correlation FS (PCFS) This is called the univariate method, which measures some type of correlation between two random variables, such as a particular feature and a target variable, and keeps the features that have a higher correlation. 123 5323 Complex & Intelligent Systems (2023) 9:5313–5332 It can be measured by correlation using Eq. (23). It can be measured by correlation using Eq. (23). detection techniques were used for comparison. The exist- ing fraud detection techniques are IFDTC4.5, SAE-GAN, CNN-SVM-KNN, and DEAL, which are illustrated. r = n i=1(Ci −¯C)(Gi −¯G) n i=1(Ci −¯C)2 n i=1(Gi −¯G)2 . (23) (23) IFDTC4.5 Askari et al. [40] built or reﬁned a decision tree using intuitionistic fuzzy logic and DT (C4.5) to detect dig- ital fraud. The fuzzy logic takes into account the cognitive characteristics of variables so that it can estimate that normal transactions are not considered fraudulent digital transac- tions, and fraudulent digital transactions are not treated as normal digital transactions. Tree generation mechanisms construct decision trees using transactional datasets that have been used and considered information gain ratio. The trained data set is then used to build the tree, which is then tuned sev- eral times until it has accurate classiﬁcation. Ci is the overall training example, ¯C is the mean value of the random variable Ci and ¯G are the average values of the random variable Gi. So, the correlation value is between + 1 and −1. A + 1 means accurate positive correlation, -1 means absolute negative correlation, and both + 1 and −1 or close to zero mean they are highly correlated. If it is closer to zero means, there is no correlation. Hence, select features that have a high positive or negative correlation. Selection-based feature selection (SBFS) For this model, use forward selection so that the features are consequently added to an empty set until an addition is made where the extra features do not reduce the criterion. At every step, based on the cross-validation score of an estimator, the estimator selects an important feature to add or remove. Frame-up: 2 CNN usually exceeds in performance when compared with other DL meth- ods such as AE, restricted Boltzman machine (RBM), and deep belief networks (DBN). They summarised the results on different datasets with MCC and AUC values. Rough set theory-based feature selection The major task performed by rough set theory is feature extraction, and it handles the certainty of the data. RST algorithm is used to reduce a set of attributes to a minimum of ‘N’ attributes. Here we calculate the indiscernibility relation speciﬁed in Eq. (6) based on the information table. Many RD’s are gen- erated from this RST with different minimal set combination and out of all these, some sets are taken into consideration that are listed in Table 1. For Example, ﬁve reducts are cho- sen at random, with the set containing a minimum of 9-13 attributes. The RD1 has 13 attributes, the RD4 and RD5 are sets of ten attributes, and the RD3 has nine attributes. DEAL Arya et al. [43] proposed DEAL is a predictive framework, i.e., deep ensemble learning to identify abnor- mal transactions in real-time data. Proposed DEAL, a novel framework for predicting fraud transactions in real credit card data streams. We represent each transaction as a tensor to reveal the latent and inherent relations between spending patterns and fraudulent transactions. By uniting the extra-tree ensemble method with deep learning (DNN) in characteristic space, the effect of highly imbalanced classes is reduced, thus yielding a better prediction accuracy of fraudulent classes. We addressed the valuable metrics for evaluating the CCFD frameworks. The proposed framework is evaluated on the basis of these valuable metrics: categorical accuracy, train- Frame-up: 2 In the case of unsupervised learning, this sequential feature selector always cares about the features (X), not the targeted outputs (Y). SAE-GAN Together, the sparse auto-encoder and generative adversarial networks outperform other methods for detect- ing fraud. SAE aids in increasing the number of features and obtaining key features [41]. They trained GAN using the optimised Generator G, Discriminator D, and loss function parameters. After the training process, the discriminator will have the capability to separate fraudulent or abnormal trans- actions, which are different from genuine transactions. The trained SAE, which is a discriminator in the GAN, is used to predict the form of a new digital transaction during the testing phase. Recursive feature elimination (RFE) RFE is one kind of greedy algorithm that works in terms of feature ranking tech- niques. RFE starts with the entire set and then removes the least-suited features one by the one, selecting the top-most appropriate features based on this ranking technique. That means the estimator is well trained on the complete feature set, and the importance of an individual feature is obtained through any particular callable attribute. CNN-SVM-KNN Raghavan et al. [42] investigated various ML and DL models on various data sets to identify abnormal transactions. The primary goal of this paper is to generate intuition about which models work best for which types of datasets. This study reveals that in detecting fraud, SVM would be the best model with larger datasets, and this could potentially be combined with CNNs to score a more predictable performance. SVM, RF, and KNNs can pro- vide better enhancement on smaller datasets. CNN usually exceeds in performance when compared with other DL meth- ods such as AE, restricted Boltzman machine (RBM), and deep belief networks (DBN). They summarised the results on different datasets with MCC and AUC values. CNN-SVM-KNN Raghavan et al. [42] investigated various ML and DL models on various data sets to identify abnormal transactions. The primary goal of this paper is to generate intuition about which models work best for which types of datasets. This study reveals that in detecting fraud, SVM would be the best model with larger datasets, and this could potentially be combined with CNNs to score a more predictable performance. SVM, RF, and KNNs can pro- vide better enhancement on smaller datasets. Evaluation metrics The confusion matrix is an important criterion for calculating the performance of a classiﬁcation algorithm, and knowing which criterion gives the summarization helps in understand- ing what the classiﬁcation model is estimating correctly and what types of errors it is making. TPOS is the number of samples that are both given the same value (i.e., positive) in actual and predicted, and TNEG is the number of instances that include both actual and predicted. Negative, FNEG, and FPOS represent the numbers in the classiﬁcation rate of errors. The given performance metrics, such as accuracy, true fraud, false fraud, speciﬁcity, and precision, are expressed in Eqs. (24)–(28). Accuracy = (TPOS + TNEG)/(TPOS +FNEG + FPOS + TNEG) (24) True fraud = TPOS/(TPOS + FNEG) (25) False fraud = FPOS/(FPOS + TNEG) (26) Speciﬁcity = TNEG/(FPOS + TNEG) (27) Precision = TPOS/(TPOS + FPOS) (28) Results for frame-up: 1 Experimental results The proposed RDQN model is efﬁcient in improving the accuracy of digital transaction fraud detection. It was done in three frames. Initially, feature selection techniques are applied to datasets, and the best technique, namely RST, is chosen due to its demonstrated performance. The RST gener- ated minimal feature subsets known as RD1, RD2,…, RD5. Second, the DQN algorithm was applied to each reduced dataset, and the weighted average of accuracy was calculated. Lastly, we compared DQN’s performance in terms of clas- sifying fraud and non-fraud transactions with other existing methodologies. To implement a model, these are the require- ments that must be taken into consideration. As a result, we used a 10th generation Intel Core i5 processor and a hybrid drive system with SSD (256 GB) and HDD (1 TB) to accel- erate the process. For our convenience in various operations, we implemented the proposed model in Python 3.8 using Scikit-Learn, Keras, and MATLAB (R2020b). And given the results, it demonstrated its superiority among all state- of-the-art methods in detecting fraudulent transactions. Frame-up: 3 In this section, we introduced the renovated DQN algorithm, which operates by agent in a bank environment. The RST derived reducts (RD1, RD2,…, RD5) are fed to DQN to form RDQN1, RDQN2,…, RDQN5. To know the perfor- mance of all RDQNs, we calculated a weighted average and compared the renovated DQN algorithm with other state- of-the-art methods. Some existing hybrid models of fraud 12 3 Complex & Intelligent Systems (2023) 9:5313–5332 5324 Table 2 Performance based on base classiﬁers Performance metrics LR NB SVM DT ANN Accuracy 0.83744651 0.85661528 0.90937335 0.88006331 0.91523536 True fraud rate 0.93798124 0.94525423 0.95487656 0.96003464 0.96157049 False fraud rate 0.62647555 0.63403477 0.45567652 0.62168979 0.49409781 Sensitivity 0.37352445 0.36596523 0.54432348 0.37831021 0.50590219 Precision 0.90017466 0.90880979 0.96194558 0.93596987 0.96243349 Error 0.16255349 0.14338472 0.09062665 0.11993669 0.08476464 F1-score 0.91868915 0.92667382 0.95839803 0.94784953 0.96200179 ing and prediction accuracy, log loss, false positives (FP), and fraud catching rate (FCR). have an appropriate transaction name, such as time, amount, and class. The rest of the attributes (V1–V28) do not contain any names for their corresponding transactions. Moreover, this is a numerical dataset that was deliberated by the Euro- pean Bank in 2013 and is protected, meaning it is data in the form of numbers with anonymous attribute names. The credit card fraud dataset provided is 100% complete and does not contain any missing values. Dataset description The dataset is available in Kaggle repository [44], and the information on the growing risk of digital transactional fraud underlinesthedifﬁcultyingettingthiskindofdata.Themajor challenge is the highly imbalanced dispersion among fraud and non-fraud target classes in millions of tuples of data. Banks are often exposed to fraudulent transactions and con- stantly improve systems to track them. The European fraud dataset contains a total of 2,84,807 samples of transactions. Out of these samples, 492 cases were identiﬁed as fraudu- lent transactions, and the remaining are treated as genuine transactions. The dataset has 31 attributes in total, with 30 attributes for input and one attribute for the target class or output. Of these attributes, only three variables are found to Table 2 Performance based on base classiﬁers Results for frame-up: 3 The reduced feature sets generated by the RSES toolbox are mentioned in Fig.6 and are thought to be of equal value to the original massive data set. It generated a number of reductions that uniquely identiﬁed the transaction as fraudulent or not. This randomly generated reduced set has fewer attributes than the original huge data set in its entirety. Based on a few runs through the RSES tool, a minimal set of 9–13 attributes was chosen from among all the reducts. Reduct table with attributes, number of rows, attribute set size, Pos.Reg, SC, and Reducts displayed. The reducts were used as input for the proposed model. The set RD1 consists with 13 attribute set i.e., RD1 = {Time, V4, V13, V14, V15, V17, V18, V21, V22, V24, V25, V26, Amount}, RD2 = {V4, V9, V11, V12, V14, V15, V22, V24, V26, Amount}, RD3 = {V4, V14, V15, V17, V19, V21, V23, V26, Amount}, RD4 = {Time, V1, V3, V28, V24, V11, V25, V12, V26, V4}, RD5 = {Time, V1, V3, V28, V24, V11, V12, V25, V26, V4, V15, V22}. The reduced feature sets generated by the RSES toolbox are mentioned in Fig.6 and are thought to be of equal value to the original massive data set. It generated a number of reductions that uniquely identiﬁed the transaction as fraudulent or not. Fig. 4 Performance metrics of base classiﬁers the model. The 70 percent data are 199,365× 31; out of these, 199,125 are non-fraud transactions, and 240 are frauds. After appropriate training and testing, all selected ANN models outperform all other speciﬁed models in terms of accuracy. SVM is among and follows. Table 2 depicts the data about the comparison of all the metrics of the speciﬁed models. The ANN model performed well, yielding the best results in terms of accuracy (91.5%), F score (96.2%), and true fraud rate. Figure4 shows the bar chart for the above performance comparison on various clas- siﬁers, which are speciﬁed in Table 2. The proposed renovated RDQN model was discussed and testedwithotherfrauddetectiontechniques,whichareshown in Table 9. Results for frame-up: 1 Table 2 depicts performance metrics that are derived from the confusion matrix shown in Fig.3. These performance metrics are required to compare the performance of different base classiﬁers. To validate the performance of these classic algorithms, a dataset was required. The dataset information is available in “Dataset description”. The dataset is formal divided into 70% for the training model and 30% for testing 123 123 123 Complex & Intelligent Systems (2023) 9:5313–5332 5325 Fig. 3 Confusion matrix for DTFD Fig. 4 Performance metrics of base classiﬁers Fig 3 Confusion matrix for DTFD The main objective of the RST is to extract important features. These features play a vital role in classiﬁcation and prediction. To explore this, tools called the Rough Set Explo- ration System Toolbox 2.2.2 (RSES) were used. The idea behind RST was to use an information table to calculate the indiscernibility relation speciﬁed in Eq. (4). Then, obviously, it would get all the reducts or reduced sets, which are useful for a better way of classifying things. Theperformanceswerecomparedbyperformancemetrics like accuracy, true-positive rate, false-positive rate, and F1 score. Generally, it was observed that RFE+NB, RFE+ANN, RSBFS+SVM, and RSBFS+ANN gave the best accuracy. Out of RFE and RSBFS, RSBFS has provided better accu- racy. That means it was given the best features. Finally, it was proved that the RSBFS+SVM model worked well. Tables 3, 4, 5, 6 and 7 are depicted clearly about feature selection- based performance among base classiﬁers and heeded how RSBFS was worked well rather than all other feature selec- tion techniques from Fig.5. So, RSBFS was Chosen as feature selection technique and was applied to proposed model to improve the performance. Furthermore, agent could easily learn the model within banking environment. Fig. 3 Confusion matrix for DTFD g Fig. 4 Performance metrics of base classiﬁers Fig. 4 Performance metrics of base classiﬁers Results for frame-up: 2 In this framework, different feature selection algorithms based on speciﬁed traditional classiﬁers were examined in Tables 3, 4, 5, 6 and 7. The selected features from different algorithms improve the model of any classiﬁer more than having all the features. Here, many ﬁlter- and wrapper-based feature selection methods were utilized, and those are dis- cussed in “Frame-up: 2”. The proposed model has achieved state-of-the-art per- formance on a highly skewed credit card fraud data set with 96.09 percent accuracy. It was able to correctly clas- sify fraudulent and non-fraudulent transactions. This high- performance model opens the door to many opportunities for exploring the scope of reinforcement learning in the ﬁeld of classiﬁcation problems and decision-making. 123 3 3 Complex & Intelligent Systems (2023) 9:5313–5332 5326 Table 3 Roughset-based performance on base classiﬁers Performance metrics RSBFS + LR RSBFS + NB RSBFS + SVM RSBFS + DT RSBFS + ANN Accuracy 0.93909373 0.94261094 0.946128 0.94250255 0.94969722 TPR 0.96379526 0.96393035 0.966543 0.96946565 0.97474747 FPR 0.43081452 0.48168007 0.490085 0.52894995 0.52324195 Sensitivity 0.56918548 0.51831993 0.509915 0.47105005 0.47675805 Precision 0.97235342 0.96639441 0.967802 0.96584067 0.97235342 Error 0.06090627 0.05738906 0.053872 0.05749745 0.05030278 F1-score 0.96805542 0.96516081 0.967172 0.96764977 0.97354898 Table 4 Chi square feature based performance on base classiﬁers Performance metrics CSFS + LR CSFS + NB CSFS + SVM CSFS + DT CSFS + ANN Accuracy 0.89331145 0.90861129 0.8984086 0.9144733 0.91482502 TPR 0.96718945 0.95483871 0.96731813 0.96794872 0.95514546 FFR 0.64285714 0.46401225 0.62686567 0.56140351 0.41666667 Sensitivity 0.35714286 0.53598775 0.37313433 0.43859649 0.58333333 Precision 0.94245156 0.96066468 0.94628813 0.95933926 0.96754511 Error 0.10668855 0.09138871 0.1015914 0.0855267 0.08517498 F1-score 0.95466028 0.95774283 0.95668757 0.96362476 0.9613053 Table 5 Pearson correlation feature based performance on base classiﬁers Performance metrics PCFS + LR PCFS + NB PCFS + SVM PCFS + DT PCFS + ANN Accuracy 0.90274928 0.90978369 0.92033531 0.92033531 0.93792133 TPR 0.96753247 0.95489691 0.96815287 0.96942675 0.96375 FFR 0.62686567 0.45567652 0.50932287 0.51951952 0.44177093 Sensitivity 0.37313433 0.54432348 0.49067713 0.48048048 0.55822907 Precision 0.94663278 0.96196287 0.96698263 0.96702459 0.9710939 Error 0.09725072 0.09021631 0.07966469 0.07966469 0.06207867 F1-score 0.95696853 0.95841687 0.9675674 0.96822418 0.96740801 Table 3 Roughset-based performance on base classiﬁers Table 5 Pearson correlation feature based performance on base classiﬁers In Fig.7, the vertical axis represents training loss and the horizontal axis represents epochs. Figure7a–f clearly show varying training losses with subsequent epochs. Table 5 Pearson correlation feature based performance on base classiﬁers Results for frame-up: 2 The main objective of the DQN agent is to reduce the training loss, whichresultsinanimprovementinaccuracy.Figure7ashows that training loss is more due to considering all the attributes in the data set, which reﬂect themselves in the training pro- cess of the model. The epochs of noise would be greater at ﬁrst, but they would be reduced with proper training provided by an agent, as shown in Fig.7. formance in terms of average weighted accuracy, which is shown in Table 8 as well as Fig.8. The maximum accuracy value elucidated the effectiveness of the classiﬁcation proce- dureofDQN,whichwascomparedwithotherfrauddetection techniques. Therefore, we compared our proposed Renovated RDQN modelwithotherfrauddetectiontechniquessuchasIFDTC4.5 [40], SAE with GAN [41], a combination of CNN, SVM, and KNN [42], and DEAL [43], as shown in Table 9 and clearly represented in Fig.9. Among all these state-of-the-art meth- ods in fraud detection, SAE+GAN and DEAL work better with 94.98 and 94.65%. According to Fig.7e, RD4 is very efﬁcient but has expen- sive features based on the accuracy and error rate of the deep learning model (DQN). As a result, it is concluded that RDQN4 is the most useful in classiﬁcation. Next to RD4, RD5 performed better with less training loss. Subsequently, we ensembled all the models (RDQN1 + RDQN2 + RDQN3 + RDQN4 + RDQN5) and then calculated a weighted aver- age, which was considered the performance metric compared to other fraud detection models. RDQN gave the better per- Table 10 shows the comparison of the proposed RDQN with the best selected traditional algorithms (SVM, ANN), feature selection techniques (RSBFS+ANN, RSBFS+SVM), and other fraud detection techniques (SAE+GAN, DEAL). Results for frame-up: 2 Our proposed classiﬁer RDQN scales higher in accuracy of 96.09with a graph, as shown in Fig.10, to determine the 123 123 Complex & Intelligent Systems (2023) 9:5313–5332 5327 Table 6 Selection-based performance on base classiﬁers Performance metrics SBFS + LR SBFS + NB SBFS + SVM SBFS + DT SBFS + ANN Accuracy 0.90386306 0.92502491 0.92502491 0.9144733 0.93674893 TPR 0.96757248 0.96958175 0.96958175 0.96794872 0.96370463 FFR 0.62686567 0.49947862 0.47769314 0.56859362 0.4523135 Sensitivity 0.37313433 0.50052138 0.52230686 0.43140638 0.5476865 Precision 0.94669713 0.9696432 0.9721075 0.95818263 0.96983437 Error 0.09613694 0.07497509 0.07497509 0.0855267 0.06325107 F1-score 0.95702098 0.96961247 0.97084298 0.96304091 0.96675979 Table 7 Recursive feature elimination performance on base classiﬁers Performance metrics RFE + LR RFE + NB RFE + SVM RFE + DT RFE + ANN Accuracy 0.9121285 0.90760068 0.91400434 0.93674893 0.92857559 TPR 0.96786632 0.97453222 0.9748589 0.96996245 0.97605316 FFR 0.56859362 0.65340407 0.65340407 0.49947862 0.56640182 Sensitivity 0.43140638 0.34659593 0.34659593 0.50052138 0.43359818 Precision 0.95807621 0.95304657 0.95363574 0.97002316 0.96894642 Error 0.0878715 0.09239932 0.08599566 0.06325107 0.07142441 F1 score 0.96294639 0.96366965 0.96413054 0.9699928 0.97248681 Table 6 Selection-based performance on base classiﬁers Fig. 5 Comparison of accuracy on speciﬁed feature selection tech- niques upon classiﬁers while evaluating the performance of the DQN classiﬁer. The number of samples in the data set connected to weights in the network determines how many states there are. The temporal complexity of training a DQN with an input layer, two hid- den layers, and one output layer (optimal policies, denoted by “P”) was investigated. The time complexity for this solution was determined as O(N Z v−1 v=0 Pv · P(v +1)). The complex- ity analysis of DQN Classiﬁer on ’M’ number of reducts, then it would be O(M N Z v−1 v=0 Pv · P(v + 1)). Conclusion and future scope The proposed renovated method RDQN is used in this research work to detect fraud in digital transactions using reinforcement learning. The second module selects the best feasible attribute set using ﬁltering and wrapping techniques based on traditional ML algorithms like LR, NB, DT, SVM, and ANN. RST provided critical properties as reducts for fur- ther processing. The third module takes reducts as inputs for DQN to classify fraud and non-fraud transactions. Now, the proposed DQN performance has been compared with other base classiﬁers, feature-based classiﬁers, and other exist- ing fraud techniques. The proposed model has a 96.09% accuracy. This is a suitable model for solving any binary classiﬁcation-related problems. In future work, there is still room for establishing other learning environments and mod- ifying the reward functions. It is also important to compare the proposed model with new research that arises in the future. Fig. 5 Comparison of accuracy on speciﬁed feature selection tech- niques upon classiﬁers accuracy of our proposed model. Conclusively, the proposed model outperforms others in digital fraud detection cases. Table 7 Recursive feature elimination performance on base classiﬁers Complexity of the model In experiments, the model has successfully distinguished between fraudulent and non-fraudulent data. To forecast a model’s potential, a complexity analysis is crucial during the design and implementation phases. The number of neurons, the passing of weights, the number of states, the number of epochs, and the actions to be taken based on the Q values were some of the limitations that were taken into account 123 12 3 3 5328 5328 Complex & Intelligent Systems (2023) 9:5313–5332 Fig. 6 Reducts randomly chosen as input to DQN Table 8 Comparison of accuracy for different DQN on reducts Performance metrics RDQN1 RDQN2 RDQN3 RDQN4 RDQN5 Avg wei accuracy Accuracy 0.93641849 0.9645114 0.95667976 0.9848174 0.96160385 0.9609004 TPR 0.96684516 0.97508144 0.98039216 0.98214286 0.98049256 0.98169839 FFR 0.48812053 0.37442783 0.54220315 0.34640523 0.4796748 0.53051643 Sensitivity 0.51187947 0.62557217 0.45779685 0.65359477 0.5203252 0.46948357 Precision 0.96833532 0.98493462 0.97686061 0.99045561 0.98198913 0.97936827 Error 0.06358151 0.0354886 0.04332024 0.0151826 0.03839615 0.0390996 F1-score 0.96758967 0.97998326 0.9786232 0.98628172 0.98124028 0.98053194 Table 9 Comparison of accuracy between existing classiﬁcation models Performance metrics IFDTC4.5 [40] SAE-GAN [41] CNN + SVM + KNN [42] DEAL [43] Accuracy 0.91761534 0.94987983 0.91175333 0.94657366 True fraud rate 0.95272653 0.98025179 0.95242259 0.98142139 False fraud rate 0.4499777 0.4796748 0.48803099 0.52486538 Speciﬁcity 0.5500223 0.5203252 0.51196901 0.47513462 Precision 0.96098975 0.98176649 0.95454604 0.97951666 Error 0.08238466 0.05012017 0.08824667 0.05342634 F1-score 0.9568403 0.98100855 0.95348313 0.9804681 Complex & Intelligent Systems (2023) 9:5313–5332 Fig. Complexity of the model 6 Reducts randomly chosen as input to DQN Table 8 Comparison of accuracy for different DQN on reducts Performance metrics RDQN1 RDQN2 RDQN3 RDQN4 RDQN5 Avg wei accuracy Accuracy 0.93641849 0.9645114 0.95667976 0.9848174 0.96160385 0.9609004 TPR 0.96684516 0.97508144 0.98039216 0.98214286 0.98049256 0.98169839 FFR 0.48812053 0.37442783 0.54220315 0.34640523 0.4796748 0.53051643 Sensitivity 0.51187947 0.62557217 0.45779685 0.65359477 0.5203252 0.46948357 Precision 0.96833532 0.98493462 0.97686061 0.99045561 0.98198913 0.97936827 Error 0.06358151 0.0354886 0.04332024 0.0151826 0.03839615 0.0390996 F1-score 0.96758967 0.97998326 0.9786232 0.98628172 0.98124028 0.98053194 Table 9 Comparison of accuracy between existing classiﬁcation models Performance metrics IFDTC4.5 [40] SAE-GAN [41] CNN + SVM + KNN [42] DEAL [43] Accuracy 0.91761534 0.94987983 0.91175333 0.94657366 True fraud rate 0.95272653 0.98025179 0.95242259 0.98142139 False fraud rate 0.4499777 0.4796748 0.48803099 0.52486538 Speciﬁcity 0.5500223 0.5203252 0.51196901 0.47513462 Precision 0.96098975 0.98176649 0.95454604 0.97951666 Error 0.08238466 0.05012017 0.08824667 0.05342634 F1-score 0.9568403 0.98100855 0.95348313 0.9804681 Table 8 Comparison of accuracy for different DQN on reducts Performance metrics RDQN1 RDQN2 RDQN3 RDQN4 RDQN5 Avg wei accuracy Accuracy 0.93641849 0.9645114 0.95667976 0.9848174 0.96160385 0.9609004 TPR 0.96684516 0.97508144 0.98039216 0.98214286 0.98049256 0.98169839 FFR 0.48812053 0.37442783 0.54220315 0.34640523 0.4796748 0.53051643 Sensitivity 0.51187947 0.62557217 0.45779685 0.65359477 0.5203252 0.46948357 Precision 0.96833532 0.98493462 0.97686061 0.99045561 0.98198913 0.97936827 Error 0.06358151 0.0354886 0.04332024 0.0151826 0.03839615 0.0390996 F1-score 0.96758967 0.97998326 0.9786232 0.98628172 0.98124028 0.98053194 Table 8 Comparison of accuracy for different DQN on reducts Table 9 Comparison of accuracy between existing classiﬁcation models Performance metrics IFDTC4.5 [40] SAE-GAN [41] CNN + SVM + KNN [42] DEAL [43] Accuracy 0.91761534 0.94987983 0.91175333 0.94657366 True fraud rate 0.95272653 0.98025179 0.95242259 0.98142139 False fraud rate 0.4499777 0.4796748 0.48803099 0.52486538 Speciﬁcity 0.5500223 0.5203252 0.51196901 0.47513462 Precision 0.96098975 0.98176649 0.95454604 0.97951666 Error 0.08238466 0.05012017 0.08824667 0.05342634 F1-score 0.9568403 0.98100855 0.95348313 0.9804681 123 123 Complex & Intelligent Systems (2023) 9:5313–5332 5329 Fig. 7 Training loss in epochs of RDQN Fig. 8 Comparison graph between RDQN1−N and weighted average of RDQN1−N Fig. 8 Comparison graph between RDQN1−N and weighted average of RDQN1−N 123 Complex & Intelligent Systems (2023) 9:5313–5332 5330 Fig. References 21. 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Complexity of the model 9 Accuracy comparison between different feature detection techniques Table 10 Accuracy of RDQN with other fraud techniques Base classiﬁers Feature based classiﬁers Other fraud detection techniques Proposed classiﬁer Algorithms SVM ANN RSBFS + ANN RSBFS + SVM SAE + GAN DEAL RDQN Accuracy 0.909373 0.915235 0.94969722 0.946128 0.94988 0.946574 0.9609004 True fraud rate 0.954877 0.96157 0.974747 0.966543 0.980252 0.981421 0.98169839 False fraud rate 0.455677 0.494098 0.523242 0.490085 0.479675 0.524865 0.53051643 Speciﬁcity 0.544323 0.505902 0.476758 0.509915 0.520325 0.475135 0.46948357 Precision 0.961946 0.962433 0.972353 0.967802 0.981766 0.979517 0.97936827 Error 0.090627 0.084765 0.050303 0.053872 0.05012 0.053426 0.0390996 F1-score 0.958398 0.962002 0.973549 0.967172 0.981009 0.980468 0.98053194 Fig. 10 Comparison graph of proposed RDQN with base, feature-based, other FDT Table 10 Accuracy of RDQN with other fraud techniques Base classiﬁers Feature based classiﬁers Other fraud detection techniques Proposed classiﬁer Algorithms SVM ANN RSBFS + ANN RSBFS + SVM SAE + GAN DEAL RDQN Accuracy 0.909373 0.915235 0.94969722 0.946128 0.94988 0.946574 0.9609004 True fraud rate 0.954877 0.96157 0.974747 0.966543 0.980252 0.981421 0.98169839 False fraud rate 0.455677 0.494098 0.523242 0.490085 0.479675 0.524865 0.53051643 Speciﬁcity 0.544323 0.505902 0.476758 0.509915 0.520325 0.475135 0.46948357 Precision 0.961946 0.962433 0.972353 0.967802 0.981766 0.979517 0.97936827 Error 0.090627 0.084765 0.050303 0.053872 0.05012 0.053426 0.0390996 F1-score 0.958398 0.962002 0.973549 0.967172 0.981009 0.980468 0.98053194 Fig. 10 Comparison graph of proposed RDQN with base, feature-based, other FDT Table 10 Accuracy of RDQN with other fraud techniques Table 10 Accuracy of RDQN with other fraud techniques Base classiﬁers Feature based classiﬁers Other fraud detection techniques Proposed classiﬁer Algorithms SVM ANN RSBFS + ANN RSBFS + SVM SAE + GAN DEAL RDQN Accuracy 0.909373 0.915235 0.94969722 0.946128 0.94988 0.946574 0.9609004 True fraud rate 0.954877 0.96157 0.974747 0.966543 0.980252 0.981421 0.98169839 False fraud rate 0.455677 0.494098 0.523242 0.490085 0.479675 0.524865 0.53051643 Speciﬁcity 0.544323 0.505902 0.476758 0.509915 0.520325 0.475135 0.46948357 Precision 0.961946 0.962433 0.972353 0.967802 0.981766 0.979517 0.97936827 Error 0.090627 0.084765 0.050303 0.053872 0.05012 0.053426 0.0390996 F1-score 0.958398 0.962002 0.973549 0.967172 0.981009 0.980468 0.98053194 Fig. 10 Comparison graph of proposed RDQN with base, feature-based, other FDT Fig. 10 Comparison graph of proposed RDQN with base, feature-based, other FDT 12 3 5331 Complex & Intelligent Systems (2023) 9:5313–5332 Data availability we believe that this data should be available for others to use and will provide the concerned material to anyone who contacts the corresponding author. 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https://openalex.org/W4394862973	https://ieeexplore.ieee.org/ielx7/6287639/6514899/10500694.pdf	English	null	MSRFSR: Multi-stage Refining Face Super-Resolution with Iterative Collaboration between Face Recovery and Landmark Estimation	IEEE access	2,024	cc-by	17,085	This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 2 Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Date of publication xxxx 00, 0000, date of current version xxxx 00, 0000. Digital Object Identifier 10.1109/ACCESS.2023.1120000 Digital Object Identifier 10.1109/ACCESS.2023.1120000 AMIR HAJIAN1, (Member, IEEE), SUPAVADEE ARAMVITH2, (Senior Member, IEEE) 1Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand 2Multimedia Data Analytics and Processing Research Unit, Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 Thailand Corresponding author: Supavadee Aramvith (supavadee.a@chula.ac.th) is research is supported by the Ratchadapiseksompotch Fund for Postdoctoral Fellowship through the Graduate School and the tchadapiseksompotch Fund Chulalongkorn University, Bangkok, Thailand. ABSTRACT Face Super-resolution (FSR) models encounter a significant challenge related to extremely low-dimensional (16×16 pixels) and degraded input images. This deficiency in crucial facial details within the low-level and intermediate levels of the FSR model presents obstacles in tasks such as face alignment, landmark detection, and consequently, difficulty in recovering high-frequency details, resulting in unfaithful and unrealistic super-resolved face images. This research proposes an innovative FSR model with strategically designed multi-attention techniques to enhance facial attribute recovery capabilities. The model incorporates a Non-local Module (NL) and residual pixel attention technique at the low-level stage of the FSR model. Simultaneously, a Spatial Feature Transfer (SFT) module refines mid-level features by leveraging spatial information through an iterative interaction process between an attentive module and a landmark estimation network. By strategically utilizing these modules under an iterative collaboration framework, our method effectively addresses challenges in facial detail recovery, demonstrating enhanced model understanding and refined representation. The proposed model is rigorously examined on CelebA, Helen, AFLW2000, and WFLW datasets at scale factors of ×8 and ×16. The results consistently demonstrate the superiority of our proposed Multi-Stage Refining Face Super-Resolution (MSRFSR) model over state-of- the-art methods through extensive quantitative and qualitative experiments on four datasets and both scales INDEX TERMS Face image super-resolution, non-local attention, residual pixel attention, spatial feature transfer. INDEX TERMS Face image super-resolution, non-local attention, residual pixel attention, spatial feature transfer. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ MSRFSR: Multi-stage Refining Face Super-Resolution with Iterative Collaboration between Face Recovery and Landmark Estimation AMIR HAJIAN1, (Member, IEEE), SUPAVADEE ARAMVITH2, (Senior Member, IEEE) 1Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand 2Multimedia Data Analytics and Processing Research Unit, Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand AMIR HAJIAN1, (Member, IEEE), SUPAVADEE ARAMVITH2, (Senior Member, IEEE) 1Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand 2Multimedia Data Analytics and Processing Research Unit, Department of Electrical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand I. INTRODUCTION Although utilizing an iterative collaboration approach be- tween the attentive module and estimated facial landmarks enhanced the performance of the FSR model to recover more global and local detail compared to the other prior guide approaches, the obstacles of poor-quality input images and consequently lack of recovering high-frequency details at mid-level of FSR model remain. Our research presents a novel approach to FSR by em- ploying multi-stage refinement techniques that harness the potential of an iterative collaboration process between the recovery and landmark estimation networks. This innovative framework enhances the model’s capability to recover higher fidelity and more detailed facial attributes compared to base- line models. We introduce three key contributions to our FSR model: To enhance the mid-level information recovery capabilities of the FSR model and achieve a more realistic texture for face images, the viable approach involves leveraging the spatial information of features. This goal is effectively addressed by incorporating the Spatial Feature Transfer [12] (SFT). Nevertheless, the sub-optimal quality of the extracted low- level features from unclear and degraded LR input hinders the SFT module’s efficient recovery of intricate facial details. To mitigate the degradation effects associated with the low- level extracted features, a feasible solution is to employ a non-local [13] module (NL) in conjunction with the resid- ual channel attention [14] technique. Integrating the non- local module captures long-range dependencies within the features, promoting a more comprehensive understanding of facial structures. Simultaneously, the residual channel atten- tion technique enhances the focus on critical facial details by selectively emphasizing informative channels. This combined approach addresses the shortcomings in the quality of low- level features, fostering a more refined and context-aware representation. By incorporating the non-local module and residual channel attention technique, we aim to significantly enhance the efficacy of the SFT module in recovering facial details, ultimately contributing to improving the capability (1) Propose an NL module at the early stage of our FSR network to reduce the noise effects of low-resolution face images and produce an enhanced feature representation. This module effectively addresses the noise degradation effect in low-quality inputs, improving feature quality. (2) Employs a residual pixel attention module on low- resolution features at the early stage of the network to capture the inter-channel relationships in low-resolution feature maps and emphasizes the importance of specific channels, enhanc- ing the model’s ability to capture intricate facial details. I. INTRODUCTION Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 1. Visual Comparison: Our MSRFSR model compared to DIC [5] on CelebA [15] and Helen [16] datasets at a scale factor of ×8 reveals distinctive performance in fidelity and yields more detailed results. non-prior guide approach, they suffer from inaccurate or even wrong prior guide information due to the lack of sharp detail in the input face images. A noticeable challenge encountered in FR models pertains to the inadequate quality of the shallow dimensions of input LR face image (often at sizes of 16×16 or 8×8 pixels). This issue manifests as a deficiency in valuable facial details within the FSR model’s intermediate levels (mid-level). The constrained information in such LR images poses a significant hurdle, impacting the model’s ability to effectively face alignment, landmark detection, and recover facial features. Additionally, the LR face images may contain some artifacts, including a lack of sharpness and any other degradation, making it difficult to recover the human face’s global and local details in a super-resolved image. Unfaithful and unrealistic super-resolved face images in different face poses are the adverse impact of this problem. FIGURE 1. Visual Comparison: Our MSRFSR model compared to DIC [5] on CelebA [15] and Helen [16] datasets at a scale factor of ×8 reveals distinctive performance in fidelity and yields more detailed results. To improve the capability of the FSR model in recovering more accurate global information from poor-quality input images, Ma et al. [5] proposed a face hallucination model comprising two recurrent networks. These networks are de- signed for iterative operation and improve the performance of facial component recovery and landmark detection tasks. Furthermore, to improve the restoration of local details, they enhanced the landmark information guidance utilizing an attentive module that aggregates the individual face attributes meticulously. FIGURE 1. Visual Comparison: Our MSRFSR model compared to DIC [5] on CelebA [15] and Helen [16] datasets at a scale factor of ×8 reveals distinctive performance in fidelity and yields more detailed results. of the attentive module to boost the guidance of landmarks and the overall improvement of our FSR model on large up- sampling scales (×8 and ×16). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION of face attribute detail. On the other hand, the SISR model focuses on enhancing a wide variety of image content without considering the features of face attributes [4]. Therefore, the face configuration is considered a highly structured object and utilized as confident prior knowledge for reconstructing the global face structure. According to the reconstructed global face attributes, the local face information is also recovered [5]. To utilize the facial prior guide approach in the FSR model, different techniques such as face parsing maps, landmarks heatmaps, Spatial attention maps, and the three-dimensional facial guide [6-11] are used in the face attributes (eyes, lips, nose, and eyebrows) recovering process. Due to the prior- guide technique utilization in FSR models, they outperform at large scales (×8 and more) compared to the SISR models. F ACE super-resolution (FSR) has attracted more attention and has been used in various image-based applications. FSR, also known as face hallucination, seeks to reconstruct a high-resolution (HR) face image from a low-resolution (LR) input. Due to the constraints in acquiring high-quality images and the influence of imaging conditions, face im- ages are almost captured in poor perceptual quality in real- world scenarios. The low-quality face image issue negatively affected the performance of face-image-based applications such as face detection [1] and face recognition [2]. FSR is a particular instance of the single image super-resolution (SISR) technique [3], and it is considered an ill-posed prob- lem because of the ambiguity in reconstructing face images’ pixels. The FSR model is designed to capture the unique characteristics of facial attributes and optimizes the recovery F Although the facial prior guide approach models outper- form in recovering global face information compared to the 1 VOLUME 11, 2023 VOLUME 11, 2023 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. I. INTRODUCTION (3) Develop an SFT module involving an affine transfor- mation that spatially adjusts the features according to facial characteristics derived from facial heatmaps. The SFT applies at the mid-level of our proposed model before the upsampling layer and improves the effectiveness of the upscaling process by ensuring relevant facial details are brought into focus. The illustration in Figure 1 showcases the effectiveness of our multi-stage refinement process in producing higher fidelity and more detailed face images. In Figure 1, Sample 2 2 VOLUME 11, 2023 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 1. Comparison of various FSR models. Model Year Innovation Accuracy Metric Dataset Scale Factor Bi-channel [25] 2015 Integrates two channels of information PSNR-SSIM Collected from the web ×4 URDGN [26] 2016 Pixel-wise L2 regularization and feedback PSNR-SSIM CelebA ×8 WSRNet [27] 2017 Wavelet-based and multiple scaling PSNR-SSIM CelebA - Helen ×8, ×16 Super-FAN [8] 2018 Facial landmark information PSNR-SSIM CelebA - Helen - WiderFace ×8 FSRNet [6] 2018 Geometry prior knowledge PSNR-SSIM-MSE CelebA - Helen ×8 FACN [29] 2020 Facial Attribute Capsule to recover detail PSNR-SSIM-IFC CelebA ×8 SPARNet [30] 2020 Face Attention Units PSNR-SSIM CelebA - Helen ×8, ×16 DIC [5] 2020 Collaboration of attentive and landmark PSNR-SSIM CelebA - Helen ×4, ×8 SISN [31] 2021 External-Internal Split Attention Group PSNR-SSIM-LPIPS-MPS FFHQ-CelebA ×4, ×8 SCTAnet [11] 2023 Spatial Attention and Multi-scale Patch PSNR-SSIM-LPIPS CelebA - Helen ×8, ×16 FishNet [32] 2023 Parsing map attention fusion block PSNR-SSIM CelebA - Helen ×8, ×16 MSGAN [33] 2023 Head pose estimation network PSNR-SSIM-LPIPS BIWI - AFLW - Widerface ×8, ×16 CTCnet [34] 2023 Multi-scale connected encoder-decoder PSNR-SSIM-LPIPS-VIF CelebA- Helen ×8 FMANet [35] 2023 Facial Mask Attention and MaskPix loss PSNR-SSIM-LPIPS-FID CelebA- Helen ×4, ×8 SFMNet [36] 2023 Frequency-spatial interaction block PSNR-SSIM-LPIPS CelebA- Helen ×4, ×8 content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 1. Comparison of various FSR models. recursive regression architecture, convex optimization based on the positional patch approach, and local smooth regression, respectively. I. INTRODUCTION (a) demonstrates a fidelity comparison of the generated SR image with closed eyes, highlighting its superior performance to the DIC [5] model. Sample 2 further illustrates the model’s capability to recover additional facial details, surpassing the performance of the DIC [5] model. Nevertheless, these approaches prove ineffective in restor- ing natural facial attributes, mainly when dealing with large up-sampling factors such as ×8. When the magnification factor is larger than ×4, these methods encounter challenges in producing confident super-resolved face images. The remaining sections of the article are structured as fol- lows. Section II briefly reviews the relevant works. Section III provides the methodology and proposes the model’s architec- ture. The implementation details, datasets, and experimental results are demonstrated in Section IV. Section V demon- strates discussion and future work. Finally, the proposed FSR research is concluded in Section VI. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ A. CONVENTIONAL FACE SUPER-RESOLUTION The idea of improving the quality of face images, known as face hallucination (face super-resolution), became a focus of interest when researchers proposed the first general Super- resolution (SR) models. In 2000, Baker et al. [17] proposed the first FSR model. Their basic model improves the resolu- tion of face images by learning from training image datasets. This model is relatively simple, producing a super-resolved face image by comprehending facial structures while ignoring the high-frequency information recovery. In 2015, the Bi-channel Convolutional Neural Network (CNN) [25] pioneered using CNN to super-resolve face im- ages by adaptively combining the two channels’ information, marking a notable milestone in this area. To address the limitation in operating in larger scale factors (beyond ×4), the URDGN [26] model introduced a discriminative generative network by resolving very low-resolution face images to ×8 by incorporating a pixel-wise L2 loss function and utilizing feedback branch from the discriminative network to enhance the recovered global information of the face image. To prevent the over-smoothed effect in large-scale factors (×8 and ×16), WSRNet [27] proposed a wavelet-based FSR network that utilized the wavelet transformation domain instead of the im- age domain and designed multiple scaling factors technique within a unified framework. This FSR model learns to predict LR’s corresponding series of HR’s wavelet coefficients and improves human faces’ global information and local texture details. The extension of the WRNet [27] model into the generative adversarial networks is detailed in [28]. g q y y Liu et al. [18] proposed a two-step FSR architecture to address this limitation. This model first restores the coarse face image using a linear network architecture in the initial stage and then retrieves high-frequency details using a non- parametric Markov technique. Although this model improved the recovery of face details compared to the previous model [17], there is still room for enhancing the retrieval of more facial details. Several FSR models have been proposed to tackle this issue in traditional deep-learning algorithms [19- 24]. To improve the generation of crucial facial details be- tween 2004 and 2007, Chang et al. [19], Wang et al. [20], and Chakrabarti et al. [21] each applied different methods including local embedding, eigen transformation, and kernel principal component, respectively. Furthermore, to boost the performance of FSR models in shallow architecture, Shi et al.. [22], Jung et al. [23], and Jiang et al.. B. DEEP LEARNING BASED FACE SUPER-RESOLUTION Due to the rapid development of deep learning in computer vision, there have been significant advancements in face hal- lucination using deep learning methods [4]. Our review fo- cuses on designing diverse network structures for FSR models and techniques for dealing with different attention techniques, facial geometry alignment, and textural and contextual infor- mation. Table 1 presents comparative comparisons of FSR models, highlighting key aspects such as the year of publi- cation, a brief overview of the methodology employed, the accuracy metric, the dataset used, and the examined scale factor. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS In 2018, SuperFan [8] and FSRNet [6] utilized face geome- try as prior knowledge of FSR models to improve the quality of resultant images by effectively recovering local informa- tion. SuperFan [8] utilized a sub-network for face alignment and detecting facial landmarks information and integrated this information into a generative adversarial network. The FSRNet [6] used facial landmark heatmaps and parsing maps without strict alignment and integrated adversarial loss into this model. To improve texture details and enhance facial structure, the External-Internal Split Attention Group proposed split- attention in a split-attention network [31] (SISN). This model simultaneously considers overall facial structure and fine tex- ture details and attempts to produce more fidelity face images. In the extension of [29], to enhance the fidelity of facial results, Bao et al. [11] proposed multi-attention modules (Residual Spatial Attention and Multi-scale Patch embedding and Spatial attention) in the SCTANet model to improve the recovery of both global and local information. The Facial Attribute Capsules Network [29] (FACN) lever- ages an integrated representation model to comprehensively encapsulate facial information and reduce the noise effect of super-resolved face images in real-world scenarios. An integrated learning strategy generates the attribute capsules in semantic, probabilistic, and facial attribute manners. The Spa- tial Attention Residual Network [30] (SPARNet) integrated a spatial attention mechanism into vanilla residual blocks to address the difficulty in recapturing finer facial details. This technique allows convolutional layers to focus specifically on essential facial structures while reducing attention to regions with fewer features. In 2023, Wang et al. [32] introduced Super-Resolving Face Image by Facial Parsing Information (Fishnet) by designing a parsing map attention fusion block (parsing map-guided approach) and integrating the parsing map information with an attention mechanism. The multi-scale refine block in this model attempts to preserve the spatial and contextual details and recover high-resolution information and the context from low-resolution features. Multi-Stage Generative Adversarial Network [33] (MS- GAN) proposed an end-to-end head-posed estimation net- work and integrated it with the FSR network. Utilizing pose- aware adversarial loss and head pose alignment feedback improves the fidelity of non-frontal face images in real-world scenarios. A. CONVENTIONAL FACE SUPER-RESOLUTION [24] employed 3 3 VOLUME 11, 2023 VOLUME 11, 2023 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS To improve the FSR model’s capability of recov- ering local and global facial detail, the CNN-Transformer Cooperation Network [34] (CTCNet) employed a multi-scale connected encoder-decoder framework as its backbone. The structure attention module, along with a Transformer block, attempts to enhance the consistency of local facial detail and global facial structure simultaneously. Ma et al. [5] introduced an iterative collaboration between two recurrent networks (DIC), focusing on recovery and land- mark estimation to improve the FSR model accuracy and quality. The iterative framework exploits prior knowledge of landmarks and progressively enhances each other’s perfor- mance. The DIC [5] model emphasizes the dynamic infor- mation exchange between the face image recovery network and the landmark estimation network. Additionally, an inno- vative attentive fusion module generates facial components and aggregates them attentively for improved high-frequency detail restoration. While this framework has shown accept- able capability in gradually producing results and utilizing an attentive fusion technique to recover more facial details, its effectiveness is hindered by using a degraded low-resolution input (16 × 16). This limitation prevents the full advantage of the approach. To increase the accuracy of the FSR model and generate better perceptual quality images from LR faces, the FMANet [35] model introduced a facial mask attention module and attempted to enhance the identity fidelity of the resolved face image. Moreover, the MaskPix loss function was introduced to emphasize pixels containing dense identity features in this model selectively. SFMNet [36] proposed the frequency- FIGURE 2. The architecture of MSRFSR model. FIGURE 2. The architecture of MSRFSR model. III. METHODOLOGY Figure 3 depicts the non-local module’s architecture.i f The Fn denotes the fusion module and it is determined as (2): Specifically, instead of attending only the neighboring pix- els, a larger neighborhood region is considered. Detecting similar intensity or texture characteristics using weighted averages in non-local technique is defined as (4): (2) Fn = MRef (ILR, ISR(n−1)) · HMSR(n−1) (2) Based on the iterative collaboration approach to reconstruct the SR image in n steps, the final SR output is defined by ISR(n) as shown in (3): zi = W(t) · yi + ILR (4) (4) where zi and ILR define the output and input of NLM, respec- tively. W(t) denotes the weights of yi. ISR(n) = MSR(Fn) + MST(HMSR(n−1)) + U(ILR) (3) (3) In continuation of this section, detailed explanations of the Multi-stage Refinement Network, Face Recovery Network, and Face Alignment Network are discussed. In continuation of this section, detailed explanations of the Multi-stage Refinement Network, Face Recovery Network, and Face Alignment Network are discussed. yi = softmax(W(θ) · W(ϕ)).W(g) (5) (5) yi is obtained by dot product of W(g) into the dot product of W(θ) and W(ϕ) after applying softmax operation. W(θ), W(ϕ) and W(g) are the weight matrices to be learned. θ, ϕ, and g are (1×1) convolution. H and T are patch sizes set to 7, and the number of channels set to 48. III. METHODOLOGY The overall MSRFSR architecture comprises three branches: a refinement network, a face recovery network, and a face alignment network (FAN), as shown in Figure 2. The LR input suffers degradation (noise, blurring, and lack of sharpness), which requires a multi-stage enhancement procedure [12-14]. The first convolution layer extracts the LR features from the LR input and up-scales them by a factor of two. The features are fed into the proposed multi-stage refinement network con- sisting of a non-local module and the residual pixel attention blocks, as depicted in Figure 2. FIGURE 3. The architecture of Non-local module (NL). FIGURE 3. The architecture of Non-local module (NL). FIGURE 4. The architecture of RCA block. The face recovery network and FAN branch interact using an iterative collaboration approach. The face recovery branch takes the refined features as the input and generates the first super-resolved face image. The generated super-resolution (SR) images are fed into the FAN branch to estimate the alignment procedure and generate facial heatmaps. These are prior guides for fusion with the attentive module in an iterative collaborative process. The first generated SR image can be computed as (1): FIGURE 4. The architecture of RCA block. ISR(1) = MSR(MRef (ILR)) + U(ILR) (1) (1) where ISR(1) and ILR denote the first reconstructed image and the LR input image, respectively. The MSR and MRef represent the recurrent SR module and multi-stage refinement network, respectively. The up-sampling module is demonstrated by U. degradation effects at the low-level extracted features in this stage of the proposed model. These enhancements optimize the recovering and alignment procedures to fully utilize the capability of iterative collaboration technique in our FSR model. Utilizing ISR(1), the FAN branch predicts the initial fa- cial landmarks (68 landmarks in the face) and produces the first facial heatmaps demonstrated by HMSR(1). The facial heatmaps include left and right eyes, lips, nose, and face shapes, utilized as prior knowledge for integrating into the attentive fusion module. For the second procedure, the atten- tive module is implemented on two inputs including HMSR(1) and MRef (ILR). The application of the non-local attention technique is con- strained to recovering the global details in LR feature maps by exploiting the interdependence of pixels [37-39]. The proposed NL is considered an attention approach to detect recurring patterns and textures across different regions of the feature maps [37]. VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 Author et al : Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 FIGURE 3. The architecture of Non-local module (NL). FIGURE 4. The architecture of RCA block. author's version which has not been fully edited and tation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS spatial interaction block based on the Fourier transform technique to achieve optimal performance in recovering the global and local facial dependencies. Exploring correlations between spatial and frequency domains improves global and local signal generation in SR images. A. MULTI-STAGE REFINEMENT NETWORKi The Multi-stage Refinement Network comprises the NL and RCA blocks as demonstrated in Figure 2. It aims to reduce the VOLUME 11, 2023 5 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ accepted for publication in IEEE Access. This is the author's version which has not been fully edited and This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 5. The structure of Spatial Feature Transform (SFT). FIGURE 5. The structure of Spatial Feature Transform (SFT). The proposed RCA blocks are a channel attention tech- nique that employs convolutional layers to increase the net- work’s capability to learn specific weights of the channel [14, 40]. As shown in Figure 4, the proposed RCA module com- prises convolutional layers with ReLU activation functions, global average pooling, and sigmoid activation functions. This approach helps recover and precisely extract channel- wise information from low-resolution feature maps. components, including the mouth, nose, right and left eyes, and jawline. The channels corresponding to each facial com- ponent are added together, and softmax operation is applied to create a facial attention heatmap for the respective facial attribute. Guided by facial attention heatmaps, the specific compo- nent features are extracted by group convolution, and the weighted features from the multi-stage refinement network are added to form attentive fused output. The values of the weights behave as an attention map, allocating higher values to more critical information and lower values to less important information for consideration. After each convolution layer, the activation function enables the deep-learning model to learn more complex channel de- pendencies. A. MULTI-STAGE REFINEMENT NETWORKi A skip connection allows the network to bypass the low-frequency information from the earlier layer and integrate it with the features from the later layer [3,40]. In the proposed multi-stage refinement network, utilizing RCA blocks after applying the non-local module is a strategic approach to emphasize inter-channel facial detail effectively. The NL enhances noise degradation at the initial stage of the refinement procedure [38,39]. At the same time, RCA blocks are specifically designed to bolster inter-channel facial detail representations on low-level feature maps [14]. This combined technique addresses noise issues. It ensures a more comprehensive and refined treatment of facial details, ulti- mately optimizing the collaboration between face recovery and face alignment networks. Based on the DIC [5] model, the super-resolution feed- back network [41] is employed in our model’s face recovery network. The proposed recurrent SR module is structured to utilize feedback connections and generate robust high-level representations. The recurrent SR module’s input has 32×32 pixels and 48 channels and passes through the convolution and feedback layers. The preservation of spatial information is essential in the context of generated face images since it entails the mainte- nance of local attributes necessary for fidelity preservation and recovering more tiny details at various spatial regions of a facial image [12,42,43,44]. Therefore, an SFT block is em- ployed, and input spatial features are produced by heatmaps to spatial-wise feature modulation with the recurrent SR fea- tures. The architecture of the proposed SFT module is demon- strated in Figure 5. The SFT module learns a spatial mapping function M to predict the modulation parameters α and β based on prior condition ψ and formulates as (6): This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ C. FACE ALIGNMENT NETWORK The face alignment process in our FSR framework involves using accurate facial landmarks to guide the face recovery network, progressively improving face image fidelity and landmark estimation. Our model continuously improves the landmark estimation at each step of the iterative approach to provide more precise auxiliary information to efficiently combine the prior guidance to the spatial feature transform operation and attentive fusion module. On the other hand, the iterative collaboration approach and feedback between the two networks’ branches enhance the performance of land- mark estimation and the face recovery network, ultimately improving the overall efficiency of the proposed FSR model. The FAN in our model is based on the DIC [5]. The proposed FAN includes pre-processing and post-processing compo- nents, as shown by P1 and P2, along with four interconnected hourglasses that incorporate recurrent feedback in the FAN architecture [45]. In the beginning (at the first iteration), there aren’t any face component maps or recurrent features. The ISR(1) reconstructs using a refined LR image by multi- stage refinement and recurrent SR networks. For the nth step in the iterative calibration approach, where n=1, ..., N, the Face Recovery Network generates face SR images ISR(n) by utilizing the FAN result and the feedback information from (n −1)th step. The training objective function utilized in our FSR model is LL1 that demonstrated as (8): LL1 = 1 N N X i=1 \|yi −ˆyi\| (8) (8) where N denotes the total number of elements in the tensors, yi and ˆyi are the ground truth (HR) image and the predicted super-resolved (SR) image, respectively. The number of iterations in our iterative super-resolution approach was set to n = 4. The batch size was set to 8. To mitigate over-fitting, we enhanced the variety of training samples in our experiment by incorporating random rotations of 90°, 180°, and 270°and horizontal flips. For training, we utilized the ADAM optimizer [48] with a bias β1 = 0.9 and The FAN takes the input from the first generated SR image (128 × 128). In the pre-processing stage, convolutions are applied to SR images, down-sampled from 128 × 128 to 32 × 32, and then fed to the recurrent hourglass network. The hourglass network estimates the facial landmarks and sends them to the P2 stage. In the post-processing stage, the detected facial landmarks are merged into five types of face component maps. A. IMPLEMENTATION SETTINGS We conducted experiments using the CelebA [15] and Helen [16] datasets, which are widely recognized in the field of Face Super-Resolution (FSR) models. To prepare the data, as the standard of other FSR models [5], we first utilized the estimated landmarks and extracted square sections from each image to remove the background. These sections were then resized to 128×128 pixels without prior alignment and used as the ground truth images. Subsequently, we reduced the size of HR face images to 16×16 pixels for a scale factor of ×8 and to 8×8 pixels for a scale factor of ×16. We employed the bicubic interpolation method for both datasets to downscale the images for model training. For the CelebA [15] dataset, our training set comprised 168,854 images, with 1,000 images reserved for testing. In the case of the Helen [16] dataset, our training set consisted of 2,005 images, while 50 were held out for testing purposes. Table 2 summarizes and compares the detailed strategy utilized in our implementation. Additionally, we conducted experiments with our model to identify its limitations. We selected two datasets, AFLW2000 [46] and WFLW [47], to assess the model’s performance across different facial image datasets. IV. EXPERIMENT AND DISCUSSIONSi (7) ˆy = SFT(F\|α, β) = αJFLβ (7) In this section, we first introduce the implementation settings of the MSRFSR model. Then, we explore the effectiveness of various components, and finally, we compare the perfor- mance of our model with that of other state-of-the-art models quantitatively and qualitatively. where F represents the feature maps with dimensions same as α and β. The J denotes element-wise multiplication and L shows element-wise addition. To maintain the spatial dimensions, the SFT module performs both feature-wise ma- nipulation and spatial-wise transformation. The model takes the condition ψ generated from the Heatmaps, and the output is the affine transformation to scale (α) and shift (β) feature maps. Implementing multiplication and addition features is a practical and feasible way to incrementally fuse two different levels of detail before up-sampling the face image. Shifting and scaling the feature map is similar to regulating the re- gional image information. This means that essential details in every region of the face images are retained, and others are ignored. B. FACE RECOVERY NETWORK The Face Recovery Network comprises an attentive fusion module, recurrent super-resolution, and SFT block, as shown in Figure 5. M : ψ→(α, β) (6) (6) The learned α and β parameters apply an affine trans- formation to feature maps located at the intermediate layer of generating super-resolved network and adaptively impact generating more fidelity face images. To be more exact, the preceding ψ is represented using the parameters α and β in an affine transformation, through a mapping function. By obtaining the α and β based on certain conditions, the The attention fusion module is a part of the face recovery branch that fuses the information between the face recovery network and the FAN. Thus, the gradients can be back- propagated to both the face recovery network and FAN part recursively and facilitate the utilization of different facial components as the landmark guidance of our FSR model.i The facial component maps are grouped into specific facial 6 6 VOLUME 11, 2023 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS transformation is applied by scaling and shifting feature maps as defined in (7): and the other is used for the spatial feature transfer operation. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ C. FACE ALIGNMENT NETWORK In our designed model, the five types of component maps are split into two paths, as demonstrated in Figure 2. One path is utilized for the attentive fusion module, TABLE 2. Hyperparameter setting. Dataset CelebA[15] Helen[16] Image for train 168,854 Images 2,005 Images Batch size 8 8 Epoch 5 × 105 5 × 105 Iterative steps (n) 4 4 Image for test 1,000 Images 50 Images Inference time 18,527 ms 1,076 ms 7 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4. This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS C. VISUAL COMPARISONS OF FACE RECOVERY IN DIFFERENT ITERATION STEPS β2 = 0.999 for the alignment loss weight in the training process. The initial learning rate commenced at 1 × 10−4 and underwent halving at 2 × 104, and 4 × 104. Our model’s training in both datasets was stopped after 5 × 105 epochs. The experiments were conducted using PyTorch [49] on an NVIDIA RTX 2080 Ti GPU with 4352 CUDA cores and 11GB of GDDR6 memory. The previous section compared various iteration steps to de- termine our model’s optimal value for n = 4. Here, we present visual comparisons of face recovery during the training mode of our FSR model for each iteration step. Figure 6, Figure 7, Figure 8, and Figure 9 depict visual comparisons of face image reconstruction, comparing them with both the HR im- age and the bicubic upsampling method. The corresponding PSNR and SSIM of the generated image for each iteration step with the HR image are displayed in the figures. Results for the CelebA dataset at a scale factor of ×8, Helen dataset at the factor of scale ×8, CelebA dataset at a scale factor of ×16, and Helen dataset at a scale factor of ×16 are shown in Fig- ures 6, Figures 7, Figures 8, and Figures 9, respectively.Upon visual inspection and quantitative evaluation of each figure, it is evident that the perceptual quality and the recovery of high-frequency details in facial attributes gradually improve at each iterative calibration step during the training phase of our FSR model. The assessment of super-resolved face images involves the use of the Peak Signal-to-Noise Ratio (PSNR), Learned Perceptual Image Patch Similarity (LPIPS) [50], Structural Similarity Index (SSIM) [51], and Frechet Inception Distance (FID) [52]. These metrics are calculated on the Y channel within the transformed YCbCr color space. B. INVESTIGATION OF DIFFERENT ITERATIVE STEPS IN TRAINING In this section, our focus revolves around the comprehensive evaluation and comparative analysis of the proposed iterative collaboration framework for the proposed model. The eval- uation metrics employed include PSNR, SSIM, and LPIPS. We present the results in Table 3 and Table 4, where each table corresponds to different aspects of our model’s per- formance. Specifically, the evaluation encompasses various iterative steps, ranging from step 1 to step 5. The CelebA [15] and Helen [16]datasets are utilized for these evaluations. The evaluations are conducted for scale factors of ×8 and ×16, with the corresponding outcomes represented in Table 3 and Table 4, respectively. FIGURE 6. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on CelebA image at a scale factor of ×8. Following a comprehensive analysis of the different itera- tion steps in Table 3 and Table 4, from Step 1 to Step 4, a con- sistent and gradual improvement is observed in PSNR, SSIM, and LPIPS. However, Step 5 has no discernible enhancement in these metrics. According to the results of Table 3 and Table 4, the optimal iteration step for our model, the best values for PSNR, SSIM, and LPIPS, is found at n = 4 for both scale factors. FIGURE 6. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on CelebA image at a scale factor of ×8. ×8. TABLE 3. The quantitative comparison among different iterative steps (n) on CelebA and Helen datasets at a scale factor of ×8. Iterative steps Scale CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ PSNR↑ SSIM↑ LPIPS↓ n = 1 ×8 23.75 0.6817 0.4913 22.99 0.6598 0.5019 n = 2 ×8 25.49 0.7119 0.2874 25.03 0.7189 0.2858 n = 3 ×8 26.75 0.7684 0.2474 25.52 0.7320 0.2365 n = 4 ×8 27.69 0.8083 0.1691 27.18 0.8092 0.1789 n = 5 ×8 27.64 0.8072 0.1709 27.15 0.8088 0.1794 TABLE 4. The quantitative comparison among different iterative steps (n) on CelebA and Helen datasets at a scale factor of ×16. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ B. INVESTIGATION OF DIFFERENT ITERATIVE STEPS IN TRAINING Visual comparison of face image reconstruction in three steps of iterative collaboration approach on Helen image at a scale factor of ×8. FIGURE 10. Landmark estimation in 4 steps of iterative collaboration approach at a scale factor of ×16, and the facial estimated landmark in heatmap shape. FIGURE 10. Landmark estimation in 4 steps of iterative collaboration approach at a scale factor of ×16, and the facial estimated landmark in heatmap shape. FIGURE 7. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on Helen image at a scale factor of ×8. FIGURE 8. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on CelebA image at a scale factor of ×16. B. INVESTIGATION OF DIFFERENT ITERATIVE STEPS IN TRAINING Iterative steps Scale CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ PSNR↑ SSIM↑ LPIPS↓ n = 1 ×16 17.35 0.4035 0.6806 17.48 0.4186 0.6737 n = 2 ×16 20.06 0.4756 0.6556 20.39 0.4996 0.6588 n = 3 ×16 21.62 0.6513 0.3358 22.42 0.6981 0.3402 n = 4 ×16 23.77 0.6903 0.2599 23.06 0.6720 0.2644 n = 5 ×16 23.75 0.6898 0.2613 23.01 0.6701 0.2669 8 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 3. The quantitative comparison among different iterative steps (n) on CelebA and Helen datasets at a scale factor of ×8 he quantitative comparison among different iterative steps (n) on CelebA and Helen datasets at a scale factor of ×16. TABLE 4. The quantitative comparison among different iterative steps (n) on CelebA and Helen datasets at a scale factor of ×16. Iterative steps Scale CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ PSNR↑ SSIM↑ LPIPS↓ n = 1 ×16 17.35 0.4035 0.6806 17.48 0.4186 0.6737 n = 2 ×16 20.06 0.4756 0.6556 20.39 0.4996 0.6588 n = 3 ×16 21.62 0.6513 0.3358 22.42 0.6981 0.3402 n = 4 ×16 23.77 0.6903 0.2599 23.06 0.6720 0.2644 n = 5 ×16 23.75 0.6898 0.2613 23.01 0.6701 0.2669 8 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ 8 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 n accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 7. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on Helen image at a scale factor of ×8. FIGURE 7. D. ITERATIVE COLLABORATION IN LANDMARK ESTIMATION This section illustrates the face alignment process in various face recovery procedures. In Figure 10, we showcase land- mark estimation at four steps within our iterative calibration framework at a scale of the factor of ×18 in the training phase of the FSR model. The final estimated landmark is visualized with a heatmap shape. The chosen sample face image in this figure is considered challenging due to non- frontal face angles and existing non-face attributes (such as a hand) in the face image. According to the results in Figure 10, alignment and land- mark estimation in challenging face pose images become more accurate with each iterative recovery of the face image. This indicates a gradual improvement in prior knowledge information, subsequently assisting the proposed FSR model in recovering more accurate and faithful face images by en- hancing the estimation of precise prior information within the iterative calibration framework. FIGURE 8. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on CelebA image at a scale factor of ×16. FIGURE 9. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on Helen image at a scale factor of ×16. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ E. ABLATION STUDY The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. NL RCA SFT CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ X X X 23.05 0.6298 0.3270 89.49 21.79 0.6115 0.3395 91.74 X ✓ X 23.19 0.6584 0.3037 85.94 22.61 0.6422 0.3102 88.97 ✓ ✓ X 23.55 0.6780 0.2983 77.46 22.77 0.6516 0.2918 84.83 X X ✓ 23.14 0.6575 0.3184 87.03 22.55 0.6418 0.3210 89.70 X ✓ ✓ 23.65 0.6830 0.2807 69.23 22.94 0.6628 0.2749 79.16 ✓ ✓ ✓ 23.77 0.6903 0.2600 64.04 23.06 0.6720 0.2644 73.02 TABLE 5. The quantitative evaluation on different configurations of attention modules at a scale factor of ×8. NL RCA SFT CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ X X X 27.37 0.7962 0.2079 80.35 26.69 0.7933 0.2998 85.41 X ✓ X 27.50 0.8017 0.1993 69.62 27.03 0.8082 0.2365 79.56 ✓ ✓ X 27.52 0.8019 0.1973 57.63 27.08 0.8086 0.1904 61.18 X X ✓ 27.47 0.7992 0.2014 78.07 26.98 0.8029 0.2649 81.54 X ✓ ✓ 27.57 0.8041 0.1714 41.19 27.13 0.8089 0.1829 49.92 ✓ ✓ ✓ 27.69 0.8083 0.1691 37.89 27.18 0.8092 0.1789 43.21 TABLE 5. The quantitative evaluation on different configurations of attention modules at a scale factor of ×8. TABLE 6. The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. TABLE 6. The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. NL RCA SFT CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ X X X 23.05 0.6298 0.3270 89.49 21.79 0.6115 0.3395 91.74 X ✓ X 23.19 0.6584 0.3037 85.94 22.61 0.6422 0.3102 88.97 ✓ ✓ X 23.55 0.6780 0.2983 77.46 22.77 0.6516 0.2918 84.83 X X ✓ 23.14 0.6575 0.3184 87.03 22.55 0.6418 0.3210 89.70 X ✓ ✓ 23.65 0.6830 0.2807 69.23 22.94 0.6628 0.2749 79.16 ✓ ✓ ✓ 23.77 0.6903 0.2600 64.04 23.06 0.6720 0.2644 73.02 F. VISUAL COMPARISONS OF CONTRIBUTING DIFFERENT ATTENTION MODULES module) consistently leads to a noteworthy improvement in PSNR, SSIM, FID, and LPIPS, compared to the baseline and other combinations. In this section, we assess the impact of each attention module on generating higher-quality face images. Figure 15 illus- trates four sample images from the CelebA [15] and Helen [16] datasets. We extract face image patches and evaluate their visual quality. Specifically, we compare the visual quality of the baseline model, the model utilizing the NL module, the combination of NL and Residual RCA modules, and finally, the combination of NL, RCA, and SFT modules. To attain a comprehensive understanding of the impact of distinct refinement modules within the FSR model and to discern their relative significance in shaping the overall performance, the evaluation results are visually presented in Figure 11 Figure 12, Figure 13, and Figure 14. These figures represent the performance metrics, including PSNR, SSIM, and LPIPS in each figure. The charts facilitate a comparative analysis of the model’s performance across various combina- tions of refinement modules at scale factors of ×8 and ×16 in the CelebA [15] and Helen datasets. Specifically, Figure 11 demonstrates PSNR, SSIM, and LPIPS in the CelebA dataset at a scale factor of ×8. Figure 12, Figure 13, and Figure 14 visualize the corresponding performances at a scale factor of ×8 for the Helen dataset, a scale factor of ×16 for the CelebA dataset, and a scale factor of ×16 for the Helen dataset, respectively. As depicted in Figure 15, the perceptual quality of the results employing the NL, RCA, and SFT refinement mod- ules significantly improves compared to other methods. The PSNR and SSIM metrics in this combination surpass those of other configurations. In other words, the proposed model successfully recovers more facial details than the baseline and other combined approaches. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ E. ABLATION STUDY A systematic investigation assesses the impact of the NL, RCA, and SFT modules demonstrated in this section. Table 5 and Table 6 showcase the performance metrics, including PSNR, SSIM, LPIPS, and FID, of the proposed FSR model equipped with different NL, RCA, and SFT configurations. The evaluations are conducted on CelebA [15] and Helen [16] datasets with scales of ×8 and ×16, as demonstrated in Tables 5 and 6. The best values are highlighted in bold. In these tables, we compared the baseline model (without any attention module). The other rows correspond to the vari- ation’s combinations in refinement modules, including the RCA module only, a combination of NL with RCA, an SFT module only, a combination of RCA and SFT, and finally, the combination of the three proposed modules. Across both scales and datasets, the combined application of all three attention modules (the NL module, RCB module, and SFT FIGURE 9. Visual comparison of face image reconstruction in three steps of iterative collaboration approach on Helen image at a scale factor of ×16. VOLUME 11, 2023 9 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 5. The quantitative evaluation on different configurations of attention modules at a scale factor of ×8. NL RCA SFT CelebA [15] Helen [16] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ X X X 27.37 0.7962 0.2079 80.35 26.69 0.7933 0.2998 85.41 X ✓ X 27.50 0.8017 0.1993 69.62 27.03 0.8082 0.2365 79.56 ✓ ✓ X 27.52 0.8019 0.1973 57.63 27.08 0.8086 0.1904 61.18 X X ✓ 27.47 0.7992 0.2014 78.07 26.98 0.8029 0.2649 81.54 X ✓ ✓ 27.57 0.8041 0.1714 41.19 27.13 0.8089 0.1829 49.92 ✓ ✓ ✓ 27.69 0.8083 0.1691 37.89 27.18 0.8092 0.1789 43.21 TABLE 6. G. COMPARISON WITH OTHER METHODS Performance comparison (PSNR/SSIM/LPIPS) of our model ith various attention module configurations at a scale factor of ×8 on e CelebA Dataset. FIGURE 12. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the Helen Dataset. FIGURE 12. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the Helen Dataset. FIGURE 11. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the CelebA Dataset. FIGURE 12. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the Helen Dataset. FIGURE 11. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the CelebA Dataset. FIGURE 12. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×8 on the Helen Dataset. Figure 18 presents visual comparisons of the proposed model at a scale factor of ×8 with other state-of-the-art models [5,6,8,26,27,31]. Sample images 1 and 2 are from the Helen [16] dataset, while samples 3 and 4 are from the CelebA [15] dataset. Based on the results in this figure, our model consistently produces higher-fidelity face images compared to the other methods. in CelebA and Helen datasets at a scale factor of ×16. Our proposed model achieves a PSNR of 23.77 (dB) and SSIM of 0.6903 in CelebA datasets, with the lowest LPIPS value of 0.2600 attributed to our model. Furthermore, our model ex- hibits the best LPIPS in the Helen dataset, highlighting its su- perior performance compared to other methods. Additionally, our model’s network parameters are less than SCTANet [11] with 27.98 million network parameters. Figure 17 presents a graphical visualization comparing the PSNR improvement achieved through the bicubic method at a scale factor of ×16. According to the figure, the PSNR improvement compared to the bicubic method at this scale is 3.43 (dB) for the CelebA dataset and 2.61 (dB) for the Helen dataset. Figure 19 illustrates visual comparisons of the proposed method at a scale factor of ×16 with other state-of-the-art models, specifically DIC [5] and SISN [31]. Image samples 1 and 2 belong to the CelebA [15] dataset, while samples 3 and 4 are from the Helen [16] dataset. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ G. COMPARISON WITH OTHER METHODS According to the visualizations in these figures, using the SFT module alone in our middle-level configuration to pre- serve spatial information contributes less to improving the accuracy of the FSR model. However, when combined with the RCA and NL attention modules and RCA, it exhibits the second-best and the best performance in the FSR model, respectively. The results suggest that the effectiveness of the SFT module at mid-level features could be improved by issues arising from the degraded and poor quality of LR face inputs. This section compares our quantitative and qualitative results with several other methods. We conduct quantitative comparisons with various models [5,6,8,11, 26,27,29,30,31,32,35,40,53.54,55,56,57,58]. Table 7 presents comparisons of PSNR, SSIM, and LPIPS in the CelebA and Helen datasets at a scale factor of ×8. Figure 16 demonstrates a graphical representation comparing the PSNR improvement based on bicubic performance at the same scale factor. The results in Table 7 indicate that our model ex- hibits the highest performance in the CelebA dataset and the second-best performance in the Helen dataset. Notably, the network parameters of our model total 24.69 million, which is lower than SCTANet’s 27.56 million parameters. According to Figure 16, the PSNR improvement compared to the bicubic method at this scale is 4.8 (dB) and 6.2 (dB) for the CelebA and Helen datasets, respectively. In contrast, employing the NL attention module to address noise and degradation at the initial stage, coupled with the application of RCA to emphasize inter-channel dependencies at a model level, significantly enhances the performance of SFT. Consequently, this combination performs best, yielding the highest PSNR and SSIM and the lowest LPIPS in both scales and datasets. Table 8 provides comparisons of PSNR, SSIM, and LPIPS Table 8 provides comparisons of PSNR, SSIM, and LPIPS 10 VOLUME 11, 2023 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS GURE 11. G. COMPARISON WITH OTHER METHODS The image labels (a) to (e) depict the LR image (8×8), the results of DIC, SISN, our 11 VOLUME 11, 2023 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 13. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×16 on he CelebA Dataset. FIGURE 14. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×16 o the Helen Dataset. FIGURE 14. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×16 o the Helen Dataset. FIGURE 14. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×16 on the Helen Dataset. FIGURE 13. Performance comparison (PSNR/SSIM/LPIPS) of our model with various attention module configurations at a scale factor of ×16 on the CelebA Dataset. model, and the HR images, respectively. Even at scale ×16, our model consistently outperforms other models, showcas- ing its superior performance in generating high-quality super- resolved images. Specifically, at this large scale factor, the proposed model demonstrates exceptional performance in reconstructing facial attributes with greater fidelity, such as eyes, lips, and nose, compared to the other state-of-the-art models depicted in this figure. model, and the HR images, respectively. Even at scale ×16, our model consistently outperforms other models, showcas- ing its superior performance in generating high-quality super- resolved images. Specifically, at this large scale factor, the proposed model demonstrates exceptional performance in reconstructing facial attributes with greater fidelity, such as eyes, lips, and nose, compared to the other state-of-the-art models depicted in this figure. VOLUME 11, 2023 VOLUME 11, 2023 12 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ accepted for publication in IEEE Access. This is the author's version which has not been fully edited and been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication Citation information: DOI 10 1109/ACCESS 2024 338970 t al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS URE 15. Visual comparisons of contributing different attention modules baseline, (NL), (NL+RCA), (NL+RCA+SFT). IGURE 15. Visual comparisons of contributing different attention modules baseline, (NL), (NL+RCA), (NL+RCA+SFT). FIGURE 15. Visual comparisons of contributing different attention modules baseline, (NL), (NL+RCA), (NL+RCA+SFT). FIGURE 15. Visual comparisons of contributing different attention modules baseline, (NL), (NL+RCA), (NL+RCA+SFT). 13 13 VOLUME 11, 2023 work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 7. Quantitative benchmark test results at a scale factor of ×8. Red indicates the best performance and blue indicates the second best. VOLUME 11, 2023 Methods Scale CelebA [15] Helen [16] Params↓ PSNR↑ SSIM↑ LPIPS↓ PSNR↑ SSIM↑ LPIPS↓ Bicubic ×8 23.61 0.6779 0.4899 20.98 0.6228 0.4912 — SRCNN [53] ×8 23.93 0.6345 0.3013 23.98 0.6670 0.3105 0.01 M URDGN [26] ×8 25.62 0.7261 0.2626 25.23 0.7205 0.2763 1.0 M VDSR [54] ×8 26.36 0.7605 0.2403 25.31 0.7266 0.2489 0.6 M WSRNet [27] ×8 26.83 0.7873 0.2297 26.02 0.7731 0.2316 71.3 M Super-FAN [8] ×8 27.08 0.7841 0.2145 26.23 0.7656 0.3104 1.3 M FSRNet [6] ×8 27.05 0.7714 0.2127 25.45 0.7364 0.3090 3.1 M FMANet [35] ×8 26.63 0.7361 0.3751 26.66 0.678 0.4402 — RCAN [40] ×8 27.24 0.7917 0.2205 25.50 0.7383 0.3437 15.0 M SPARNet [30] ×8 27.28 0.7932 0.1995 26.43 0.7839 0.2674 10.0 M ELAN [55] ×8 27.23 0.7899 0.1915 26.46 0.7204 0.2553 9.76 M SwingIR [56] ×8 27.41 0.7987 0.2001 26.53 0.7856 0.2644 12.44 M FACN [29] ×8 27.22 0.7802 0.1828 25.06 0.7189 0.3113 — SAN [57] ×8 27.43 0.7826 0.2080 25.46 0.7360 0.3260 15.99 M DIC [5] ×8 27.37 0.7969 0.2079 26.69 0.7933 0.2999 20.8 M HAN [58] ×8 27.47 0.7838 0.2087 25.40 0.7347 0.3247 3.23 M FishFSRNet [32] ×8 27.54 0.8072 0.1807 26.86 0.7984 0.1948 25.13 M SCTANet [11] ×8 27.62 0.8041 0.1695 27.67 0.8194 0.1693 27.56 M MSRFSR (Ours) ×8 27.69 0.8083 0.1691 27.18 0.8092 0.1789 24.69 M ark test results at a scale factor of ×8. Red indicates the best performance and blue indicates the second best. TABLE 7. Quantitative benchmark test results at a scale factor of ×8. Red indicates the best performance and blue ind FIGURE 16. Comparison of performance improvement (PSNR) at a scale factor of ×8 on CelebA and Helen Datasets. FIGURE 16. Comparison of performance improvement (PSNR) at a scale factor of ×8 on CelebA and Helen Datasets. VOLUME 11, 2023 14 VOLUME 11, 2023 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. VOLUME 11, 2023 Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 8. Quantitative benchmark test results at a scale factor of ×16. Red indicates the best performance and blue indicates the second best. TABLE 8. Quantitative benchmark test results at a scale factor of ×16. Red indicates the best performance and blue indicates the second best. Methods Scale CelebA [15] Helen [16] Params↓ PSNR↑ SSIM↑ LPIPS↓ PSNR↑ SSIM↑ LPIPS↓ Bicubic ×16 20.34 0.4961 0.6479 20.45 0.5024 0.6438 — SRCNN [53] ×16 20.54 0.4972 0.3601 20.73 0.5088 0.3667 0.01 M URDGN [26] ×16 22.29 0.5786 0.3387 21.68 0.5452 0.3393 1.0 M VDSR [54] ×16 22.42 0.5942 0.3341 21.35 0.5417 0.3486 0.6 M WSRNet [27] ×16 23.13 0.6343 0.3349 22.00 0.5763 0.3369 71.5 M FSRNet [6] ×16 23.04 0.6293 0.3297 22.05 0.5820 0.3359 3.1 M Super-FAN [8] ×16 23.42 0.6515 0.3275 22.54 0.5991 0.3381 1.3 M DIC [5] ×16 23.05 0.6298 0.3270 21.79 0.6115 0.3395 20.8 M RCAN [40] ×16 23.28 0.6624 0.3289 22.91 0.6706 0.3428 15.0 M SPARNet [30] ×16 23.31 0.6634 0.3283 22.95 0.6709 0.3457 10.0 M SwingIR [56] ×16 23.52 0.6744 0.3006 22.51 0.6636 0.3139 12.44 M SISN [31] ×16 23.42 0.6743 0.3114 22.55 0.6037 0.3242 21.66 M ELAN [557] ×16 23.30 0.6618 0.3319 22.81 0.6617 0.3519 9.77 M FishFSRNet [32] ×16 23.68 0.6784 0.2612 22.85 0.6346 0.2649 25.94 M SCTANet [11] ×16 23.67 0.6857 0.2603 23.32 0.6927 0.2644 27.98 M MSRFSR (Ours) ×16 23.77 0.6903 0.2600 23.06 0.6720 0.2644 24.69 M chmark test results at a scale factor of ×16. Red indicates the best performance and blue indicates the second best. TABLE 8. Quantitative benchmark test results at a scale factor of ×16. Red indicates the best performance and blue ind FIGURE 17. Comparison of performance improvement (PSNR) at a scale factor of ×16 on CelebA and Helen Datasets. FIGURE 17. Comparison of performance improvement (PSNR) at a scale factor of ×16 on CelebA and Helen Datasets. 15 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. VOLUME 11, 2023 This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 e has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 20. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×8. FIGURE 21. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×16. FIGURE 20. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×8. FIGURE 20. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×8. FIGURE 20. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×8. FIGURE 21. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×16. FIGURE 21. Performance and network complexity evaluated on the CelebA and Helen datasets at a scale factor of ×16 H. COMPARISON OF NETWORK COMPLEXITY AND PERFORMANCE model [5], our model demonstrates significant improvements in performance in both datasets at this scale. Figure 20 and Figure 21 illustrate our model’s network com- plexity and performance, comparing them with other state-of- the-art models across the CelebA and Helen datasets at scale factors of ×8 and ×16, respectively. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 11, 2023 This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 18. Visual comparison of our model with other state-of-the-art methods scale factor of ×8. (a): Bicubic, (b): URDGN, (c): WSRNet, (d): SuperFAN, (e): FSRNet, (f): DIC, (g): SISN, (h): Ours, (i): HR. FIGURE 18. Visual comparison of our model with other state-of-the-art methods scale factor of ×8. (a): Bicubic, (b): URDGN, (c): WSRNet, (d): SuperFAN, (e): FSRNet, (f): DIC, (g): SISN, (h): Ours, (i): HR. FIGURE 18 Visual comparison of our model with other state of the art methods scale factor of 8 (a): Bicubic (b): URDGN (c): WSRNet (d): SuperFAN FIGURE 18. Visual comparison of our model with other state-of-the-art methods scale factor of ×8. (a): Bicubic, (b): URDGN, (c): WSRNet, (d): SuperFAN, (e): FSRNet, (f): DIC, (g): SISN, (h): Ours, (i): HR. 16 VOLUME 11, 2023 VOLUME 11, 2023 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ or et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 19 Visual comparison with other methods scale factor of ×16 FIGURE 19. Visual comparison with other methods scale factor of ×16. l h h h d l f f FIGURE 19. Visual comparison with other methods scale factor of ×16. 17 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Author et al : Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. I. USER STUDY Citation information: DOI 10.1109/ACCESS.2024.3389702 l.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS TABLE 9. User study evaluation at a scale factor of ×8. Dataset Image FishNet [32] DIC [5] SCTANet [11] CTCNet [34] Ours CelebA [15] 201518 2.1 1.7 3.6 3.2 4.3 201469 2.1 1.9 3.8 3.2 4 201989 2 2.2 3.1 3.8 3.9 201434 2.4 1.4 3.2 3.5 4.5 Helen [16] 3227182502-1 2.6 1.9 3.2 3.2 4.1 3217492469-1 2.3 2.1 3.9 2.5 4.2 3261640996-1 2.5 2 3.8 2.9 3.8 3249818146-1 2.1 2.2 3.1 3.6 4 Average Score 8 Images 2.26 1.92 3.46 3.23 4.1 TABLE 9. User study evaluation at a scale factor of ×8. TABLE 9. User study evaluation at a scale factor of ×8. TABLE 10. User study evaluation at a scale factor of ×16. TABLE 10. User study evaluation at a scale factor of ×16. Dataset Image FishNet [32] DIC [5] SCTANet [11] CTCNet [34] Ours CelebA [15] 201418 2.5 1.4 3.3 3.7 4.1 201475 2.9 1.7 3.2 3.3 3.9 201641 2.7 1.1 3 3.2 4 201481 2.5 1 3.7 3.6 4.2 Helen [16] 3220402975-1 2 1 3.9 3.9 4.2 3222262589-1 1.8 1.2 3.9 3.7 4.4 3247810519-1 2 1 3.7 3.8 4.5 3217491791-1 1.9 1.1 4 4 4 Average Score 8 Images 2.16 1.18 3.58 3.65 4.16 quality performance among other models. model and DIC [5] at a scale factor ×8. Samples 1 and 2 belong to AFLW2000 [46], while Samples 3 and 4 belong to WFLW [47]. Labels a to e represent the LR image (16×16), bicubic, DIC [5], our model, and the HR image, respectively. Our model exhibits superior performance in all demonstrated samples compared to the baseline in recovering more facial details and generating higher fidelity results. J. QUANTITATIVE AND QUALITATIVE COMPARISON ON AFLW2000 AND WFLW DATASETS We conduct quantitative comparisons with the baseline model [5] on the AFLW2000 [46] and WFLW [47] datasets. Tables 11 and 12 present PSNR, SSIM, LPIPS, and FID at scale factors of ×8 and ×16, respectively. The examined models are trained on the CelebA and Helen datasets, as shown in the tables. Based on these tables, our model outperforms the DIC [5] model in PSNR, SSIM, LPIPS, and FID for both datasets under the same training conditions. Furthermore, the performance of our model and the baseline model in the trained CelebA dataset surpasses that of the models trained on the Helen dataset. Figure 23 compares our model and DIC [5] at a scale factor of ×16. Samples 1 and 2 are from AFLW2000, while Samples 3 and 4 are from WFLW. Labels a to e indicate the LR image (8×8), bicubic, DIC, our model, and the HR image, respectively. Across all the samples, our model outperforms the baseline by recovering more facial details and producing higher-quality results. Figure 22 demonstrates the visual comparison between our TABLE 11. The quantitative evaluation on different configurations of attention modules at a scale factor of ×8. Method Scale AFLW2000 [46] WFLW [47] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ Bicubic ×8 19.17 0.5347 0.4981 89.81 22.37 0.6657 0.5017 87.35 DIC (CelebA) ×8 24.29 0.7046 0.1992 56.22 27.38 0.7847 0.1999 58.92 DIC (Helen) ×8 23.68 0.6801 0.1979 56.47 26.68 0.7638 0.2043 59.32 MSRFSR (CelebA) ×8 24.31 0.7084 0.1917 54.98 27.60 0.7948 0.1949 57.19 MSRFSR (Helen) ×8 23.77 0.6824 0.1932 55.13 27.04 0.7712 0.1991 58.71 TABLE 11. The quantitative evaluation on different configurations of attention modules at a scale factor of ×8. TABLE 12. The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. Method Scale AFLW2000 [46] WFLW [47] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ Bicubic ×16 18.80 0.5302 0.6848 95.83 20.35 0.5697 0.6907 93.38 DIC (CelebA) ×16 20.07 0.5421 0.3081 78.91 22.98 0.6254 0.3010 82.71 DIC (Helen) ×16 19.79 0.5492 0.2996 79.12 22.71 0.6139 0.2998 83.02 MSRFSR (CelebA) ×16 20.28 0.5593 0.2901 77.22 23.38 0.6398 0.2975 81.14 MSRFSR (Helen) ×16 19.98 0.5532 0.2894 78.05 23.09 0.6375 0.2914 82.69 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ TABLE 12. The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. TABLE 12. I. USER STUDY Table 9 and Table 10 present our user study evaluation results for our model at scale factors of ×8 and ×16, respectively. We aimed to collect detailed feedback from participants about their preferences and perceptions of the FSR models’ outputs. Using a user study approach, we sought to gather comprehen- sive feedback from participants regarding their preferences and perceptions of the FSR models’ outputs. We ensured variability and representation for participants by using ran- dom images from the datasets, which contributed to the ro- bustness and generalizability of our findings. We conducted a user study involving four robust FSR models [5,11,32,34] evaluated on the CelebA and Helen datasets. We recruited ten individuals, including both experts and non-experts and included four random images from each dataset. The comparison in Figure 20 shows that WSRnet [27] exhibits the highest network parameters, with PSNR values of 26.83 dB and 36.02 dB for the CelebA and Helen datasets, respectively. Notably, our model’s performance in the CelebA and Helen datasets improves to 27.69 dB and 27.18 dB, re- spectively, while the network complexity decreases by around 2.7 times compared to WSRnet [27] at this scale. Compared to the DIC [5] model, our model contains 4 million more parameters, achieving an increased PSNR performance in both datasets compared to the DIC [5] model. Based on Figure 21, the network complexity of our model is lower than that of FishFSRNet [32] and SCTANet [11]. In contrast, the performance of our model surpasses both FishFSRNet [32] and SCTANet [11] in the CelebA dataset. In the Helen dataset, our model’s performance falls within the same range as SCTANet (with only a 0.26 dB difference). In contrast, the complexity of our model is 3.20 million parameters less than SCTANet [32]. Compared to the baseline Our evaluation criteria were designed to capture subjective preferences across visual quality, naturalness, and perceptual similarity to HR images. Participants were asked to rate the face images using a 5-point scale, ranging from "Poor" to "Excellent," with "1" indicating the lowest score and "5" the highest. The highest score in the two tables shows the best 18 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. J. QUANTITATIVE AND QUALITATIVE COMPARISON ON AFLW2000 AND WFLW DATASETS (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. J. QUANTITATIVE AND QUALITATIVE COMPARISON ON AFLW2000 AND WFLW DATASETS The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. Method Scale AFLW2000 [46] WFLW [47] PSNR↑ SSIM↑ LPIPS↓ FID↓ PSNR↑ SSIM↑ LPIPS↓ FID↓ Bicubic ×16 18.80 0.5302 0.6848 95.83 20.35 0.5697 0.6907 93.38 DIC (CelebA) ×16 20.07 0.5421 0.3081 78.91 22.98 0.6254 0.3010 82.71 DIC (Helen) ×16 19.79 0.5492 0.2996 79.12 22.71 0.6139 0.2998 83.02 MSRFSR (CelebA) ×16 20.28 0.5593 0.2901 77.22 23.38 0.6398 0.2975 81.14 MSRFSR (Helen) ×16 19.98 0.5532 0.2894 78.05 23.09 0.6375 0.2914 82.69 TABLE 12. The quantitative evaluation on different configurations of attention modules at a scale factor of ×16. 19 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al : Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 22. Visual comparison of our model with baseline at a scale factor of ×8 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. FIGURE 22. Visual comparison of our model with baseline at a scale factor of ×8 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. FIGURE 22. Visual comparison of our model with baseline at a scale factor of ×8 on AFLW2000 and WFLW datasets. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ V. DISCUSSION AND FUTURE WORK leveraging spatial information. However, it is noted that using the SFT module alone in the middle-level configuration con- tributes less to improving the accuracy of the FSR model, pri- marily due to issues arising from degraded and poor-quality LR face inputs. In contrast, employing the NL attention mod- ule to address noise and degradation at the initial stage, cou- pled with the application of RCA to emphasize inter-channel dependencies, significantly enhances the performance of the To enhance fidelity and detail in generating face images at large scale factors, the NL module and RCA technique at the low-level stage focus on critical facial details and effectively mitigate shortcomings in feature quality, leading to a re- fined and context-aware representation. Additionally, the de- signed SFT module at the mid-level architecture significantly contributes to recovering high-frequency facial attributes by VOLUME 11, 2023 20 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 2 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 23. Visual comparison of our model with baseline at a scale factor of ×16 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS FIGURE 23. Visual comparison of our model with baseline at a scale factor of ×16 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. SFT module at the mid-level. Consequently, this innovative combination of multi-attention techniques demonstrates the best performance in generating faithful and detailed face im- ages compared to previous FSR models, yielding the highest PSNR, SSIM, LPIPS, and VIF scores across large scales and four datasets. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VI. CONCLUSION This research introduces a Multi-stage Refining Face Super- resolution model, establishing a novel paradigm through it- erative collaboration between landmark estimation and an attentive recovery network. The formidable challenge posed by the degraded low-dimensional (16×16 pixels or 8×8 pixels) input images restricts gaining the full capability of the iterative collaboration framework to generate more de- tailed and accurate face images. The proposed Multi-stage Refining model utilizes an SFT module for mid-level feature refinement and incorporates NL and a residual pixel attention technique at the low-level stage. The NL module captures long-range dependencies within the low-level features. At the same time, the RCA technique enhances focus on critical facial details by selectively emphasizing informative chan- nels. This approach effectively addresses shortcomings in feature quality, fostering a more refined and context-aware representation. Consequently, it significantly enhances the efficacy of the SFT module at the mid-level stage, enabling the recovery of more faithful facial details. Applying the proposed refinement approach demonstrates improvement in both the accuracy and perceptual quality of super-resolved face images, surpassing the performance of baseline mod- els. Empirical evaluations conducted on CelebA and Helen datasets at scale factors of ×8 and ×16 demonstrate note- worthy enhancements in terms of PSNR, SSIM, and LPIPS. Visual comparisons further underscore the model’s superior- ity, showcasing significant advancements compared to other state-of-the-art models. [8] Bulat, A. and Tzimiropoulos, G., ‘‘Super-fan: Integrated facial landmark localization and super-resolution of real-world low resolution faces in arbitrary poses with gans,’’ in Proceedings of the IEEE conference on computer vision and pattern recognition, 2018, pp. 109–117. [9] Kim, D., Kim, M., Kwon, G. and Kim, D.S., ‘‘Progressive face super-resolution via attention to facial landmark,’’ in arXiv preprint arXiv:1908.08239, 2019. [10] Hu, X., Ren, W., LaMaster, J., Cao, X., Li, X., Li, Z., Menze, B. and Liu, W., ‘‘Face super-resolution guided by 3d facial priors,’’ in Computer Vision–ECCV 2020: 16th European Conference, Glasgow, UK, August 23–28, 2020, Proceedings, Part IV 16 , 2020, pp. 763–780. Springer International Publishing. [11] Bao, Q., Liu, Y., Gang, B., Yang, W. and Liao, Q., ‘‘SCTANet: A Spatial Attention-Guided CNN-Transformer Aggregation Network for Deep Face Image Super-Resolution,’’ in IEEE Transactions on Multimedia, 2023. [12] Gu, J., Lu, H., Zuo, W. and Dong, C., ‘‘Blind super-resolution with iter- ative kernel correction,’’ in Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition , 2019, pp. 1604–1613. [1] Zhou Y, Liu D, Huang T, ‘‘Survey of face detection on low-quality images,’’ in 2018 13th IEEE international conference on automatic face & gesture recognition (FG 2018), 2018, pp. 769–773. ACKNOWLEDGMENT We would like to express our sincere gratitude to the De- partment of Electrical Engineering, Faculty of Engineering, Chulalongkorn University. [21] Chakrabarti, A., Rajagopalan, A.N. and Chellappa, R., ‘‘Super-resolution of face images using kernel PCA-based prior,’’ in IEEE Transactions on Multimedia, 9(4), 2007, pp. 888–892.i [22] Shi, J. and Zhao, G., ‘‘Face hallucination via coarse-to-fine recursive kernel regression structure,’’ in IEEE Transactions on Multimedia, 21(9), 2019, pp. 2223–2236. pp [2] Li, P., Prieto, L., Mery, D. and Flynn, P, ‘‘Face recognition in low quality images: A survey,’’ in arXiv preprint arXiv:1805.11519, 2018. VI. CONCLUSION [13] Shen, L., Zhao, B., Li, Q., Zhang, C., Sun, X. and Peng, B., ‘‘Local to non- local: Multi-scale progressive attention network for image restoration,’’ in Computer Vision and Image Understanding, 233,, 57(11), 2020, pp. 9277– 9289. [14] Haut, J.M., Fernandez-Beltran, R., Paoletti, M.E., Plaza, J. and Plaza., ‘‘Remote sensing image superresolution using deep residual channel at- tention,’’ in IEEE Transactions on Geoscience and Remote Sensing, 2023, pp. 103725. [15] Liu, Z., Luo, P., Wang, X. and Tang, X, ‘‘Deep learning face attributes in the wild,’’ in Proceedings of the IEEE international conference on computer vision, 2016, pp. 3730–3738. [16] Le, V., Brandt, J., Lin, Z., Bourdev, L. and Huang, T.S., ‘‘Interactive facial feature localization,’’ in Computer Vision–ECCV 2012: 12th European Conference on Computer Vision, Florence, Italy, October 7-13, 2012, Proceedings, Part III 12, 2012, 679–692, Springer Berlin Heidelberg. [17] Baker, S. and Kanade, T., ‘‘Hallucinating faces,’’ in Proceedings Fourth IEEE international conference on automatic face and gesture recognition (Cat. No. PR00580), 2000, pp. 83–88. [18] Liu, C., Shum, H.Y. and Zhang, C.S., ‘‘A two-step approach to halluci- nating faces: global parametric model and local nonparametric model,’’ in Proceedings of the 2001 IEEE Computer Society Conference on Computer Vision and Pattern Recognition. CVPR 2001, (Vol. 1, pp. I-I), 2001. In future work, we will integrate the proposed FSR model developed with real-time face recognition systems. This ex- tension aims to explore the potential impact of our model on enhancing recognition accuracy in practical scenarios. [19] Chang, H., Yeung, D.Y. and Xiong, Y., ‘‘Super-resolution through neighbor embedding,’’ in Proceedings of the IEEE Computer Society Conference on Computer Vision and Pattern Recognition, 2004, pp. 1–8. [20] Wang, X. and Tang, X. , ‘‘Hallucinating face by eigentransformation,’’ in IEEE transactions on systems, man, and cybernetics, part C (applications and reviews), 35(3), 2005, pp. 425–434. Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS images, where only partial facial features, such as one eye, one side of the nose, and a portion of the mouth, are visible. This constraint restricts the model’s applicability in scenarios where profile images are prevalent or necessary. [3] Hajian, A. and Aramvith, S, ‘‘Fusion Objective Function on Progres- sive Super-Resolution Network,’’ in Journal of Sensor and Actuator Net- works,12(2):26, 2023. [4] Jiang, J., Wang, C., Liu, X., Ma, J., ‘‘Deep learning-based face super- resolution: A survey,’’ in ACM Computing Surveys (CSUR),55(1), 2021, pp. 1–36. Future research endeavors will address this limitation by tuning the FAN module to accommodate profile face im- ages and incorporating additional training datasets containing profile face images. This effort could enhance the model’s robustness and broaden its utility in real-world applications, thus advancing the effectiveness and applicability of FSR techniques in diverse practical settings. [5] Ma, C., Jiang, Z., Rao, Y., Lu, J. and Zhou, J., ‘‘Deep face super-resolution with iterative collaboration between attentive recovery and landmark esti- mation,’’ in Proceedings of the IEEE/CVF conference on computer vision and pattern recognition, 2020, pp. 5569–5578. [6] Chen, Y., Tai, Y., Liu, X., Shen, C. and Yang, J., ‘‘Fsrnet: End-to-end learning face super-resolution with facial priors,’’ in Proceedings of the IEEE conference on computer vision and pattern recognition, 2018, pp. 2492–2501. [7] Yu, X., Fernando, B., Ghanem, B., Porikli, F. and Hartley, R., ‘‘Face super- resolution guided by facial component heatmaps,’’ in Proceedings of the European conference on computer vision (ECCV), 2018, pp. 217–233. V. DISCUSSION AND FUTURE WORK This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 V. DISCUSSION AND FUTURE WORK Although the proposed model excels in generating faithful and detailed full-face images across diverse angles, its per- formance could be improved when tasked with generating profile face images. This limitation arises due to the focus of the FAN module, which was tuned exclusively for the frontal view of the face, and the training datasets (CelebA and Helen) contain only full-face images. Consequently, the model must be more accurate in accurately reconstructing profile face FIGURE 23. Visual comparison of our model with baseline at a scale factor of ×16 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. FIGURE 23. Visual comparison of our model with baseline at a scale factor of ×16 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. FIGURE 23. Visual comparison of our model with baseline at a scale factor of ×16 on AFLW2000 and WFLW datasets. (a): LR, (b): Bicubic, (c): DIC, (d): Ours, (e): HR. SFT module at the mid-level. Consequently, this innovative combination of multi-attention techniques demonstrates the best performance in generating faithful and detailed face im- ages compared to previous FSR models, yielding the highest PSNR, SSIM, LPIPS, and VIF scores across large scales and four datasets. SFT module at the mid-level. Consequently, this innovative combination of multi-attention techniques demonstrates the best performance in generating faithful and detailed face im- ages compared to previous FSR models, yielding the highest PSNR, SSIM, LPIPS, and VIF scores across large scales and four datasets. and detailed full-face images across diverse angles, its per- formance could be improved when tasked with generating profile face images. This limitation arises due to the focus of the FAN module, which was tuned exclusively for the frontal view of the face, and the training datasets (CelebA and Helen) contain only full-face images. Consequently, the model must be more accurate in accurately reconstructing profile face Although the proposed model excels in generating faithful 21 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. 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[45] Lai, H., Xiao, S., Pan, Y., Cui, Z., Feng, J., Xu, C., Yin, J. and Yan, S., ‘‘Deep recurrent regression for facial landmark detection,’’ in IEEE Transactions on Circuits and Systems for Video Technology, 28(5), 2016, pp.1144-1157. 23 VOLUME 11, 2023 VOLUME 11, 2023 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 This article has been accepted for publication in IEEE Access. This is the author's version which has not been fully edited and content may change prior to final publication. Citation information: DOI 10.1109/ACCESS.2024.3389702 Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS Author et al.: Preparation of Papers for IEEE TRANSACTIONS and JOURNALS SUPAVADEE ARAMVITH (Senior Member, IEEE) received a B.S. (first class honors) degree in Computer Science from Mahidol University in 1993. She received her M.S. and Ph.D. in Electrical Engineering from the University of Washington, Seattle, USA, in 1996 and 2001, respectively. She joined Chulalongkorn University in June 2001. She is an associate professor at the Department of Electrical Engineering specializing in image and video signal processing. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ REFERENCES She has successfully advised 14 Ph.D., 30 Master’s, and 41 Bachelor’s Graduates. She published over 130 papers in International Conference Proceedings and Journals with 4 International Book Chapters. She has rich project management experience as the project leader and former technical committee chair to the Thailand Government bodies in Telecommunications and ICT. She is very active in the international arena with leadership positions in the global network, such as JICA Project for AUN/SEED-Net, NICT ASEAN IVO, and professional organizations such as IEEE, IEICE, APSIPA, and ITU. SUPAVADEE ARAMVITH 24 VOLUME 11, 2023 VOLUME 11, 2023 VOLUME 11, 2023
https://openalex.org/W2802582357	https://europepmc.org/articles/pmc5982241?pdf=render	English	null	A Double Dwell High Sensitivity GPS Acquisition Scheme Using Binarized Convolution Neural Network	Sensors	2,018	cc-by	9,435	Received: 18 February 2018; Accepted: 25 April 2018; Published: 9 May 2018 Received: 18 February 2018; Accepted: 25 April 2018; Published: 9 May 2018 Abstract: Conventional GPS acquisition methods, such as Max selection and threshold crossing (MAX/TC), estimate GPS code/Doppler by its correlation peak. Different from MAX/TC, a multi-layer binarized convolution neural network (BCNN) is proposed to recognize the GPS acquisition correlation envelope in this article. The proposed method is a double dwell acquisition in which a short integration is adopted in the ﬁrst dwell and a long integration is applied in the second one. To reduce the search space for parameters, BCNN detects the possible envelope which contains the auto-correlation peak in the ﬁrst dwell to compress the initial search space to 1/1023. Although there is a long integration in the second dwell, the acquisition computation overhead is still low due to the compressed search space. Comprehensively, the total computation overhead of the proposed method is only 1/5 of conventional ones. Experiments show that the proposed double dwell/correlation envelope identiﬁcation (DD/CEI) neural network achieves 2 dB improvement when compared with the MAX/TC under the same speciﬁcation. Keywords: GPS acquisition; binarized convolution neural network; high sensitivity; double dw sensors sensors sensors A Double Dwell High Sensitivity GPS Acquisition Scheme Using Binarized Convolution Zhen Wang, Yuan Zhuang , Jun Yang, Hengfeng Zhang, Wei Dong, Min Wang, Luchi Hua, Bo Liu and Longxing Shi Zhen Wang, Yuan Zhuang , Jun Yang, Hengfeng Zhang, Wei Dong, Min Wang, Luchi Hua, Bo Liu and Longxing Shi National ASIC System Engineering Research Center, Southeast University, 2 Sipailou, Nanjing 210096, China; 230119228@seu.edu.cn (Z.W.); dragon@seu.edu.cn (J.Y.); zhhf@seu.edu.cn (H.Z.); dongwei_seu@seu.edu.cn (W.D.); wangmin93@seu.edu.cn (M.W.); hualuchi@gmail.com (L.H.); liubo_cnasic@seu.edu.cn (B.L.); lxshi@seu.edu.cn (L.S.) * Correspondence: zhy.0908@gmail.com; Tel.: +86-25-8379-3265 National ASIC System Engineering Research Center, Southeast University, 2 Sipailou, Nanjing 210 230119228@seu.edu.cn (Z.W.); dragon@seu.edu.cn (J.Y.); zhhf@seu.edu.cn (H.Z.); dongwei_seu@seu.edu.cn (W.D.); wangmin93@seu.edu.cn (M.W.); hualuchi@gmail.com (L.H.); liubo_cnasic@seu.edu.cn (B.L.); lxshi@seu.edu.cn (L.S.) * Correspondence: zhy.0908@gmail.com; Tel.: +86-25-8379-3265 Sensors 2018, 18, 1482; doi:10.3390/s18051482 1. Introduction GPS signal deteriorates greatly in urban environments, and the received signal power often degrades to −143 dBm or lower, resulting in acquisition difﬁculties. As the mostly used acquisition method, the MAX/TC has been thoroughly studied to balance false alarm probability and detection probability [1–3]. However, increasing the detection probability for weak signals in GPS acquisition is still an unsolved problem. Currently, there are two ways to improve the detection probability for weak GPS signals: • Enhancing the quality of correlation (often characterized by signal–noise-ratio) by extending the signal integration time; • Enhancing the quality of correlation (often characterized by signal–noise-ratio) by extending the signal integration time; signal integration time; • Optimizing the acquisition decision strategy by advanced techniques. However, the performance of the ﬁrst method is not good enough to acquire weak GPS signals, since the signal integration time is limited by the transition of modulated navigation data. Conventional studies of high sensitivity acquisition focus on improving the coherent integration, in which the coherent integration time has been extended to 10 ms by using large and complex computation operations [4–7]. Due to the large hypothesis parameter space existing in the cold-start environment, Sensors 2018, 18, 1482; doi:10.3390/s18051482 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 11 Sensors 2018, 18, 1482 the computational overhead of current algorithms is fairly large, which cause them to be impractical for civilian receivers. Lacking an efﬁcient processing technique, the correlation envelope feature has been ignored in GPS acquisition for a long time. In recent years, convolution neural network (CNN) has attracted attention for its remarkable performance in identiﬁcation and classiﬁcation [8]. Motivated by its great performance, correlation envelope identiﬁcation (CEI) with CNN has been introduced to compress the GPS acquisition hypothesis parameter space by identifying the envelope in a low-quality correlation. With the narrowed hypothesis parameter space, a long integration can be adopted in the follow-up acquisition to achieve a high-quality correlation. Considering the demand of huge multiplications in traditional CNN, binarized convolution neural network (BCNN) [9] is used to reduce the number of multiplications in the proposed algorithm. The BCNN weights are only +1/−1 in the network training. Moreover, the fully connected layers, which require huge computation, are replaced. It further simpliﬁes the network complexity and reduces the computation overhead. 2. DD/CEI GPS Acquisition Scheme 2.1. MAX/TC Acquisition Method 1. Introduction To the best of our knowledge, this article introduces CNN to global navigation satellite system (GNSS) signal acquisition for the ﬁrst time, and no other researchers have discussed the CNN-based GNSS acquisition in the past years. The main contributions of this article are as follows: • CNN is ﬁrstly introduced in GPS correlation envelop identiﬁcation. Motivated by image recognition, the GPS acquisition correlation envelop can be detected with CNN. • CNN is ﬁrstly introduced in GPS correlation envelop identiﬁcation. Motivated by image recognition, the GPS acquisition correlation envelop can be detected with CNN. • The neural network is optimized to binary CNN to reduce the computation overhead. Considering the huge calculations introduced by CNN, a binary CNN whose weights are constrained by +1/−1 is used in this article. • The neural network is optimized to binary CNN to reduce the computation overhead. Considering the huge calculations introduced by CNN, a binary CNN whose weights are constrained by +1/−1 is used in this article. • The performance of the proposed algorithm is validated by the ﬁeld test, and it shows that the acquisition sensitivity is improved by 2 dB under the same speciﬁcation. On the other hand, in the case of the same acquisition sensitivity, the computation overhead of the proposed algorithm is only around 1/5 of conventional ones because the long integration only exists in narrowed parameter space. The rest of this article is organized as follows. Section 2.1 states the problem which exists in conventional acquisition methods. Section 2.2 elaborates the proposed double dwell acquisition scheme. Section 2.3 presents the correlation envelope identiﬁcation decision strategy based on BCNN. The test results are shown in Section 3 and this article is concluded in Section 4. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme As shown in Figure 2, the double dwell acquisition scheme includes two steps: As shown in Figure 2, the double dwell acquisition scheme includes two steps: 2.2. Double Dwell Acquisition Scheme As shown in Figure 2 the double dwell acquisition scheme includes two steps: 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; g , q p 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; g , q p 1. Recognizing the most possible envelope around the correlation peak by using correlation envelope identify network (CEI) and giving the most possible parameter space; p y g g p p p 2. Detecting the signal in the most narrowed parameter space filtered by step 1. 2. Detecting the signal in the most narrowed parameter space ﬁltered by step 1. envelope identify network (CEI) and giving the most possible parameter space 2 Detecting the signal in the most narrowed parameter space filtered by step 1 p y g g p p p 2. Detecting the signal in the most narrowed parameter space filtered by step 1. 2. Detecting the signal in the most narrowed parameter space ﬁltered by step 1. envelope identify network (CEI) and giving the most possible parameter space 2. Detecting the signal in the most narrowed parameter space filtered by step 1. Correlation Frequency Mix Integration(short) Correlation Matrix (NCA X Nf) CEI/CNN Correlation Frequency Mix Integration(long) Narrowed Hypothesis Parameter Space (Nf X Nf) MAX/TC ... ... Correlation Envelope 0 1 1 2 2 CA CA f f f d f N Bin N N Bin f Bin     − −     − ( , ) df  Received Signal ( , ) df  Hypothesis Parameter First Dwell Second Dwell Correlation Matrix (Nf X Nf) Figure 2. 2.1. MAX/TC Acquisition Method Figure 1 Normalized correlation results in different signal powers Although it is a significant method to extend the integration time to promote the performance of GPS acquisition, a long integration time with a large hypothesis parameter space will introduce high computation complexity and overhead. Therefore, apart from extending the coherent integration time, compressing the hypothesis parameter space is the inevitable choice to further improve the performance of GPS acquisition. Although it is a signiﬁcant method to extend the integration time to promote the performance of GPS acquisition, a long integration time with a large hypothesis parameter space will introduce high computation complexity and overhead. Therefore, apart from extending the coherent integration time, compressing the hypothesis parameter space is the inevitable choice to further improve the performance of GPS acquisition. Although it is a significant method to extend the integration time to promote the performance of GPS acquisition, a long integration time with a large hypothesis parameter space will introduce high computation complexity and overhead. Therefore, apart from extending the coherent integration time, compressing the hypothesis parameter space is the inevitable choice to further improve the performance of GPS acquisition. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme 2.1. MAX/TC Acquisition Method GPS is a code division multiple access (CDMA) system in which signals are modulated by binary phase shift key (BPSK). Therefore, GPS signal acquisition is the detection and estimation of code phase and Doppler frequency based on the cross-ambiguity function (CAF) [10], which is expressed as the correlation between the incoming signal and the locally generated signal. R(eτ, efd) =\| N−1 ∑ n=0 c(nTs −eτ)sinc( efdTs)e−j2π efdnTs + w(n)\| (1) (1) where N is the integration time, TS is the code period of the signal, c(nTS −eτ) is the local replica of the C/A code, eτ and efd are the code delay and the Doppler shift included in the hypothesis parameter space, and w(n) is the additive white Gaussian noise (AWGN). The MAX/TC acquisition method simply selects the largest correlation peak and compares it with a preset threshold. The acquisition correlations with 2 ms coherent integration for weak GPS signals are shown in Figure 1. In Figure 1, the round point marks the correlation generated by the correct parameters, while the tetragon point marks the largest correlation. The correlation peak occurs 3 of 11 3 of 11 3 of 11 Sensors 2018, 18, 1482 Sensors 2018, 18, x FOR Sensors 2018, 18, x FOR in the correct code phase when the signal power is strong. However, the correlation peak occurs in an incorrect code phase in the case of weak signals. Therefore, the MAX/TC acquisition method based on the correlation peak is impractical for weak GPS signals. p g p g p occurs in the correct code phase when the signal power is strong. However, the correlation peak occurs in an incorrect code phase in the case of weak signals. Therefore, the MAX/TC acquisition method based on the correlation peak is impractical for weak GPS signals. correct parameters, while the tetragon point marks the largest correlation. The correlation peak occurs in the correct code phase when the signal power is strong. However, the correlation peak occurs in an incorrect code phase in the case of weak signals. Therefore, the MAX/TC acquisition method based on the correlation peak is impractical for weak GPS signals Figure 1. Normalized correlation results in different signal powers. Figure 1. Normalized correlation results in different signal powers. Figure 1 Normalized correlation results in different signal powers Figure 1. Normalized correlation results in different signal powers. Figure 1. Normalized correlation results in different signal powers. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme Sensors 2018, 18, x FOR PEER REVIEW 4 of 11 In the second dwell, a long coherent integration is generated by correlating between the f d h h i d h i d i l Th ibl d d In the second dwell, a long coherent integration is generated by correlating between the preferred hypothesis parameters and the received signal. The most possible parameters are detected by the MAX/TC method based on the results of the high-quality correlation. Sensors 2018, 18, x FOR PEER REVIEW 4 of 11 In the second dwell, a long coherent integration is generated by correlating between the f d h th i t d th i d i l Th t ibl t d t t d The proposed acquisition method and its conditional probability are described in Figure 3. The probability PC D is the recognition probability that the neural network recognizes the correlation envelope. If the correct envelope is recognized, there are three cases in the second dwell: (1) the real code/Doppler is detected, and its probability is deﬁned as detection probability in the second dwell, PM D1; (2) the fake code/Doppler is detected, and its probability is deﬁned as false alarm probability in the second dwell, PM P1; (3) misses: that is, the correlation peak does not exceed the threshold, and its probability is 1 −PM D1 −PM P1. If the neural network has recognized the false envelope, there are two cases in the second dwell: (1) the fake code/Doppler is detected, and its probability is deﬁned as false alarm probability in the second dwell, PM F2; (2) misses: that is, the correlation peak does not exceed the threshold, and its probability is 1 −PM F2. preferred hypothesis parameters and the received signal. The most possible parameters are detected by the MAX/TC method based on the results of the high-quality correlation. The proposed acquisition method and its conditional probability are described in Figure 3. The probability 𝑃𝐷 𝐶 is the recognition probability that the neural network recognizes the correlation envelope. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme If the correct envelope is recognized, there are three cases in the second dwell: (1) the real code/Doppler is detected, and its probability is defined as detection probability in the second dwell, 𝑃𝐷1 𝑀; (2) the fake code/Doppler is detected, and its probability is defined as false alarm probability in the second dwell, 𝑃𝑃1 𝑀; (3) misses: that is, the correlation peak does not exceed the threshold, and its probability is 1 −𝑃𝐷1 𝑀−𝑃𝑃1 𝑀. If the neural network has recognized the false envelope, there are two cases in the second dwell: (1) the fake code/Doppler is detected, and its probability is defined as false alarm probability in the second dwell, 𝑃𝐹2 𝑀; (2) misses: that is, the correlation peak does not exceed the threshold, and its probability is 1 −𝑃𝐹2 𝑀. 1st Dwell (short) 2nd Dwell (long) Detection Success 2nd Dwell (long) MAX False Alarm Detection Miss MAX C DP C DP - 1 M D P 1 M F1 P M F1 M D P - P - 1 1 M F2 P M F2 P - 1 Neural Networks D P F P M P Figure 3. Double dwell/correlation envelope identification (DD/CEI) conditional probability graph. Figure 3. Double dwell/correlation envelope identiﬁcation (DD/CEI) conditional probability graph. 1st Dwell (short) Figure 3. Double dwell/correlation envelope identification (DD/CEI) conditional probability graph. Figure 3. Double dwell/correlation envelope identiﬁcation (DD/CEI) conditional probability graph. From the overall acquisition perspective, the detection probability 𝑃𝐷, the false alarm probability 𝑃𝐹, and the missed detection probability 𝑃𝑀 are presented as follows. C M P P P =  (2) From the overall acquisition perspective, the detection probability PD, the false alarm probability PF, and the missed detection probability PM are presented as follows. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme Double dwell acquisition architecture. I h fi d ll h i d i l i l d i h ll h ibl Correlation Frequency Mix Integration(short) Correlation Matrix (NCA X Nf) CEI/CNN Correlation Frequency Mix Integration(long) Narrowed Hypothesis Parameter Space (Nf X Nf) MAX/TC ... ... Correlation Envelope 0 1 1 2 2 CA CA f f f d f N Bin N N Bin f Bin     − −     − ( , ) df  Received Signal ( , ) df  Hypothesis Parameter First Dwell Second Dwell Correlation Matrix (Nf X Nf) Figure 2. Double dwell acquisition architecture. Figure 2. Double dwell acquisition architecture. g q i d i l i l d i h ll h Figure 2. Double dwell acquisition architecture. Figure 2. Double dwell acquisition architecture. g p p g the correlation matrix, whose size is the product of code bins 𝑁𝐶𝐴 and frequency bins 𝑁𝑓. Then, the matrix is processed by the CEI neural network and the 𝑁𝐶𝐴 /𝑁𝑓 possibilities are output. Finally, the most likely parameter block including the right parameters is located and the possible parameter space is significantly reduced. In the first dwell, the received signal is correlated with all the possible parameters to generate the correlation matrix, whose size is the product of code bins 𝑁𝐶𝐴 and frequency bins 𝑁𝑓. Then, the matrix is processed by the CEI neural network and the 𝑁𝐶𝐴 /𝑁𝑓 possibilities are output. Finally, the most likely parameter block including the right parameters is located and the possible parameter space is significantly reduced. In the ﬁrst dwell, the received signal is correlated with all the possible parameters to generate the correlation matrix, whose size is the product of code bins NCA and frequency bins Nf . Then, the matrix is processed by the CEI neural network and the NCA /Nf possibilities are output. Finally, the most likely parameter block including the right parameters is located and the possible parameter space is signiﬁcantly reduced. Sensors 2018, 18, 1482 4 of 11 In the second dwell, a long coherent integration is generated by correlating between the preferred hypothesis parameters and the received signal. The most possible parameters are detected by the MAX/TC method based on the results of the high-quality correlation. 2.3. Correlation Envelope Identify Network 2.3. Correlation Envelope Identify Network GPS acquisition is a two-dimensional code/Doppler correlation, and the correlation can be regarded as a grayscale “image” whose brightness represents its correlation value. When local code and Doppler are aligned with the satellite signal, there is a peak in the correlation and there is a bright block in the “image”. However, due to the weak signal, the correlation peak is not obvious, and the block is not bright enough. Conventional acquisition decision strategies only recognize the correlation peak and ignore the important envelope characteristic around the correlation peak as shown in Figure 4. In this article, the aim of the proposed neural network decision strategy is to recognize the envelope (i.e., bright block) in the “image”. GPS acquisition is a two-dimensional code/Doppler correlation, and the correlation can be regarded as a grayscale “image” whose brightness represents its correlation value. When local code and Doppler are aligned with the satellite signal, there is a peak in the correlation and there is a bright block in the “image”. However, due to the weak signal, the correlation peak is not obvious, and the block is not bright enough. Conventional acquisition decision strategies only recognize the correlation peak and ignore the important envelope characteristic around the correlation peak as shown in Figure 4. In this article, the aim of the proposed neural network decision strategy is to recognize the envelope (i.e., bright block) in the “image”. Figure 4. Cross-ambiguity function (CAF) envelope feature. Figure 4. Cross-ambiguity function (CAF) envelope feature. Figure 4. Cross-ambiguity function (CAF) envelope feature. Figure 4. Cross-ambiguity function (CAF) envelope feature. The input data of the proposed network is the result of the short integration correlation within the whole hypothesis parameter space, whose size is 𝑁𝐶𝐴∙𝑁𝑓. Considering the complexity and graininess of the CEI neural network, correlation generated with 𝑁𝑓∙𝑁𝑓 parameters is regarded as a basic block, the CAF envelope is deemed to exist within one of the basic blocks. Thus, 𝑁𝐶𝐴/𝑁𝑓 output ports are included in our network, and each output port represents the probability that the CAF envelope exists within the corresponding basic block. The structure of the envelope identification neural network is demonstrated in Figure 5. The convolution layer extracts data features by using the convolution operation between input data and middle layer data. 2.3. Correlation Envelope Identify Network 2.3. Correlation Envelope Identify Network In this article, we use three convolutional layers to extract the correlation peak due to the CNN powerful noise filtering capability, whose number is the balance of precision and computation overhead. We also speed up the training convergence by batch normalization layers in the first and second convolutional layers. It is noteworthy that there is no fully connected layer in our network: the reason for this we will discuss in the next section. The detailed design parameters are described in Table 1. The stride controls how the filter convolves around the input volume, and the stride is set in a way to ensure that the output volume is an integer and not a fraction. The size and number of convolution kernel are set in a way to ensure that the network obtains a high precision in the practical test. Finally, padding is the added data around the feature map to control the size of the output. The input data of the proposed network is the result of the short integration correlation within the whole hypothesis parameter space, whose size is NCA·Nf . Considering the complexity and graininess of the CEI neural network, correlation generated with Nf ·Nf parameters is regarded as a basic block, the CAF envelope is deemed to exist within one of the basic blocks. Thus, NCA/Nf output ports are included in our network, and each output port represents the probability that the CAF envelope exists within the corresponding basic block. The structure of the envelope identiﬁcation neural network is demonstrated in Figure 5. The convolution layer extracts data features by using the convolution operation between input data and middle layer data. In this article, we use three convolutional layers to extract the correlation peak due to the CNN powerful noise ﬁltering capability, whose number is the balance of precision and computation overhead. We also speed up the training convergence by batch normalization layers in the ﬁrst and second convolutional layers. It is noteworthy that there is no fully connected layer in our network: the reason for this we will discuss in the next section. The detailed design parameters are described in Table 1. The stride controls how the ﬁlter convolves around the input volume, and the stride is set in a way to ensure that the output volume is an integer and not a fraction. 2.2. Double Dwell Acquisition Scheme 2.2. Double Dwell Acquisition Scheme 2 2 Double Dwell Acquisition Scheme 1 D D D P P P  (2) 1 2 (1 ) C M C M F D F D F P P P P P − =  +  (3) 1 1 2 (1 ) (1 ) (1 ) C M M C M M D D F D D P P P P P P  − −  − = + − (4) –(4) show that the acquisition performance of the proposed algorithm is dominated f it d d ll if th iti 𝑃𝐶i hi h h Ob i l th PD = PC D × PM D1 (2) PF = PC D × PM F1 + (1 −PC D) × PM F2 (3) PM = PC D × (1 −PM D1 −PM F1) + (1 −PC D) × (1 −PM D2) (4) (2) (2) (3) (4) by the performance of its second dwell if the recognition accuracy 𝑃𝐷 𝐶 is high enough. Obviously, the second dwell acquisition has a better performance due to a smaller hypothesis parameter space and the results of the high-quality correlation. The overall computation overhead of the proposed method is dominated by its first dwell; and, therefore, promoting the recognition probability and decreasing computation overhead in its first dwell are both efficient methods to improve the acquisition performance. Equations (2)–(4) show that the acquisition performance of the proposed algorithm is dominated by the performance of its second dwell if the recognition accuracy PC D is high enough. Obviously, the second dwell acquisition has a better performance due to a smaller hypothesis parameter space and the results of the high-quality correlation. The overall computation overhead of the proposed method is dominated by its ﬁrst dwell; and, therefore, promoting the recognition probability and decreasing computation overhead in its ﬁrst dwell are both efﬁcient methods to improve the acquisition performance. 5 of 11 5 of 11 Sensors 2018, 18, 1482 Sensors 2018, 18, x FOR 2.3.1. The Binarized Convolutional Layer 2.3.1. The Binarized Convolutional Layer CNN has shown significant performance improvements in several applications including characteristics identification and computer vision. However, CNN-based recognitions require large amounts of memory and computational power. Therefore, they are unsuitable for smaller devices such as cell phones and embedded electronics [11]. BCNN is a network that effectively reduces the computational complexity and memory overhead. During the forward pass, BCNN drastically replaces most arithmetic calculations with bit-wise operations, which substantially improves the power-efficiency [9]. During the training, the weight is binarized to +1 or −1 for the forward pass and the calculation of gradient during the backpropagation. When updating the weight, the calculation uses the floating-point format weight. After the update, the weight will be constrained to the range of [−1, +1] by clipping. As shown in Equation (5), most calculation of the traditional CNN inference is matrix multiplication, which causes huge computation overhead. With +1/−1 weights, BCNN converts the multiplication into the XOR operation to reduce the computation overhead. 𝑊00 ⋯ 𝑊0𝑛 wb=𝑆𝑖𝑔𝑛(𝑤) ={ +1,𝑖𝑓 𝑥≥0 −1 𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒 𝑛 (5) CNN has shown signiﬁcant performance improvements in several applications including characteristics identiﬁcation and computer vision. However, CNN-based recognitions require large amounts of memory and computational power. Therefore, they are unsuitable for smaller devices such as cell phones and embedded electronics [11]. BCNN is a network that effectively reduces the computational complexity and memory overhead. During the forward pass, BCNN drastically replaces most arithmetic calculations with bit-wise operations, which substantially improves the power-efﬁciency [9]. During the training, the weight is binarized to +1 or −1 for the forward pass and the calculation of gradient during the backpropagation. When updating the weight, the calculation uses the ﬂoating-point format weight. After the update, the weight will be constrained to the range of [−1, +1] by clipping. As shown in Equation (5), most calculation of the traditional CNN inference is matrix multiplication, which causes huge computation overhead. With +1/−1 weights, BCNN converts the multiplication into the XOR operation to reduce the computation overhead. [ 𝑊𝑚0 ⋯ 𝑊𝑚𝑛 ] [𝑥0 𝑥1, , 𝑥𝑛] [∑ ±𝑥𝑖 𝑖=0 ] 2.3.2. The Batch Normalization Layer During each stochastic gradient descent (SGD), the corresponding activation is normalized by the mini-batch. Therefore, the mini-batch data has a mean value of 0 and a variance of 1, which is called batch normalization. In deep networks, internal covariant shift (ICS) makes training slow and complex. 2.3. Correlation Envelope Identify Network 2.3. Correlation Envelope Identify Network The size and number of convolution kernel are set in a way to ensure that the network obtains a high precision in the practical test. Finally, padding is the added data around the feature map to control the size of the output. Table 1. Envelope recognition binarized convolution neural network (BCNN) parameters. Table 1. Envelope recognition binarized convolution neural network (BCNN) parameters. Table 1. Envelope recognition binarized convolution neural network (BCNN) parameters. Name Layer Stride Convolution Kernel Number Padding Convolution Kernel Size Convolution Layer 1 1 32 2 5 × 5 2 1 16 1 3 × 3 3 16 1 0 16 × 16 Batch Normalizatio n Layer Epsilon Gamma Beta 1 0.0004 1 0 2 0.0004 1 0 Relu Layer Formal 1 Relu(x) = max(x,0) 2 Table 1. Envelope recognition binarized convolution neural network (BCNN) parameters. Name Layer Stride Convolution Kernel Number Padding Convolution Kernel Size Convolution Layer 1 1 32 2 5 × 5 2 1 16 1 3 × 3 3 16 1 0 16 × 16 Batch Normalization Layer Epsilon Gamma Beta 1 0.0004 1 0 2 0.0004 1 0 Relu Layer Formal 1 Relu(x) = max(x,0) 2 6 of 11 11 Sensors 2018, 18, 1482 Conv1 Conv2 Conv3 5 5 32 16 1 1 3 3 Stride of 2 Stride of 1 Correlation Results 1616368 Input data Layer1 Layer2 Output Feature Map 161636832 Feature Map 161636816 Probability 102311 Stride of 0 16 16 Figure 5. Envelope recognition by BCNN. Figure 5. Envelope recognition by BCNN. Input data Figure 5. Envelope recognition by BCNN. Figure 5. Envelope recognition by BCNN. 2.3.1. The Binarized Convolutional Layer 2.3.1. The Binarized Convolutional Layer Batch normalization takes a step towards reducing the ICS, and dramatically accelerates the training of deep neural networks It accomplishes this goal via a normalization step that fixes the   W00 · · · W0n ... ... ... Wm0 · · · Wmn  × [x0, x1, . . . , xn] wb = Sign(w) = { +1, if x ≥0 −1, otherwise −−−−−−−−−−−−−→ h ∑ n i=0 ± xi i (5) 2.3.2. The Batch Normalization Layer (5) p complex. Batch normalization takes h i i f d l k 2.3.2. The Batch Normalization Layer 2.3.4. The Training Process In this work, 20,000 randomly generated correlation results are used as input data for training, and 8000 correlation results are used as the test dataset. This work uses the SGD to update the network weight. During the forward pass and the calculation of gradient, the weight is binarized to +1 or −1, while the weight is updated in ﬂoating point form. The weights are initialized using the Glorot weight initialization method, which is proposed in work [14]. The pseudo code of the training process is shown below. Algorithm 1. Pseudocode of the training process. Input: Training period p; Output: Network’s test accuracy; network’s test loss 1: Let datasettraining denote the signal data generated for training; 2: Let datasettest denote the signal data generated for test; 3: Let Wreal denote the network’s weight which initialized randomly; 4: for i = 1 to p do Wbinary = Binarized (Wreal); loss = ForwardProcess (datasettraining, Wbinary); grad = BackforwardProcess (loss, Wreal); Wnew_real = UpdateWeight (Wreal, grad); Wbinary = ClipWhileExceed (Wnew_real); 5: Wbinary = Binarized (Wreal); 6: [accuracy, loss] = TestForward (Wbinary, datasettest); 7: return accuracy and loss Algorithm 1. Pseudocode of the training process. Output: Network’s test accuracy; network’s test loss 1: Let datasettraining denote the signal data generated for training; g 2: Let datasettest denote the signal data generated for test; test g g ; 3: Let Wreal denote the network’s weight which initialized randomly; 4: for i = 1 to p do Wbinary = Binarized (Wreal); loss = ForwardProcess (datasettraining, Wbinary); grad = BackforwardProcess (loss, Wreal); Wnew_real = UpdateWeight (Wreal, grad); Wbinary = ClipWhileExceed (Wnew_real); 5: Wbinary = Binarized (Wreal); 6: [accuracy, loss] = TestForward (Wbinary, datasettest); 7: return accuracy and loss 3: Let Wreal denote the network’s weight which initialized randomly; 4: for i = 1 to p do Wbinary = Binarized (Wreal); loss = ForwardProcess (datasettraining, Wbinary); grad = BackforwardProcess (loss, Wreal); Wnew_real = UpdateWeight (Wreal, grad); Wbinary = ClipWhileExceed (Wnew_real); y 5: Wbinary = Binarized (Wreal); y 6: [accuracy, loss] = TestForward (Wbinary, datasettest); 7: return accuracy and loss To decrease the computation overhead in the ﬁrst dwell, 2 ms coherent integration is adopted for the envelope recognition. In order to make the trained neural network suitable for different GPS signal scenes, the training set in our study is GPS signals with random power, Doppler frequency, and code phase. 2.3.3. The Rectiﬁed Linear Unit and Fully Connected Layer We refer to neurons with this nonlinearity as rectiﬁed linear units (ReLUs). The rectiﬁer function is rectiﬁer (x) = max (0, x). The activation function allows a network to easily obtain sparse representations [13]. Usually, a typical CNN network contains a few fully connected layers. Compared with the convolutional layer, the weight of the fully connected layer is not shared. The parameter size grows with the feature map size in a square relationship. In the problem of correlation envelope identiﬁcation, the size of the feature map is one hundred thousand orders of magnitude, which will lead to huge memory overhead. In this work, we directly use the convolution layer instead of the fully connected layer as the output layer because the output probability is equivalent to the relative value of the feature vector in envelope identiﬁcation, which signiﬁcantly reduces the weight size and computation complexity. p complex. Batch normalization takes th t i i f d l t k 2.3.2. The Batch Normalization Layer the training of deep neural networks. It accomplishes this goal via a normalization step that fixes the mean values and variances of layer inputs. Batch normalization also has a beneficial effect on the gradient flow through the network, by reducing the dependence of gradients on the scale of the parameters or of their initial values [12]. Furthermore, batch normalization can reduce the requirement of dropout operations. The batch normalization’s formula is shown below (Equation (6)). Here, β is the bias constant to be trained and the initial value is 0; ε is a very small value used to prevent the denominator During each stochastic gradient descent (SGD), the corresponding activation is normalized by the mini-batch. Therefore, the mini-batch data has a mean value of 0 and a variance of 1, which is called batch normalization. In deep networks, internal covariant shift (ICS) makes training slow and complex. Batch normalization takes a step towards reducing the ICS, and dramatically accelerates the training of deep neural networks. It accomplishes this goal via a normalization step that ﬁxes the mean values and variances of layer inputs. Batch normalization also has a beneﬁcial effect on the gradient ﬂow through the network, by reducing the dependence of gradients on the scale of the parameters or of their initial values [12]. Furthermore, batch normalization can reduce the requirement of dropout operations. The batch normalization’s formula is shown below (Equation (6)). Here, β is the bias Sensors 2018, 18, 1482 7 of 11 constant to be trained and the initial value is 0; ε is a very small value used to prevent the denominator from zero, and the initial value is 0.0001; x is the current batch normalization layer’s input, and y is the output. During the training, µ and σ2 are deﬁned to be the mean and variance of the current input mini-batch, and during the inferring, they are replaced with average statistics over the training data. y = x −µ √ σ2 + ε + β (6) (6) 2.3.3. The Rectiﬁed Linear Unit and Fully Connected Layer 3. Performance Validation 3. Performance Validation Several field tests were conducted to evaluate the performance of the proposed CEI neural network and its acquisition method, and one of them is selected in this article for illustration purposes. The parameters of correlation and signal model are presented in Table 2. Considering the environment of weak signals, the code bin is 1/16 chip for high sensitivity GPS acquisition. In the first dwell, the Doppler bin is 500 Hz and the coherent integration time is 2 ms for envelope identification. In the second dwell, the Doppler bin is narrowed to 100 Hz and the coherent integration time is 10ms for acquisition. The length of GPS navigation data is 20 ms. When the coherent integration time is no more than 10 ms, navigation data transition has a small influence on the envelope characteristics of GPS signals. This research uses the coherent integration time of 2 ms and 10 ms to avoid the effect of bit jump on the acquisition results. Therefore, navigation data transition does not affect our proposed acquisition algorithm. The CEI neural network was trained by Adam with the batch size of 32 examples whose data was from the signal model. The learning rate was initialized at 0.001 and decayed every epoch where decay rate was 0.98. We trained the network for roughly 30 epochs through the training set which took about one day on one NVIDIA Tesla K80 Several ﬁeld tests were conducted to evaluate the performance of the proposed CEI neural network and its acquisition method, and one of them is selected in this article for illustration purposes. The parameters of correlation and signal model are presented in Table 2. Considering the environment of weak signals, the code bin is 1/16 chip for high sensitivity GPS acquisition. In the ﬁrst dwell, the Doppler bin is 500 Hz and the coherent integration time is 2 ms for envelope identiﬁcation. In the second dwell, the Doppler bin is narrowed to 100 Hz and the coherent integration time is 10 ms for acquisition. The length of GPS navigation data is 20 ms. When the coherent integration time is no more than 10 ms, navigation data transition has a small inﬂuence on the envelope characteristics of GPS signals. This research uses the coherent integration time of 2 ms and 10 ms to avoid the effect of bit jump on the acquisition results. 2.3.4. The Training Process With the trained weights, a set of correlation results of −146 dBm GPS signal with 2 ms coherent integration is inferenced with the envelope identiﬁcation and its feature map is shown in Figure 6. Obviously, the input correlation data is a low-quality data set, and its regularity cannot be recognized. 8 of 11 8 of 11 Sensors 2018, 18, 1482 S 2018 18 FO With the noise reduction of our proposed network, the local feature occurs in its last layer feature map, which reveals the essential reason that the proposed network can recognize the envelope from low-quality correlation results. feature map, which reveals the essential reason that the proposed network can recognize the envelope from low-quality correlation results. With the noise reduction of our proposed network, the local feature occurs in its last layer feature map, which reveals the essential reason that the proposed network can recognize the envelope from low-quality correlation results. feature map, which reveals the essential reason that the proposed network can recognize the envelope from low-quality correlation results. Figure 6 An example of the feature map in CEI network Figure 6. An example of the feature map in CEI network. Fi 6 A l f th f t i CEI t k Figure 6. An example of the feature map in CEI network. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Signal Power (dBm) PCD (%) −143 98.7 −144 97.3 −145 96.0 −146 94.2 −147 89.5 −148 82.4 Signal Power (dBm) PCD (%) −143 98.7 −144 97.3 −145 96.0 −146 94.2 −147 89.5 −148 82.4 Signal Power (dBm) PCD (%) −143 98.7 −144 97.3 −145 96.0 −146 94.2 −147 89.5 −148 82.4 Signal Power (dBm) PCD (%) −143 98.7 −144 97.3 −145 96.0 −146 94.2 −147 89.5 −148 82.4 For simplicity, the MAX/TC acquisition method was adopted in the second dwell, whose coherent integration was 10 ms. Since the conventional double dwell acquisition method uses the second dwell to verify the ﬁrst dwell acquisition result, it leads to a low false alarm rate with weak signals. Thus, the conventional MAX/TC acquisition method was validated simultaneously by the same experimental environment, which adopts the 10 ms coherent integration time. Results of both methods are shown in Figure 7. For simplicity, the MAX/TC acquisition method was adopted in the second dwell, whose coherent integration was 10 ms. Since the conventional double dwell acquisition method uses the second dwell to verify the first dwell acquisition result, it leads to a low false alarm rate with weak signals. Thus, the conventional MAX/TC acquisition method was validated simultaneously by the same experimental environment, which adopts the 10ms coherent integration time. Results of both methods are shown in Figure 7. -148 -147 -146 -145 -144 -143 0 20 40 60 80 100 Probability (%) Signal Power Level (dBm) Detection (DD/CEI) False Alarm (DD/CEI) Missed (DD/CEI) Detection (MAX/TC) False Alarm (MAX/TC) Missed (MAX/TC) acquisition sensitivity of MAX/TC acquisition sensitivity of DD/CEI Figure 7. Comparison with Max selection/threshold crossing (MAX/TC). Figure 7. Comparison with Max selection/threshold crossing (MAX/TC). Figure 7. Comparison with Max selection/threshold crossing (MAX/TC). Figure 7. Comparison with Max selection/threshold crossing (MAX/TC). In the test, the proposed DD/CEI acquisition method adopted the same constant false alarm threshold criterion as the conventional method. Thus, the false alarm probability of this method was similar to the conventional one. When the GPS satellite signal power was −143 dBm, the detection probability of this method and the traditional method were both close to 100%. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. When the GPS satellite signal power was −146 dBm, the detection probability of the proposed method was 82%, which was double of the traditional method. Without loss of generality, the definition of acquisition sensitivity was the satellite power level in which its detection probability was equal to missed detection probability, that was the curve cross point of detection probability vs. missed detection probability. Therefore, the DD/CEI scheme at the condition of 2 ms integration achieved −147.6 dBm which was 2 dB improvement when compared to the MAX/TC acquisition method. Benefit from the deep optimization on the proposed DD/CEI the computation overhead brought In the test, the proposed DD/CEI acquisition method adopted the same constant false alarm threshold criterion as the conventional method. Thus, the false alarm probability of this method was similar to the conventional one. When the GPS satellite signal power was −143 dBm, the detection probability of this method and the traditional method were both close to 100%. When the GPS satellite signal power was −146 dBm, the detection probability of the proposed method was 82%, which was double of the traditional method. Without loss of generality, the deﬁnition of acquisition sensitivity was the satellite power level in which its detection probability was equal to missed detection probability, that was the curve cross point of detection probability vs. missed detection probability. Therefore, the DD/CEI scheme at the condition of 2 ms integration achieved −147.6 dBm which was 2 dB improvement when compared to the MAX/TC acquisition method. Benefit from the deep optimization on the proposed DD/CEI, the computation overhead brought by CEI neural network is from the addition of binarized convolution. In this network, the current feature map value is from the convolution of last feature map. Thus, the addition number of each feature map is the multiplication of feature map size, convolution kernel size, and the convolution kernel number. The addition number of our network is given as follows. Beneﬁt from the deep optimization on the proposed DD/CEI, the computation overhead brought by CEI neural network is from the addition of binarized convolution. In this network, the current feature map value is from the convolution of last feature map. Thus, the addition number of each feature map is the multiplication of feature map size, convolution kernel size, and the convolution kernel number. The addition number of our network is given as follows. 3. Performance Validation 3. Performance Validation Therefore, navigation data transition does not affect our proposed acquisition algorithm. The CEI neural network was trained by Adam with the batch size of 32 examples whose data was from the signal model. The learning rate was initialized at 0.001 and decayed every epoch where decay rate was 0.98. We trained the network for roughly 30 epochs through the training set, which took about one day on one NVIDIA Tesla K80. Table 2. Signal and correlation parameters. Parameter Value Sample Frequency (MHz) 16.368 Noise Bandwidth (MHz) 4.092 Doppler Bin (Hz) 500/100 Frequency Channel (Nf) 16 Code Bin (Chip) 1/16 Code Channel (NCA) 16,368 Table 2. Signal and correlation parameters. Parameter Value Sample Frequency (MHz) 16.368 Noise Bandwidth (MHz) 4.092 Doppler Bin (Hz) 500/100 Frequency Channel (Nf) 16 Code Bin (Chip) 1/16 Code Channel (NCA) 16,368 Table 2 Signal and correlation parameters Table 2. Signal and correlation parameters. The training data was generated uniformly, with its signal power typically ranging from −143 dBm to −148 dBm. The accuracy of training is 94.17%. To check the performance in the real environment, the proposed method is validated by a field test in which the signal data was collected from Spirent GPS Constellation Simulator. With 2ms integration in the first dwell, the recognition accuracy of the proposed CEI BCNN is shown in Table 3. It shows that the recognition accuracy is high enough for the second dwell, which is 98.7%@−143 dBm and 82.4%@−148 dBm. It also can be seen that the network maintains a certain accuracy under different noises, which means the network has good generalization ability. The training data was generated uniformly, with its signal power typically ranging from −143 dBm to −148 dBm. The accuracy of training is 94.17%. To check the performance in the real environment, the proposed method is validated by a ﬁeld test in which the signal data was collected from Spirent GPS Constellation Simulator. With 2 ms integration in the ﬁrst dwell, the recognition accuracy of the proposed CEI BCNN is shown in Table 3. It shows that the recognition accuracy is high enough for the second dwell, which is 98.7%@−143 dBm and 82.4%@−148 dBm. It also can be seen that the network maintains a certain accuracy under different noises, which means the network has good generalization ability. 9 of 11 9 of 11 Sensors 2018, 18, 1482 Sensors 2018, 18, x FOR Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. 2 1 add k k k f CA k S KS KN FN N N = =      (7) Sadd = 3 ∑ k=1 KSk 2 · KNk · FNk · Nf · NCA (7) (7) (7) Sensors 2018, 18, 1482 where 2 KS is th 10 of 11 FN is where KSk2 is the kth convolution kernel size, KNk is the kth convolution kernel number, FNk is value number of the kth feature map, and Sadd is about 5k·Nf ·NCA. value number of the kth feature map, and add S is about 5𝑘∙𝑁𝑓∙𝑁𝐶𝐴. In the traditional GPS acquisition, its computation overhead is from the integration of signal, in which the multiplication is replaced XOR Therefore the computation overhead of integration is the where KSk2 is the kth convolution kernel size, KNk is the kth convolution kernel number, FNk is value number of the kth feature map, and Sadd is about 5k·Nf ·NCA. value number of the kth feature map, and add S is about 5𝑘∙𝑁𝑓∙𝑁𝐶𝐴. In the traditional GPS acquisition, its computation overhead is from the integration of signal, in which the multiplication is replaced XOR Therefore the computation overhead of integration is the In the traditional GPS acquisition, its computation overhead is from the integration of signal, in which the multiplication is replaced XOR. Therefore, the computation overhead of integration is the addition and the computation overhead of the coherent integration is expressed as follows: which the multiplication is replaced XOR. Therefore, the computation overhead of integration is the addition and the computation overhead of the coherent integration is expressed as follows: s int CA add f S F T N N =    (8) Sadd = Fs · Tint · NCA · Nf (8) 𝑖𝑛𝑡 is the integration time, and add S is 1.6M ∙𝑁𝑓∙𝑁𝐶𝐴 for typical (8) pical where Fs is the sampling rate and Tint is the integration time, and Sadd is 1.6M·Nf ·NCA for typical weak signal acquisition. Therefore, the computation overhead of integration in the traditional GPS acquisition is far more than the proposed CEI neural network. weak signal acquisition. Therefore, the computation overhead of integration in the traditional GPS acquisition is far more than the proposed CEI neural network. Table 3. Performance of BCNN decision policy. Table 3. Performance of BCNN decision policy. Moreover, the computation overhead of the second dwell signal integration can be ignored h d t it fi t d ll th d d ll t b i l 1/1023 f th fi t Moreover, the computation overhead of the second dwell signal integration can be ignored when compared to its ﬁrst dwell as the second dwell parameter number is only 1/1023 of the ﬁrst dwell. Therefore, the total computation overhead of the proposed method is mainly decided by the ﬁrst dwell, which is only 1/5 of conventional MAX/TC method as shown in Figure 8. when compared to its first dwell as the second dwell parameter number is only 1/1023 of the first dwell. Therefore, the total computation overhead of the proposed method is mainly decided by the first dwell, which is only 1/5 of conventional MAX/TC method as shown in Figure 8. 10msFSNCANf 2msFSNCANf Sensitivity: -147.6dBm Sensitivity: -146dBm Sensitivity: -143dBm DD/CEI MAX/TC (long) MAX/TC (short) Computation Overhead CEI/CNN 5kNCANf 1st Dwell 2msFSNCANf 2nd Dwell 10msFSNCA*Nf/1023 Figure 8. Computation overhead. Figure 8. Computation overhead. Sensitivity: -147.6dBm Sensitivity: -143dBm Figure 8 Computation overhead Figure 8. Computation overhead. Predictably, the proposed method will achieve a better acquisition performance if a longer integration is used for its second dwell, which will not significantly increase the computation overhead due to its small hypothesis parameter space. Predictably, the proposed method will achieve a better acquisition performance if a longer integration is used for its second dwell, which will not signiﬁcantly increase the computation overhead due to its small hypothesis parameter space. 4. Conclusions 4. Conclusions conceived and designed the experiments, Z.W., Y.Z., J.Y., H.Z., W.D., M.W., L.H. and B. L. performed the experiments, analyzed the data, and wrote the article. Funding: This research was funded by National Natural Science Foundation of China (Grant No. 61404028) and National High Technology Research and Development Program of China (No. 2015AA016601). Funding: This research was funded by National Natural Science Foundation of China (Grant No. 61404028) and National High Technology Research and Development Program of China (No. 2015AA016601). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions 4. Conclusions This article proposed a novel DD/CEI neural network for high sensitivity GPS acquisition to reduce the acquisition computation overhead and promote the acquisition sensitivity. The hypothesis parameter space was significantly narrowed by the proposed neural network in the first dwell. Then, the long integration and conventional MAX/TC decision strategy were applied to the narrowed hypothesis space in its second dwell to improve the acquisition accuracy. Since the short integration was conducted on the initial hypothesis space, the computation overhead was significantly decreased. Moreover, the extra computation overhead brought by neural network was greatly reduced by weight binarization and structure optimization. Finally, field tests were conducted to evaluate the performance of the proposed algorithm. Experiment results showed that the proposed method could promote the acquisition sensitivity by 2 dB when compared with the MAX/TC under the same specification, and the computation overhead of the proposed algorithm was only around 1/5 of the MAX/TC under the same sensitivity This article proposed a novel DD/CEI neural network for high sensitivity GPS acquisition to reduce the acquisition computation overhead and promote the acquisition sensitivity. The hypothesis parameter space was signiﬁcantly narrowed by the proposed neural network in the ﬁrst dwell. Then, the long integration and conventional MAX/TC decision strategy were applied to the narrowed hypothesis space in its second dwell to improve the acquisition accuracy. Since the short integration was conducted on the initial hypothesis space, the computation overhead was signiﬁcantly decreased. Moreover, the extra computation overhead brought by neural network was greatly reduced by weight binarization and structure optimization. Finally, ﬁeld tests were conducted to evaluate the performance of the proposed algorithm. Experiment results showed that the proposed method could promote the acquisition sensitivity by 2 dB when compared with the MAX/TC under the same speciﬁcation, and the computation overhead of the proposed algorithm was only around 1/5 of the MAX/TC under the same sensitivity. 11 of 11 Sensors 2018, 18, 1482 Author Contributions: All authors contributed considerably to this article. The article was conceived and structured by all the authors. Z.W., J.Y. and L.S. conceived and designed the experiments, Z.W., Y.Z., J.Y., H.Z., W.D., M.W., L.H. and B. L. performed the experiments, analyzed the data, and wrote the article. Author Contributions: All authors contributed considerably to this article. The article was conceived and structured by all the authors. Z.W., J.Y. and L.S. References 1. Corazza, G.E. On the MAX/TC criterion for code acquisition and its application to DS-SSMA systems. IEEE Trans. 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In Proceedings of the Position Location and Navigation Symposium, Monterey, CA, USA, 26–29 April 2004; pp. 461–470. [CrossRef] 5. Ziedan, N.I.; Garrison, J.L. Unaided acquisition of weak GPS signals using circular correlation or double-block zero padding. In Proceedings of the Position Location and Navigation Symposium, Monterey, CA, USA, 26–29 April 2004; pp. 461–470. [CrossRef] 6. Yang, C.; Han, S. Block-accumulating coherent integration over extended interval (BACIX) for weak GPS signal acquisition. In Proceedings of the GNSS 19th International Technical Meeting of the Satellite Division, Fort Worth, TX, USA, 26–29 September 2006; pp. 2427–2440. 6. Yang, C.; Han, S. Block-accumulating coherent integration over extended interval (BACIX) for weak GPS signal acquisition. In Proceedings of the GNSS 19th International Technical Meeting of the Satellite Division, Fort Worth, TX, USA, 26–29 September 2006; pp. 2427–2440. 7. Zhang, W.; Ghogho, M. Improved fast modiﬁed double-block zero-padding (FMDBZP) algorithm for weak GPS signal acquisition. In Proceedings of the 18th European Signal Processing Conference, Aalborg, Denmark, 23–27 August 2010; pp. 1617–1621. 8. Krizhevsky, A.; Sutskever, I.; Hinton, G.E. ImageNet classiﬁcation with deep convolutional neural networks. In Proceedings of the International Conference on Neural Information Processing Systems, Lake Tahoe, NV, USA, 3–6 December 2012; Volume 60, pp. 1097–1105. [CrossRef] 9. Courbariaux, M.; Hubara, I.; Soudry, D.; El-Yaniv, R.; Bengio, Y. Binarized neural networks: Training deep neural networks with weights and activations constrained to +1 or −1. arXiv, 2016. 0. Kay, S.M. Volume II: Detection Theory. References In Fundamentals of Statistical Signal Processing; PTR Prentice H Upper Saddle River, NJ, USA, 1993; pp. 465–466. 11. Rastegari, M.; Ordonez, V.; Redmon, J.; Farhadi, A. Xnor-net: Imagenet classiﬁcation using binary convolutional neural networks. In Proceedings of the European Conference on Computer Vision, Amsterdam, The Netherlands, 11–14 October 2016; pp. 525–542. [CrossRef] 12. Ioffe, S.; Szegedy, C. Batch Normalization: Accelerating Deep Network Training by Reducing Internal Covariate Shift. In Proceedings of the International Conference on Machine Learning, Lille, France, 6–11 July 2015; Volume 37, pp. 448–456. 13. Glorot, X.; Bordes, A.; Bengio, Y.; Glorot, X.; Bordes, A.; Bengio, Y. Deep Sparse Rectiﬁer Neural Networks. In Proceedings of the International Conference on Artiﬁcial Intelligence and Statistics, Fort Lauderdale, FL, USA, 11–13 April 2011; Volume 15, pp. 315–323. 14. Glorot, X.; Bengio, Y. Understanding the Difﬁculty of Training Deep Feedforward Neural Networks. J. Mach. Learn. Res. 2010, 9, 249–256. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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https://openalex.org/W2978143923	https://centaur.reading.ac.uk/86553/1/The%20complete%20plastome%20of%20Daphne%20laureola%20L%20Thymelaeaceae.pdf	English	null	The complete plastome of <i>Daphne laureola</i> L. (Thymelaeaceae)	Mitochondrial DNA. Part B. Resources	2,019	cc-by	1,438	The complete plastome of Daphne laureola L. (Thymelaeaceae) Published Version Creative Commons: Attribution 4.0 (CC-BY) Creative Commons: Attribution 4.0 (CC-BY) Könyves, K., Yooprasert, S., Culham, A. ORCID: https://orcid.org/0000-0002-7440-0133 and David, J. (2019) The complete plastome of Daphne laureola L. (Thymelaeaceae). Mitochondrial DNA Part B, 4 (2). pp. 3364- 3365. ISSN 2380-2359 doi: https://doi.org/10.1080/23802359.2019.1674201 Available at https://centaur.reading.ac.uk/86553/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1080/23802359.2019.1674201 To link to this article DOI: http://dx.doi.org/10.1080/23802359.2019.1674201 It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . Published version at: http://dx.doi.org/10.1080/23802359.2019.1674201 To link to this article DOI: http://dx.doi.org/10.1080/23802359.2019.1674201 p g o link to this article DOI: http://dx.doi.org/10.1080/23802359.2019.167420 Publisher: Taylor & Francis All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur Mitochondrial DNA Part B Resources ISSN: (Print) 2380-2359 (Online) Journal homepage: https://www.tandfonline.com/loi/tmdn20 Central Archive at the University of Reading Reading’s research outputs online Mitochondrial DNA Part B ABSTRACT ARTICLE HISTORY Received 10 September 2019 Accepted 23 September 2019 The complete plastome sequence for the type species of Daphne, Daphne laureola, was assembled and annotated in this study. The total length of the D. laureola plastome is 171,613 bp and comprises the large single copy (LSC) spanning 85,316 bp, the small single copy (SSC) spanning 2855 bp, and two inverted repeat (IR) regions each of 41,721bp length. The SSC contains only two genes: ndhF and rpl32. This sequence extends the list of Thymelaeaceae plastomes to be used in future systematic stud- ies of the family and is the first European species to be sampled. KEYWORDS Daphne; Thymelaeaceae; plastome; SSC the IR and identifying the point where the mapped reads diverged. Daphne is a genus of small shrubs, popular with gardeners for their fragrant flowers. The systematics of Daphne, and its relationship to Wikstroemia, is poorly understood (Wang et al. 2007). Daphne laureola L. is an evergreen shrub native to Europe and North Africa and is the type species of the genus. Establishing the level of genetic divergence within a phylo- genetic framework will help understand taxonomic bounda- ries within the genus and between related genera. Here we report the complete plastome sequence of D. laureola. The mean coverage of the finished assembly is 521. The complete plastome was annotated by transferring the anno- tations from D. kiusiana (KY991380) using Geneious v11.1.5 and corrected by comparing it with other published annota- tions. The D. laureola plastome sequence was aligned to 13 published Malvales plastome sequences plus Arabidopsis thaliana (outgroup) using MAFFT v7.388 (Katoh and Standley 2013). A maximum likelihood estimate of phylogeny was con- ducted with RAxML v8.2.11 (Stamatakis 2014) within Geneious v11.1.5 using model GTR þ G and 1000 boot- strap replicates. Leaf material was collected in silica gel from D. laureola grown at RHS Garden Wisley, UK (51.312695 N, 0.476724 W). The sample was further verified as D. laureola by a BLAST search using the 3’-end of ndhF. A herbarium voucher was deposited at WSY (WSY0148635). Total genomic DNA was extracted using a CTAB protocol (Doyle and Doyle 1987). Library development and 150 bp PE sequencing using Illumina HiSeq were done by Novogene Corporation (Beijing, China). Raw sequence data were deposited in SRA (accession PRJNA555571). The plastome was assembled with Fast-Plast v1.2.6 (McKain and Wilson 2017) and NovoPlasty v2.7.0 (Dierckxsens et al. 2017). ABSTRACT Fast-Plast assemblies were run with all 29.4 M reads. Reads were trimmed to remove NEB-PE adapter sequences. The Bowtie reference index was built with Malvales plastomes included in Fast-Plast. For the NovoPlasty assembly, a matK sequence of D. laureola (JN894952) was used as starting seed and memory was limited to 8 Gb. All other parameters were unchanged. The large single copy (LSC), the small single copy (SSC), and two inverted repeat (IR) regions were identified in the final Fast-Plast contig and NovoPlasty assemblies and the circular plastome was assembled by hand using Geneious v11.1.5 (http://www.geneious.com, Kearse et al. 2012). Junctions of the single copy and inverted repeat regions were confirmed by mapping the reads to the ends of The plastome sequence of D. laureola (MN201546) is 171,613 bp, comprises the LSC spanning 85,316 bp, the SSC spanning 2,855 bp, and two IR regions each of 41,721 bp length. The IRs have expanded to encompass most of the genes normally in the SSC, which contained only two genes ndhF and rpl32. This structure is congruent with the pub- lished Daphne plastomes. The Thymelaeaceae samples form a clade with D. laureola sister to the remaining Daphne species (Figure 1). CONTACT Kalman K€onyves kalmankonyves@rhs.org.uk Royal Horticultural Society Garden Wisley, Woking, Surrey GU23 6QB, UK 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MITOCHONDRIAL DNA PART B 2019, VOL. 4, NO. 2, 3364–3365 https://doi.org/10.1080/23802359.2019.1674201 MITOCHONDRIAL DNA PART B 2019, VOL. 4, NO. 2, 3364–3365 https://doi org/10 1080/23802359 Disclosure statement The authors report no conflict of interest and are alone responsible for the content and writing of the paper. The complete plastome of Daphne laureola L. (Thymelaeaceae) Kalman K€onyvesa , Sawita Yooprasertb , Alastair Culhamb and John Davida aRoyal Horticultural Society Garden Wisley Woking UK; bHerbarium School of Biological Sciences University of Reading Whiteknights UK l Society Garden Wisley, Woking, UK; bHerbarium, School of Biological Sciences, University of Reading, Whitekn aRoyal Horticultural Society Garden Wisley, Woking, UK; bHerbarium, School of Biological Sciences, University of Kálmán Könyves, Sawita Yooprasert, Alastair Culham & John David To cite this article: Kálmán Könyves, Sawita Yooprasert, Alastair Culham & John David (2019) The complete plastome of Daphnelaureola L. (Thymelaeaceae), Mitochondrial DNA Part B, 4:2, 3364-3365 To cite this article: Kálmán Könyves, Sawita Yooprasert, Alastair Culham & John David (2019) The complete plastome of Daphnelaureola L. (Thymelaeaceae), Mitochondrial DNA Part B, 4:2, 3364-3365 © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 04 Oct 2019. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmdn20 ORCID Kalman K€onyves http://orcid.org/0000-0002-8303-1544 Sawita Yooprasert http://orcid.org/0000-0002-7898-1465 Alastair Culham http://orcid.org/0000-0002-7440-0133 John David http://orcid.org/0000-0003-0557-9886 Kalman K€onyves http://orcid.org/0000-0002-8303-1544 Sawita Yooprasert http://orcid.org/0000-0002-7898-1465 Alastair Culham http://orcid.org/0000-0002-7440-0133 John David http://orcid.org/0000-0003-0557-9886 MITOCHONDRIAL DNA PART B 3365 MITOCHONDRIAL DNA PART B 3365 MITOCHONDRIAL DNA PART B 33 Figure 1. RAxML output tree with bootstrap consensus values based on 15 complete plastome sequences. The numbers at each node indicate bootstrap support. GenBank accession numbers are given in brackets. Text in bold shows the plastome developed in this study. Families of the sampled taxa are shown on the right. Figure 1. RAxML output tree with bootstrap consensus values based on 15 complete plastome sequences. The numbers at each node indicate bootstrap support. GenBank accession numbers are given in brackets. Text in bold shows the plastome developed in this study. Families of the sampled taxa are shown on the right. organization and analysis of sequence data. Bioinformatics. 28: 1647–1649. organization and analysis of sequence data. Bioinformatics. 28: 1647–1649. References Dierckxsens N, Mardulyn P, Smits G. 2017. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 45:e18. Dierckxsens N, Mardulyn P, Smits G. 2017. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 45:e18. McKain M, Wilson M. 2017. Fast-Plast: Rapid de novo assembly and fin- ishing for whole chloroplast genomes. Available from: https://github. com/mrmckain/Fast-Plast Doyle JA, Doyle JL. 1987. A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochem Bull. 19:11–16. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 30:1312–1313. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft- ware version 7: improvements in performance and usability. Mol Biol Evol. 30:772–780. Wang Y, Gilbert MG, Mathew BF, Brickell C, Nevling LI. 2007. Thymelaeaceae. In: Wu ZY, Raven PH, Hong DY, editors. Flora of China Vol 13 (Clusiaceae through Araliaceae). Beijing: Science Press and St. Louis: Missouri Botanical Garden Press; p. 213–250. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, et al. 2012. Geneious Basic: an integrated and extendable desktop software platform for the
https://openalex.org/W3161927304	https://www.macrothink.org/journal/index.php/ijch/article/download/18490/14440	English	null	Identity Lost and Regained: A Postcolonial Analysis of Don Shirley’s Dilemma in Green Book	International journal of culture and history	2,021	cc-by	6,738	Received: April 2, 2021 Accepted: April 29, 2021 Published: May 14, 2021 doi:10.5296/ijch.v8i1.18490 URL: https://doi.org/10.5296/ijch.v8i1.18490 Received: April 2, 2021 Accepted: April 29, 2021 Published: May 14, 2021 doi:10.5296/ijch.v8i1.18490 URL: https://doi.org/10.5296/ijch.v8i1.18490 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 Identity Lost and Regained: A Postcolonial Analysis of Don Shirley’s Dilemma in Green Book Kaiju Chen (Corresponding author) Center of Business Culture and Philosophy of Culture, Institute of Hermeneutics, Guangdong University of Foreign Studies Guangzhou, China E-mail: 200010931@oamail.gdufs.edu.cn Received: April 2, 2021 Accepted: April 29, 2021 Published: May 14, 2021 doi:10.5296/ijch.v8i1.18490 URL: https://doi.org/10.5296/ijch.v8i1.18490 Abstract Images of black men in movies have been depicted negatively as brutal rioters or silly clowns at large for centuries, consolidating the stereotype that black man is inferior and subordinate compared with the white. Green Book, an Oscar-winning movie in 2019, subverts the traditional images of black men and white men, successfully portraying a well-educated black intellectual and a vulgar white driver. The present study aims to decode its success in characterizing positive images of black men, and reveal the psychological activity and identity dilemma of the hero, Don Shirley. Based on Fanon’s psychoanalysis and Bhabha’s hybridity theory, this paper probes into the following questions: Firstly, what kind of psychological trauma did the black hero suffer due to his black identity? Secondly, what leads to the hybrid identity of Don Shirley? The result shows that the black hero experienced the psychological trauma of inferiority complex and delusion of becoming white. He first mistook himself as a white man, then he lost his identity and finally regained a hybrid identity. Hybrid identity is attributed to cultural assimilation and otherness, which is a common phenomenon under the backdrop of colonialism. By an in-depth analysis of Don Shirley’s http://ijch.macrothink.org 115 1. Introduction Movies closely demonstrate the interplay and convergence between social ideology and artworks. It mirrors public desires and social trends by character setting and plots, just as Siegfried Kracauer points out “films as popular art provide insight into the unconscious motivations and fantasies of a nation.” (Kracauer, 1947: 147). Furthermore, the dissemination of movies changes and shapes public opinions, imperceptibly influence people’s thinking and behavior. Concepts and ideas that films convey in the virtual world subtly influence how people view their lives (Wu Yajun, 2011:1). Thus, the study of films can reveal social ideology and problems to some degree, helping us better understand critical social issues. Notwithstanding some improvements in black men’s social status, there remain many problems unsolved in the black movie industry. Due to the profit-chasing nature of movie producers, Hollywood structurally relegates black cinema, along with other minority-audience cinemas, to marginal status (Guerrero,1993:7). The proportion of black man main character is far smaller than the white counterpart, and their remuneration contrasted are lower too (Wu Yajun, 2011:47, Fan Qingyun, 2009). Moreover, images of black men have long been solidified and otherized, portrayed as violent, evil, and brutal rioters, or as “clowns” who have an aptitude for entertainment without intelligence (Wu Yajun, 2011:47). The negative images of the black men in movies reflect problems in society, and inevitably, have a bad impact on racial equality and social harmony. There are some recent attempts to change these stereotyping figures in Hollywood, among which, Green Book is a well-received and successful film that honored as the best picture in Oscar. It subverts the traditional expectation of black and white, depicting an intelligent, elegant, and well-educated black pianist, as well as a white driver who behaves discourteously and ignorantly. The movie unfolds the subtle identity struggle of the black intellectuals, who find himself lingering between the white world and the black world, unaware of his ethnic identity. The present study aims to decode the success of Green Book in characterization and provide some insight into the construction of positive images of black men. Based on Fanon’s psychoanalysis and Bhabha’s hybridity theory, this paper probes into the following questions: Firstly, what kind of psychological trauma does the black hero suffer due to his black identity? Secondly, what leads to the hybrid identity of the major character? International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 identity dilemma, this paper hopes to shed light on racial discrimination from the perspective of post-colonialism. identity dilemma, this paper hopes to shed light on racial discrimination from the perspective of post-colonialism. Keywords: psychoanalysis, hybrid identity, post-colonialism, inferiority complex, culture Keywords: psychoanalysis, hybrid identity, post-colonialism, inferiority complex, culture International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 deep insight into black psychology and colonial issues. One of his most prominent books, Black Skin, White Marks, marks Fanon’ s huge contribution to the incisive disclosure of negroes’ psychological trauma and the interdependency between the colonizer and the colonized. Unprecedentedly, Fanon probes into the negroes’ psychological trauma due to their black identity, and discovers complicated inferiority complex and delusion of becoming white. The colonized suffered from inferiority complex due to the culture (Fanon, 1952:9). They lost confidence in the history and future of the black, believing that they are inferior to the white. The colonized thought they were battered down by tom-toms, cannibalism, intellectual deficiency, fetishism, racial defects, and they took themselves far off from their own presence (Fanon, 1952:85). This can be proven by their imitation of the white culture, and by the fact that they viewed fitting into the white society as one of the most significant pursuit in their lives. They believed that white men’s language could give them honorary citizenship. Moreover, the desire of becoming white overwhelmed them and thrust them into a neurotic situation (Yu Wenxiu, 2004: 25). The black women dreamed to marry white men and the black men dreamed to marry white women (Fanon, 1952:28). They hoped to enter the white world by marriage. Just as what happened in the dream of a Fanon’s negro patient: “I had been walking for a long time, I was extremely exhausted, I had the impression that something was waiting for me…In this second room there were white men, and I found that I too was white.” (Fanon, 1952:74). Fanon believes that his patient is suffering from an inferiority complex and he is overwhelmed by the wish to be white (Fanon, 1952:74). Fanon argues that the negroes’ psychological trauma can largely attribute to the society where the black is made inferior. To reinforce the dominant position, the colonizer assimilated and controlled negroes explicitly or implicitly, which undermined the black’ mental health and caused stunning inexplicable psychological trauma (Yu Wenxiu, 2004: 25). The colonizer controlled the negroes by monopolizing colonial discourse, framing the supreme white and the subordinate and inferior black. 2.1 Psychopathy of the Colonized As a pioneer of post-colonial cultural criticism, Fanon paves the way for psychoanalysis in colonialism (Xi Huanhuan, 2011: 9). Working as a physician and psychiatrist, he was a French Indian who suffered from the French colonialism. Most of his works demonstrate a 116 http://ijch.macrothink.org http://ijch.macrothink.org 2.2 Hybridity of Identity Homi Bhabha’s criticism of colonialism aims at unearthing the possibility for resistance in the colonial text and discourse (He Yugao, 2012:99). Among his theoretical contributions, the hybridity theory put forward in the 1980s, is deemed as the most innovative and influential idea of Homi Bhabha, which mainly deals with identity dilemma (He Yugao, 2012:5). Hybridity can be interpreted as the wreckage of wholeness, or to be understood in an adversarial way, against what is not hybrid (He Yugao, 2012: 42). Huddart further explains hybridity refers to the fact that cultures are not a discrete phenomenon; instead, they are always in contact with one another, and this contact leads to cultural mixed-ness (Huddart, 2005:4). Intrinsically, the ambiguity and uncertain nature of social discourse cause hybridity. The colonizer endeavors to oppress and exploit the colonized physically and mentally with the help of monopolistic discourse. However, the intrinsic attributes that language incline to be ambiguous and indefinite, along with cultural differences, prevent the complete transcription of the black language, thus producing a mixture of the indigenous language and targeted language (Bhabha, 1996:22). The hybridity of language causes the hybridity of culture. Hybridity in culture shapes indefinite and uncertain identity, and more importantly, nurtures potent strength to break the ethnic boundary (Wang Ning, 2002:50). The process that gives rise to hybridity is identified as mimicry, where the colonized learn and imitate such things as culture, language, the legal system of the civilized country in an awkward way. According to Bhabha, mimicry “is the desire for a deformed, recognizable other, as a subject of a difference that is almost the same, but no quite.”(Bhabha, 1996:86). In other words, mimicry as Bhabha understands is an exaggerated copying of language, culture, manners, and ideas. This exaggeration means that mimicry is repetition with difference, so it is not evidence of the colonized’s servitude. In fact, this mimicry also is a form of mockery (Huddart, 2005:39). Hybridity is produced when the colonized mistakenly imitate the culture in the civilized country, which adds personal assumption and interpretation into the translation of culture. Mimic man, who partially absorbs others’ culture, faces an identity dilemma of unaware of which ethnic group he belongs to. Particularly, black intellectuals with white education background suffer most from multi-layer identity and ambiguous cultural recognition. They have the feeling of nostalgia for the colonized homeland and desire for a civilized world (Bhabha, 2013:304). “The Negro is bad, the Negro is mean, the Negro is ugly…Mama, the nigger’s going to eat me up.” (Fanon, 1952: 86). This kind of discourse was embedded in people’s mindset, changing how society perceived black man, who shifted from an ordinary person to a human eater. The monopolization of language was, at the same time, the monopolization of culture, ideology, and identity. Fanon points out that the perpetuation of colonialism roots in how the society portrait black man, who symbolize savage, bad man and devil in the media and the textbooks (Fanon, 1952:113). The black identity is stereotyped, so no matter what language they speak, what they do will not change how they are perceived by white men. Colonialism was never satisfied with inflicting its own culture on others, it subverted the content and organization of other nations, denied and defamed other’s history and culture (Fanon, 2007: 9). They depicted a backward society of negro, implying that their culture should be abandoned to accept the new one. Under these circumstances, a sense of inferiority and discontent emerged in the black. The interdependency between the colonized and the colonizer is also profoundly revealed in 117 http://ijch.macrothink.org International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 Fanon’s work. The black acting as “other” develops the superiority of the white, while the presence of the white proves the existing value of the black. The essential relationship of these two racial groups is mutually exclusive and mutually interdependent (Liu Haijing, 2013:33). 2.2 Hybridity of Identity Although those black intellectuals have internalized western values, they bear a distorted and conflicting affection toward the west since ethnic discrimination is the consequence of the prevalence of western cultures (He Yugao, 2012:177). In Bhabha’s opinion, the colonial ideology is full of cracks, so the colonized can resist the 118 http://ijch.macrothink.org http://ijch.macrothink.org 3.1 Psychoanalysis of Don Shirley Using Fanon's psychoanalysis as a guideline, an in-depth study of Green Book is conducted to demonstrate the thinking pattern and identity dilemma of black intellectuals, with the hope to shed light on black psychology. The backdrop of the film is set in a dark history for the black. In the 1950s, black men in south America suffered from serious racial discrimination, which prevented black travelers from living and eating in south America even for a short period of time. Therefore, a Green Book aiming to help black men find safe hotels and restaurants was launched by a black mailer Victor Green (Liu Zhiyu et al, 2020:2). This book provided detailed information for northern black visitors in terms of living, eating, and traveling, and it symbolizes the dark time when black segregation prevailed. At that time, sundown town where black was forbidden to enter after sunset still existed, which reflected the inveterate white supremacy and white hegemony (Liu Zhiyu et al, 2020:132). Under such historical background, the film Green Book portrays how the black pianist, Don Shirley, managed to perform in south America with the accompany of the white driver, Tony Vallelonga. Don Shirley was a typical black intellectual. Born in a prestigious and relatively affluent family, Dr. Don Shirley was a well-educated black pianist who behaved elegantly in north America. Distinguished and prominent as Don Shirley was, the discriminating environment he lived in negatively impacted how he perceived himself as a black and how he reacted to black cultures. His psychology was an epitome of black intellectuals. They suffered most from serious cultural alienation and mental trauma, due to the white domination, cultural aggression, and racial discrimination brought by colonial rule (Yu Wenxiu, 2004:25). The inferiority complex of Don Shirley was implicit. Fanon claims “every colonized people in his soul has an inferiority complex.” The song they sing, the magazine they read, the historic book they learn are full of discriminating language, depicting evil, foolish, and vicious black men (Fanon, 1952:114). When asked about the ivory ornaments, which imply the slavery history, in the house, he avoided being projected with the stereotype of African Americans. He refused to eat fried chicken, refused to black jazz music, and avoided black pronunciation, showing that he felt shameful toward his own culture, language, and things symbolizing black. Moreover, Don Shirley felt perplexed toward this own identity and yearned for becoming white. International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 prevalence of colonialism by imitating the culture of the colonizer, by redeveloping those cultures in his own way, to create the zone of hybridity. Hybridity makes it harder to distinguish the boundary between the black and the white, the colonial dichotomy is disintegrated, and the imagined “other” disappear (Bhabha, 1996:23). Moreover, as the white lose the mirrored “other”, they will be overwhelmed by a sense of worry that their supremacy over the black may fade away. 3.1 Psychoanalysis of Don Shirley Witnessing Tony gambled with a gang of black men, he endeavored to distinguish himself from them and said that those black man’s birth determines their destiny. On his way http://ijch.macrothink.org http://ijch.macrothink.org 119 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 back, when he stood in front of a group of black men working in the field, he behaved uncomfortably and awkwardly since he as a black man lived a far more well-off life than them. These details demonstrate he did not regard himself as a black man. Furthermore, deep down in his heart, he aspired to fit into the white world. The education he received prodded him to act like a white man, eat like a white man, and speak like a white man because social norms and values were established by the white. Therefore, he made strict regulations on himself, listening to white music, behaving elegantly and gently, being strict with his pronunciation to make sure that it was consistent with that of the white. However, he belonged to neither group, only finding himself a wretched lonely object in this dazzling world. Just as Don Shirley confessed, “So if I am not black enough, and if I am not white enough, then tell me, what am I?” The reason behind this lies in the interdependent relationship between the colonized and the colonizer. Fanon believes that “when a black man contacts with the white world he goes through an experience of sensitization. His ego collapses. His self-esteem evaporates” (Fanon, 1952:8). The white dominate social discourse and construct images for themselves. As the “other” of this contrived system, the black represent impoverished and backward people. The existence of the enlightened and pure white relies on the presence of the black standing for evil and backward. On the contrary, the black lose their meaning for existence without the white (Liu Haijing, 2013:33). The divide between black skin and white mark is not a neat division, but instead is “a doubling, dissembling image of being in at least two places at once.” (Nagy-Zekmi, 2007:5). This kind of feeling is typical of black intellectuals. Fanon’s split subject should not be read as the paradigmatic colonized subject: the psychic dislocations he points out are most likely to be felt by native elites or those colonized individuals who have been educated within and, to some extent, invited to be mobile within the colonial system rather than by those who were at the margins (Nagy-Zekmi, 2007:5). 3.2 Hybrid Identity of Don Shirley 3.2 Hybrid Identity of Don Shirley 3.2 Hybrid Identity of Don Shirley 3.2.1 Mistaking of Identity At first, Don Shirley recognized himself more of a white man than a black man and tried his best to imitate the white's manners and behavior. Notwithstanding his black skin, the education Don Shirley received is typical of the white society and white culture. He was keen on playing the piano rather than jazz, drinking wine rather than beer, being well-restricted and courteous. He knew more about the white culture than the black culture, just as Tony said "I think I am more of a black than you." When he was living in north America, his preoccupation was how to improve his musical performance and where to perform. The reputation and admiration from others convinced him that he was an elegant pianist fit into the white supreme world rather than the black society. He lived a totally different life as an African American, far from the afflicting discriminated experience his ethnic groups had suffered. beginning of the film shows, Don Shirley lived a prestigious and peaceful life that 120 http://ijch.macrothink.org http://ijch.macrothink.org International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 belonged to elites with high social status. His house was well-furnished with all kinds of precious collection and treasures, he hired a housekeeper to clean his house and a secretary to handle some work, which all showed that he was affluent and of high social standing. When Dr. Don Shirley was eating in the canteen, people around felt comfortable with his presence, which was contrasted with the discriminated treatment he received later. Besides, despite his black skin, he didn't have an inferiority complex when it came to the white, rebutting Tony when he made mistakes in the name of the music, asking Tony to stop smoking in the car, and sometimes despise Tony's bad manners. His relationship with Tony, the white, was rather equal. All these indicate his wrong judgement of his true ethnic identity. In his opinion, since he enjoyed the same well-off life as the white elite, he belonged to the white society and should be respected in the same way. Furthermore, Dr. Don Shirley's recognition of his black ethnic identity was vague and dubious. He did not regard himself as a part of the black society. When asked about the ivory ornaments, which imply the slavery history, in the house, he avoided being projected with the stereotype of African Americans. Seeing Tony gamble with a group of impoverished black men, Shirley tried to distinguish himself from them, remarking that their birth determined their destiny. This shows that Don tried every means to distinguish himself from his own ethnic group, for one thing, black always symbolizes backward and poor, and for another, he wanted to escape form the truth that he was a black man. To some extent, the mimicry was implied in this film. The culture, language, and ideology of the white was assimilated by Don Shirley. The adaption of white culture strengthens the superior position of the white by attaching the white to all the wonderful things in the world e.g. purity, civilization, and intelligence. Don Shirley regarded himself as a white so he restricted himself to act like a white man. In fact, he was trying to mimic the white. International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 those farmers saw Don Shirley, who wore a tidy suit, accompanied by a personal driver, they stared at him with confusion and curiosity, as if looking at something they had never known before. Don Shirley avoided looking back directly in his eyes, maybe because he was embarrassed by their starring eyes, or because it was a harsh reality that he and those poor black farmers shared the same ethics. He should be like them but he wasn't. Moreover, his habits and hobbies betrayed him. Rather than jazz and fried chicken, he preferred piano and top-quality food. He was careful with his pronunciation, his dressing, his manner, and talk back to Tony when he threw rubbish out of the window and he gambled with a gang of black men. He never behaved, talked, and spoke like a black man, but the discrimination always reminded him that he was nothing more than a black. After being distained in the sundown town, he was nearly at the edge of collapse, and he confessed to Tony that he had to endure teasing and contempt from the white for his entire life. Tony told Don that “You don’t know anything about your own people, what they eat, how they talk, how they live”. Although Don did not agree with what Tony said and argued back, he clearly knew Tony was right, he could hardly fit in the black world. The argue between them drove Don to despair and misery, and Tony’s words further stroke him, so he commanded Tony to pull over and walked in the rain alone. He told Tony what confused him, that he did not know who he was. At this point, Don realized that the rich white people paid him to play the piano because this made them feel cultured. As soon as he stepped off the performing stage, he turned into being another nigger to them. He said “Because that is their true culture”. And he suffered that contempt alone, because he was not accepted by his own people, because he was not like them either. 3.2.2 Identity Lost Although Don Shirley recognized himself as the white, in his visit to the south, he awoke to the reality and found that he belonged to neither the white world nor the black world. He lost his perceived identity as a result of the discrimination he received. Don Shirley came to realize he wasn't accepted by the white. His tour south witnessed a great shift in how black intellectuals were treated. In the bar, Shirley was challenged by a gang of white men and bit without any reason, and they told Shirley to “get out of my place”. He didn't fit into the white world, and he was rejected by a suit shop, despised by the police, and forbiden to use the toilet for his color. One day, Tony drove into the sundown town where the black man was supposed to leave after sunset, and they were ill-treated and detained in the police office. The discrimination he received always reminded him that he was not accepted by the white, which contradicted with his perceived identity and drove him into antagonization. On the other hand, Don Shirley did not belong to the black. He was perplexed about the black community because he and the black didn’t share identical educational background, thinking pattern, and behavior. On his way back, he saw a group of black farmers working laboriously in the field, from their face one can tell they lived a wretched and impoverished life. When http://ijch.macrothink.org 121 http://ijch.macrothink.org International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 accepted as a white for his color. These unexpected obstacles encountered by Dr. Don Shirley, whose established identity was undermined, coerced him to seek and explore a new stage mentally. In the last scene, Dr. Shirley accepted his color and the meaning it implied. He played black music in the bar, within a group of black men. People around felt delighted at the music, and it was the first time he felt so fulfilling playing the piano. He felt at ease with eating fried chicken with hands, which he could hardly accept before. These symbolize that although he was different from those black people, he can still manage to get along well with them. Besides, Don was invited to Tony’s home to celebrate Christmas eve, and Tony’s family felt comfortable with the presence of Don Shirley. Tony' s wife expressed her gratitude to Don for his help in writing those romantic letters. It all shows there is possibility that the two ethnic groups view each other equally and get along well with each other. Don understood the society defined who he was by his color, rather than his talent and capability. It was unnecessary to deliberately pretend to be white, because all people in the world should be equal, he just need to be himself. The hybrid identity still existed, but he found a position in between, realizing that ethnic equality was what we should strive for, instead of crowding into the white world. The position of black intellectuals is called the third space by Bhabha. According to Homi Bhabha, in this limited space, cultural differences give rise to certain connections, combinations, and imaginative constructions of cultural and national identity (Jin Chunying & Tian Yao, 2020: 171) Dr. Shirley was in the third space where was an intermediate position between the black world and the white world, and he regained his identity by recognizing the existence of such space. As is suggested by Bhabha, identities are incomplete, indicated by psychoanalysis, no matter it is individual or collective identities. This incompleteness is not a problem to be solved, and principally there is no full or complete identity. Instead, the incompleteness of identity needs to be acknowledged (Huddart, 2005:6). He cried out “So if I am not black enough, and if I am not white enough, and if I am not man enough, then tell me, Tony, what am I?”. Those sentiment had been suppressed for a long time, and the ultimate outbreak demonstrated the inner fear in Don, that he felt lost in terms of his identity. The loss of identity was not only the consequence of the discriminated treatment he received from the white, but also because of his feeling of being estrange and unfamiliar toward his own ethnic group. Also, the distinct contrast between him and Tony enlightened his location of his own identity. Tony said he knew exactly who he was, he was the guy who’s lived on the same neighborhood in the Bronx with his mother, his father, his brother, and now his wife and kids. This answer prodded Don to reflect on his own identity, who he was and where he belonged to. Finally, he was trapped in the dilemma of finding himself alone, without clear identity. In fact, Don Shirley has a hybrid identity. The hybridity of identity is the consequence of colonialism. To assimilate the colonized people, the colonizers ceaselessly pour cultures, ideology, and beliefs into the former’s mind. They try to establish their status by portraying the opposite as the inferior, the weaker. The black intellectuals, however, lose their identity in this process of learning and adapting. They internalize European culture, but due to the discrimination attached to this culture, they cannot fully agree with this culture, which leads to the hybridity of identity and the emerge of contradictory feelings (He Yugao, 2012:102). 3.2.3 Identity Regained 3.2.3 Identity Regained What Don Shirley had been through in the south convinced him that he would not be 122 http://ijch.macrothink.org http://ijch.macrothink.org 4. Racial Discrimination: From Don Shirley to George Floyd Green Book symbolizes the undeniable and cruel racial oppression in America back in the 1950s. In the movie, Don Shirley was discriminated as a black man, prohibited from entering sundown town publicly, rejected from using the same toilet of the white man, and hit out of no reason in the bar. With the development of modern civilization, racial discrimination seems to have been mitigated, but in fact, it still permeates in every facet of culture and hard to eradicate. This inequal side of the society only changed superficially in the past few decades, and continue to exist in a more subtle and covert way. Devah Pager and Hana Shepherd (2008:1) point out that unlike in the pre–civil rights era, when racial prejudice and discrimination were overt and widespread, today discrimination is less readily identifiable. Some racial inequities are under the disguise of institution. A 2001 survey, for example, found that more than one-third of blacks and nearly 20% of Hispanics and Asians reported that they had personally been passed over for a job or promotion because of their race or ethnicity (Schiller, 2004:3). Educational institution also witnesses racial discrimination that black students are subject to comparatively less opportunity to enter top universities. These kinds of problems are hard to discover and prove, since they can be disguised easily by other excuses, e.g. the company chooses not to hire the black may argue that the black ones are not capable enough or they just do not fit in. Law enforcement faces the same problem. Black Americans are disproportionately arrested, convicted, and incarcerated on drug charges (Fellner, 2009:1). The reason behind is difficult to tell, whether it is simply because larger proportion of Black Americans are drug addicts, or racial discrimination involves in the process of law enforcement. Until recently, the killing of George Floyd by a policeman brought racial issue in law enforcement back under the spotlight. George Floyd, a black man, was arrested for being accused of buying with a bogus bill in Minneapolis. A white policeman knelt on Floyd’s neck for more than nine minutes, with his hands tied on handcuffed and lying on the ground, during which time Floyd stopped breathing and died. It stirred up local protests against inequality and policemen’s abuse, and then they spread worldwide in over 60 countries, with the slogan of “Black Live Matter”. This acknowledgement of identity is different from the traditional ways of thinking. Traditional idea about the world is full of perpetuate inequalities between countries and peoples. It stresses that the relationship between one’s self and others, between two subjects or objects are different. Cultures are viewed as stable, discrete and the division of cultures are distinct. However, Bhabha abandons this way of thinking and proposes that the division of one’s self and other is obscure and indirect. The hybrid identity eliminates the clear boundary between one ethnic and another, thus creating an opportunity to diminish inequality. Back in the movie, Don Shirley began to take himself as a part of the black ethnic with white educational background, though he may seem outstanding. He was just an epitome of a large group of black intellectuals in the society, and the number is growing as the society develops. They belong to mimic man in the third space, with hybrid identity. The mimicry of the black causes anxiety in the colonizer, because the distinction between them become obscure, undermining the dominant ideology that the colonized are inferior than the colonizers (Huddart, 2005 :4). In face of the same capability, same education, same decent manner of black intellectuals, the white start to be perplexed of their presumed superiority. They keep questioning themselves whether the differences still exist and where the boundary is. This http://ijch.macrothink.org http://ijch.macrothink.org 123 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 tension between the illusion of difference and the reality of sameness leads to anxiety, and may serve as a break for the inequality (Huddart, 2005:4). Bhabha points out, this anxiety opens a gap in colonial discourse—a gap that can be exploited by the colonized, the oppressed. This anxiety enables the colonized to resist colonial authority through mimicry, a strategy of doubling or repetition. This implies that, firstly, Don Shirley can regain his own identity by recognizing the third space that lies in between two ethnic groups; secondly, the existence of hybridity is important since they can be exploited by the oppressor to resist the unsatisfactory reality. http://ijch.macrothink.org 4. Racial Discrimination: From Don Shirley to George Floyd This shows a worldwide pursuit of racial equity as well as the reformation and defund of police departments. Reflecting on the tremendous and widespread impact George Floyd’s death has on the society, one can say that racial problems deserve more attention from the public and the government. It is essential to a peaceful and civilized society. The root of racial inequity always is the culture of the society, just as what Fanon and Bhabha have argued. From the perspective of http://ijch.macrothink.org 124 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 International Journal of Culture and History ISSN 2332-5518 2021, Vol. 8, No. 1 psychopathology, Fanon explains how the white culture persecutes the black people, inflicting great mental damage on their bodies. The society portrays black men as savages, bad men, devil in the text books and media, thus strengthening the oppression and perpetrating the inequity (Fanon, 1952:113). Homi Bhabha extends Fanon’s research, and specifies the cultural elements behind identity dilemma of the black. The ambiguity and uncertain nature of social discourse cause a mixture of the indigenous language and targeted language, which shapes the hybridity of culture (Bhabha, 1996: 86). Bhabha’s writing emphasizes the hybridity of cultures, which on one level simply refers to the mixed-ness, or even ‘impurity’ of cultures—so long as we don’t imagine that any culture is pure (Huddart, 2005: 4). This idea provides us with an unprecedented view to resist discrimination, that is to mimic the colonizer. The mimicry causes anxiety in the colonized who believe they are superior than the black, because the boundary between them becomes blur as they try to imitate one another. The death of George Floyd alarms us of the severity of racial problems, and the study of Fanon and Bhabha do enlighten us from the perspective of culture. Therefore, Movie as an important form of culture, has a special role to play. We should pay more attention to the construed world in films, where different ethnic group should be equal and avoid stereotyping model of black men. Also, the proportion of positive images of black men should be increased to show a whole picture that the black not only involves rape, crime, and drug, but also includes such good characteristics as kind, intelligent and assiduous. Though the change of culture takes time, action always counts. 5. Conclusion Green Book is a milestone in movies of black men, for it successfully subverts the expected images for the black, revealing the psychological conflicts of a black intellectual and depicting his identity dilemma. Black intellectuals suffer from the inferiority complex and delusion of becoming white, because of the assimilation strategy in colonialism and the penetration of otherness. Black man's hybrid identity attributes to the adaptation of the white culture, and he may go through the processes of identity lost and regained. Besides, in comparison with the mimic man, white man feel anxiety as they can't find the inferior other. This serves as a gap for undermining the discriminated culture, and sheds light on the improvement in the modern society. Despite some criticisms about such film (Ju Wei, 2019; Hao Luxuan, 2019), Green Book is a meaningful attempt in changing the stereotype of black images, which is in line with the racial equality movement in contemporary society. The subversion of movie characters provides people with the possibility of constructing positive images of black people. To some extent, this movie instills the idea of equality into people's mindset, though it may seem slight, can be accumulated as a great leap in the future. Bhabha, H. K. (1996). The location of culture. Routledge Classics. Bhabha, H. K. (Ed.). (2013). Nation and narration. Routledge. https://doi.org/10.4324/9780203388341 Fan, Q. Y. (2009). Explore the cultural factors of racial discrimination in Hollywood films. References an, Q. Y. (2009). 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https://openalex.org/W2139148747	https://europepmc.org/articles/pmc4161859?pdf=render	English	null	An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein	Virology journal	2,014	cc-by	7,406	Open Access Open Access * Correspondence: adam.rash@aht.org.uk 1Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK Full list of author information is available at the end of the article Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein Adam Rash1, Alana Woodward1, Neil Bryant1, John McCauley2 and Debra Elton1 Adam Rash1, Alana Woodward1, Neil Bryant1, John McCauley2 and Debra Elton1 © 2014 Rash et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background for HA, which makes it difficult to study the evolution of either the other gene segments or the virus as a whole. It is also known that reassortment between the different line- ages of EIV has occurred [4-8,15,16] but the full extent is unknown due to a lack of data available. Avian H3N8 influ- enza viruses have been shown to frequently exchange in- ternal gene segments, and it has been suggested that the extensive reassortment within the H3 subtype poses a threat to human and animal health [17]. Next generation sequencing technologies have made it easier to sequence whole viral genomes, however these technologies are not readily available to all as considerable investment in equip- ment and bioinformatics expertise are needed. We aimed to develop a simple and robust method to sequence whole EIV genomes from all H3N8 lineages using Sanger dideoxy- nucleotide sequencing technology. Equine influenza virus (EIV) is an influenza A virus belonging to the Orthomyxoviridae family. These viruses have a negative sense, single-stranded RNA genome consisting of eight viral gene segments [1]. Originally thought to have transmitted from birds, H3N8 EIV was first isolated during a widespread outbreak in the United States in 1963 [2], and has since spread worldwide caus- ing multiple major outbreaks of disease in horses. Dur- ing the 1980s the virus diverged into two antigenically distinct lineages [3], American and Eurasian, and since then the American lineage has further evolved into the Florida sublineage clades 1 and 2, which continue to co- circulate today [4]. These lineages have historically been based on antigenic and genetic data for HA. A phylogen- etic study by Murcia et al. [5] showed that phylogenetic trees produced for each of the viral gene segments also supported division into the American and Eurasian lineages, and all but segment 7 divided into the two clades of the Florida sublineage. However, less than 100 complete viral genomes covering the 46 years from 1963 to 2008 were available at the time of the study. Each of the eight influenza virus gene segments con- tains two non-coding regions (NCRs), one at the 5′ terminus containing 13 conserved nucleotides, and the other at the 3′ terminus, which contains 12 nucleotides [18]. Unlike the 5′ end, the 3′ terminus exhibits vari- ation at the fourth nucleotide. Abstract Background: H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA), one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and determine the level of reassortment occurring between sub-lineages. Methods: To facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the design of novel primers for an N8 neuraminidase segment. Results: Two field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07, representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length from 400–600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates. Conclusions: These methods can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR products than previously reported methods for influenza A viruses, the techniques used are affordable and the equipment required is available in most research laboratories. The adoption of these methods will hopefully allow for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better understanding of EIV evolution. Keywords: Equine influenza virus, H3N8, Genome sequencing, Non-coding regions, M13, PA-X Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Page 2 of 9 Background This variation in the fourth nucleotide has been shown to affect the rescue of virus from a reverse genetics system [19]. A second ob- jective, to implement a method previously described for sequencing the NCRs of influenza viruses [20], was adapted and carried out on an EIV, as well as an N8 sub- type neuraminidase, for the first time. Other groups have studied the evolution of individual influenza A virus genes. H3N8 EIV PB2 [6] and matrix proteins [7] were found to belong to the same lineage as North American avian strains, whilst PB1, PA, HA and NP were found to have evolved independently from other influenza A viruses [8-10]. Equine NS was sug- gested as being restricted to subtype [11], as the NS seg- ments of the H3N8 viruses were close to one another but not to that of the H7N7 viruses, however very lim- ited numbers of EIV genomes were available at the time these studies were performed, and in some cases only two different EIV strains were used. More recently a study found that the internal genes of the 1963 EIV pan- demic virus were of western hemispheric avian influenza origin [12]. This study also showed that the virus shared a most recent common ancestor with avian influenza vi- ruses from South America shortly before its emergence. The time to most recent common ancestor for avian/ equine NP was calculated as being 1954, which agreed with the hypothesis that the virus emerged in South America prior to its introduction into the USA in 1963 by horses imported by air from Argentina [12]. Here we describe the genome sequencing method and highlight the sequence differences found between repre- sentatives of the two circulating clades of the Florida sublineage. Results q g q At the time of writing, only 81 full genome sets were available from the NCBI Influenza Virus Resource for EIV and only one or two gene segment sequences had been published for the majority of strains. To address the lack of available genomes, a method to sequence the ge- nomes of equine influenza viruses belonging to both clades of the currently circulating Florida sublineage, using an EIV specific primer set for PCR and M13 primers for sequencing, was developed. A/equine/Richmond/1/07 was selected as a representative of recent Florida sublineage clade 2 (FC2) viruses, as well as being a current OIE recom- mended vaccine strain. A/equine/Lincolnshire/1/07 was chosen because it was the first virus belonging to clade 1 of the Florida sublineage isolated in the UK [4]. Published nucleotide sequences were aligned for each segment and primers were designed to conserved regions (data not shown) to amplify products of 400–600 nucleotides (Figure 1). Each specific primer was elongated at the 5′ end by adding either M13 forward or M13 reverse primer Following an extensive outbreak in 1989 affecting a highly vaccinated population of racehorses in the UK, it be- came clear that, like human influenza virus, EIV undergoes antigenic drift and therefore vaccine strains need to be kept up to date [13,14]. A formal process for vaccine strain se- lection, overseen by the World Organisation for Animal Health (OIE) was put in place. This process relies heavily on surveillance data collected from the field, of which most is focussed solely on the HA gene and the protein it en- codes. Therefore the majority of published sequences are Page 3 of 9 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 PB2, PB1, PA & HA: M13 AF M13 BF M13 CF M13 DF M13 AR M13 BR M13 CR M13 DR M13 BF M13 BR NP & NA: A B C D A B C A B M & NS: M13 AF M13 AR M13 CF M13 CR M13 AF M13 AR M13 BF M13 BR Figure 1 Schematic representation of PCR primer design for genome sequencing EIV. Primers were tagged with M13 forward or reverse sequences for use in the sequencing stage. Results Segments 1–4 (PB2, PB1, PA and HA) were divided into four sections, segments 5 & 6 (NP and NA) into three sections and segments 7 & 8 (M and NS) into two sections, each of approximately 400–600 nucleotides in length. B NP & NA: B M & NS: B A Figure 1 Schematic representation of PCR primer design for genome sequencing EIV. Primers were tagged with M13 forward or reverse sequences for use in the sequencing stage. Segments 1–4 (PB2, PB1, PA and HA) were divided into four sections, segments 5 & 6 (NP and NA) into three sections and segments 7 & 8 (M and NS) into two sections, each of approximately 400–600 nucleotides in length. There were a total of 279 nucleotide differences between the two viruses, which resulted in 65 amino acid changes (Table 2). Approximately 45% (29) of the total amino acid changes were observed within the polymerase and nucleoprotein segments, with 45% (13) of these occur- ring within PA alone. The two glycoprotein segments, HA and NA, contained 40% (26) of the total amino acid differences, of which 54% (14) were within HA and 46% (12) within NA. sequences (Table 1), as described in a method for sequen- cing swine influenza genomes [21] which facilitated effi- cient sequencing. Four amplicons were produced for the larger genome segments, 1 to 4, three amplicons for seg- ments 5 and 6, and two amplicons for segments 7 and 8 (Figure 2). A total of 26 PCR products were amplified successfully from RNA extracted from allantoic fluid for both virus strains, A/equine/Richmond/1/07 and A/equine/ Lincolnshire/1/07. The nucleotide sequences of the PCR products were determined on both strands and the com- plete viral genome was assembled successfully without the need for further specific primers to complete gaps. The PCR products overlapped by approximately 100 nucleotides at each end and sequences were edited, so that the final coding sequence contained no primer-derived sequences. This method has since been applied to a further 17 strains of EIV, all primers worked well for Florida clade 1 and clade 2 strains from 2009–2013, and sequences were made avail- able on the GISAID (Global Initiative on Sharing Avian In- fluenza Data) EpiFlu database [22] (see Additional file 1). Assembled sequences for each segment of A/equine/ Richmond/1/07 and A/equine/Lincolnshire/1/07 were also uploaded onto GenBank, accession numbers indicated in additional material (see Additional file 2). Results The two smaller segments, M and NS, contained a total of 10 amino acid changes within the four predicted polypeptides (M1, M2, NS1 and NS2/NEP) that they encode. Nucleotide changes in three segments resulted in dif- ferent lengths for their predicted polypeptides. A dupli- cation of six nucleotides in A/equine/Richmond/1/07 resulted in a two amino acid insertion within the puta- tive signal peptide of the precursor HA protein extend- ing its length from 15 to 17 amino acids, as observed in recent FC2 isolates [4]. In contrast, when compared to earlier EIV isolates from 1963–2000 the predicted amino acid sequences for the NS1 protein from both viruses were truncated by 11 amino acids, as seen for other re- cent isolates [4]. This was caused by a premature stop codon at position 220, resulting in a predicted polypep- tide length of 219 amino acids. The open reading frame The gene segment sequences for each of the two vi- ruses were aligned against one another for comparison. Rash et al. Results Virology Journal 2014, 11:159 Page 5 of 9 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Page 5 of 9 Page 5 of 9 Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV (Continued) Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV (Continued) NA/CR GC AACAGCTATGACCATG AGTAGAAACAAGGAGTT M/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGTAGATATTTAAAG 1-654 50 M/AR GC AACAGCTATGACCATG CTAGCCTTACTAGCAAC M/BF GC GTAAAACGACGGCCAGT CAGTACCACGGCTAAAG 571-1027 50 M/BR GC AACAGCTATGACCATG AGTAGAAACAAGGTAGTTTTTTAC NS/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTGACAAAAAC 1-492 50 NS/AR GC AACAGCTATGACCATG CTGCTCCTTCTTCGGTG NS/BF GC GTAAAACGACGGCCAGT CATCATACTTAAAGCAAAC 407-890 50 NS/BR GC AACAGCTATGACCATG AGTAGAAACAAGGTAGTGTTTTTTAT Table 1 Primer sequences and annealing temperatures used to sequence the genome o (252 amino acids) or are truncated by 19 amino acids. To investigate further and to study the evolution of the truncation, the PA-X region of segment 3 from an add- itional 29 EIV isolated in the UK between 2005 and 2013, including 9 from 2007, were sequenced using the method described here (see Additional file 3). The 42 amino acid truncation in A/equine/Richmond/1/07 PA- of NS2/NEP however, was unaffected by this nucleotide substitution. In addition, a novel truncation in the re- cently discovered PA-X gene was identified in A/equine/ Richmond/1/07, caused by an early stop codon at pos- ition 20 of the +1 reading frame. The truncation of PA- X by 42 amino acids has not been described before, with the majority of strains having either a full length version 600bp 800bp 1kb 400bp A B C D A B C D A B C D Segment 1 (PB2) Segment 2 (PB1) Segment 3 (PA) 600bp 800bp 1kb 400bp Segment 4 (HA) A B C D A B C Segment 5 (NP) A B C Segment 6 (NA) 600bp 800bp 1kb 400bp A B Segment 7 (M) A B Segment 8 (NS) Figure 2 Agarose gel electrophoresis analysis of genome segment PCR products. Agarose gel (1%) showing PCR fragments A, B, C & D of segments 1–4 (PB2, PB1, PA and HA), A, B & C of segments 5 and 6 (NP and NA), and A & B of segments 7 and 8 (M and NS) of EIV Northamptonshire/1/13. Figure 2 Agarose gel electrophoresis analysis of genome segment PCR products. Results Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Page 4 of 9 Page 4 of 9 Table 1 Primer sequences and annealing temperatures used to sequence the genome Primer name Primer sequence (5′-3′) Approximate coverage (5′-3 PB2/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG 1-655 PB2/AR GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT PB2/BF GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA 569-1335 PB2/BR GC AACAGCTATGACCATG CCTCAAGAGTTGATG PB2/CF GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC 1216-1874 PB2/CR GC AACAGCTATGACCATG ATTATTTGAGCAGTATC PB2/DF GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT 1793-2341 PB2/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC PB1/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG 1-719 PB1/AR GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG PB1/BF GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA 577-1301 PB1/BR GC AACAGCTATGACCATG GGTTTAATATGGATACACC PB1/CF GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC 1222-1863 PB1/CR GC AACAGCTATGACCATG CATTTTAAACAAACTTC PB1/DF GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG 1789-2341 PB1/DR GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC PA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG 1-615 PA/AR GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC PA/BF GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG 529-1255 PA/BR GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC PA/CF GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG 1149-1785 PA/CR GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC PA/DF GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG 1700-2233 PA/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG HA/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT 1-515 HA/AR GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG HA/BF GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G 428-1032 HA/BR GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC HA/CF GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC 939-1336 HA/CR GC AACAGCTATGACCATG GAGCCACCAGCAATTCT HA/DF GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA 1251-1733 HA/DR GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC NP/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC 1-570 NP/AR GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC NP/BF GC GTAAAACGACGGCCAGT GACACCACATACCAAAC 480-1075 NP/BR GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT NP/CF GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG 1012-1569 NP/CR GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC NA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT 1-508 NA/AR GC AACAGCTATGACCATG GCCCTATTTTGACACTC NA/BF GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC 417-1049 NA/BR GC AACAGCTATGACCATG CCGAAACCTTTTACACCG NA/CF GC GTAAAACGACGGCCAGT CACAGTTGGATATTTGTG 951-1461 Table 1 Primer sequences and annealing temperatures used to sequence the genome of H3N8 EIV Primer name Primer sequence (5′-3′) Approximate nucleotide coverage (5′-3′) Annealing temperature used (°C) PB2/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG 1-655 50 PB2/AR GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT PB2/BF GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA 569-1335 60 PB2/BR GC AACAGCTATGACCATG CCTCAAGAGTTGATG PB2/CF GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC 1216-1874 45 PB2/CR GC AACAGCTATGACCATG ATTATTTGAGCAGTATC PB2/DF GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT 1793-2341 50 PB2/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC PB1/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG 1-719 50 PB1/AR GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG PB1/BF GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA 577-1301 50 PB1/BR GC AACAGCTATGACCATG GGTTTAATATGGATACACC PB1/CF GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC 1222-1863 50 PB1/CR GC AACAGCTATGACCATG CATTTTAAACAAACTTC PB1/DF GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG 1789-2341 50 PB1/DR GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC PA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG 1-615 50 PA/AR GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC PA/BF GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG 529-1255 50 PA/BR GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC PA/CF GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG 1149-1785 50 PA/CR GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC PA/DF GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG 1700-2233 50 PA/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG HA/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT 1-515 50 HA/AR GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG HA/BF GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G 428-1032 50 HA/BR GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC HA/CF GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC 939-1336 50 HA/CR GC AACAGCTATGACCATG GAGCCACCAGCAATTCT HA/DF GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA 1251-1733 50 HA/DR GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC NP/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC 1-570 50 NP/AR GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC NP/BF GC GTAAAACGACGGCCAGT GACACCACATACCAAAC 480-1075 45 NP/BR GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT NP/CF GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG 1012-1569 55 NP/CR GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC NA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT 1-508 45 NA/AR GC AACAGCTATGACCATG GCCCTATTTTGACACTC NA/BF GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC 417-1049 45 NA/BR GC AACAGCTATGACCATG CCGAAACCTTTTACACCG Table 1 Primer sequences and annealing temperatures used to sequence the genome Primer name Primer sequence (5′-3′) Approximate coverage (5′- PB2/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGCAAATATATTCAATATG 1-655 PB2/AR GC AACAGCTATGACCATG CTCTTTCTAGCATGTAT PB2/BF GC GTAAAACGACGGCCAGT CACAACTAACAATAACCAA 569-1335 PB2/BR GC AACAGCTATGACCATG CCTCAAGAGTTGATG PB2/CF GC GTAAAACGACGGCCAGT GCAATAATTGTAGCC 1216-1874 PB2/CR GC AACAGCTATGACCATG ATTATTTGAGCAGTATC PB2/DF GC GTAAAACGACGGCCAGT GAAGCCAATACAGCGGT 1793-2341 PB2/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TCGTTTTTAAACAATTC PB1/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGG CAAACCATTTGAATGG 1-719 PB1/AR GC AACAGCTATGACCATG CAGCGTCCTTGGTCATTG PB1/BF GC GTAAAACGACGGCCAGT CTTCCAACGAAAGAGAA 577-1301 PB1/BR GC AACAGCTATGACCATG GGTTTAATATGGATACACC PB1/CF GC GTAAAACGACGGCCAGT GCGGCTTCACTGAGTCCTGGC 1222-1863 PB1/CR GC AACAGCTATGACCATG CATTTTAAACAAACTTC PB1/DF GC GTAAAACGACGGCCAGT CAAAGACTGGTCTACTG 1789-2341 PB1/DR GC AACAGCTATGACCATG AGTAGAAACAAGG CATTTTTTCATGAAGATC PA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGG TACTGATCCAAAATGG 1-615 PA/AR GC AACAGCTATGACCATG GCCTCTCTCGGACTGAC PA/BF GC GTAAAACGACGGCCAGT GCCAGAATCAAGACCAGG 529-1255 PA/BR GC AACAGCTATGACCATG CTCACTTGGAATCCAACTTGC PA/CF GC GTAAAACGACGGCCAGT GAGAGAAAGTGGATTTTGAGGATTG 1149-1785 PA/CR GC AACAGCTATGACCATG CTGAAGGAGGCAGCGCC PA/DF GC GTAAAACGACGGCCAGT GACCCATGTTTTTGTATG 1700-2233 PA/DR GC AACAGCTATGACCATG AGTAGAAACAAGG TACTTTTTTGGACAG HA/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGGACGATATT 1-515 HA/AR GC AACAGCTATGACCATG GATTTGTTAGCCAATTCAG HA/BF GC GAAAACGACGGCCAGT CAGGTGTCACTCAAAAC G 428-1032 HA/BR GC AACAGCTATGACCATG GGATTTGCTTTTCTGGTAC HA/CF GC GTAAAACGACGGCCAGT GGTTACATATGGAAAATGCC 939-1336 HA/CR GC AACAGCTATGACCATG GAGCCACCAGCAATTCT HA/DF GC GTAAAACGACGGCCAGT GAAGGAAGAATTCAGGA 1251-1733 HA/DR GC AACAGCTATGACCATG GAGTAGAAACAAGGGTGTTTTTAAC NP/AF GC GTAAAACGACGGCCAGT AGCGAAAGCAGGGTAGATAATC 1-570 NP/AR GC AACAGCTATGACCATG CCGTGGGAGGGTTGAGCC NP/BF GC GTAAAACGACGGCCAGT GACACCACATACCAAAC 480-1075 NP/BR GC AACAGCTATGACCATG CTCTCAGGTCCTCAAAT NP/CF GC GTAAAACGACGGCCAGT CCAGCACACAAGAGCCAG 1012-1569 NP/CR GC AACAGCTATGACCATG AGTAGAAACAAGGGTATTTTTC NA/AF GC GTAAAACGACGGCCAGT AGCAAAAGCAGGAGTTT 1-508 NA/AR GC AACAGCTATGACCATG GCCCTATTTTGACACTC NA/BF GC GTAAAACGACGGCCAGT CACACAGGGCTCATTAC 417-1049 NA/BR GC AACAGCTATGACCATG CCGAAACCTTTTACACCG NA/CF GC GTAAAACGACGGCCAGT CACAGTTGGATATTTGTG 951-1461 eratures used to sequence the genome of H3N8 EI Rash et al. Results Agarose gel (1%) showing PCR fragments A, B, C & D of segments 1–4 (PB2, PB1, PA and HA), A, B & C of segments 5 and 6 (NP and NA), and A & B of segments 7 and 8 (M and NS) of EIV Northamptonshire/1/13. Page 6 of 9 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07 Segment Nucleotide changes Protein Amino acid changes Amino acid changes from Richmond/1/07 to Lincolnshire/1/07 1 35 PB2 4 A105T, K251R, I398V, K660R 2 48 PB1 8 F94L, M119V, V149I, M179I, R329Q, E377D,D618E, K621R 3 47 PA 13 D64E, I86M, M210A, K237E, G240E, P259S, N321S, L348I, S409N, I465V, T476A, I500V, R626K 4 47a HA1† 11b K-14T, F-11L, I-9a1, F-9b1, N7G, R62K, V78A, D104N, A138S, N159S, E291D HA2 3 T43A, E50G, I198V 5 30 NP 4 I131M, T257I, A359T, S450N 6 37 NA‡ 12 T9A, S12F, V35A, E40K, G42D, H66Y, P78S, I191V, N235D, S337N, I410V, G416E 7 24 M1 3 I15V, I80V, K95R M2 2 S86D, 290G 8 11 NS1 4 I48S, I84V, Y207H, G210R NS2/NEP 1 M52I aIncludes a 6 nucleotide duplication resulting in btwo additional amino acids in A/equine/Richmond/1/07. †Numbering starting after the putative signal sequence, ‡numbering starting from start codon 1Two amino acid insertion in Richmond/1/07 HA, not present in Lincolnshire/1/07. Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07 Segment Nucleotide changes Protein Amino acid changes Amino acid changes from Richmond/1/07 to Lincolnshire/1/07 Table 2 Nucleotide and amino acid differences between A/equine/Richmond/1/07 and A/equine/Lincolnshire/1/07 aIncludes a 6 nucleotide duplication resulting in btwo additional amino acids in A/equine/Richmond/1/07. †Numbering starting after the putative signal sequence, ‡numbering starting from start codon 1Two amino acid insertion in Richmond/1/07 HA, not present in Lincolnshire/1/07. segment 6, and both the 5′ and 3′ of segment 7 were not visible by gel electrophoresis, as shown in Figure 3. Despite bands of the correct size not being visible for these products, sequence covering the NCR regions of interest were successfully determined for all three. Sub- sequent sequencing of the amplified NCRs revealed that the 13 nucleotides of the 5′ end were identical in all 8 viral gene segments, as well as the 12 bases of the 3′ end of the vRNA except for the fourth nucleotide. Results The three polymerase segments (PB2, PB1 and PA) all con- tained a cytosine, whilst the remaining segments (HA, NP, NA, M and NS) contained a uracil at the fourth nu- cleotide position. X was identified in three of the isolates, one from an- other horse in the same outbreak that A/equine/Rich- mond/1/07 was isolated from, and the other two from a separate outbreak in 2007. The remaining isolates did not share the truncation, and all had a full length PA-X of 252 amino acids (61 amino acids following the +1 frameshift) (see Additional file 3). Sequencing of the non-coding regions of equine influenza viruses Previous studies have shown that there are discrepancies in the segments that contain a cytosine at nucleotide position four of the 3′ NCR, and that a cytosine at this position is not restricted to the polymerase segments [23]. This variation has been implicated in the differing levels of vRNA and mRNA synthesis observed during the virus replication cycle whereby a uracil at this pos- ition increased mRNA production and delayed vRNA synthesis [23]. Another study showed that the fourth nu- cleotide of the NCR at the 3′ end of influenza vRNA segments could influence rescue of viruses using reverse genetics [19]. As one of our future aims was to generate a reverse genetics system for A/equine/Richmond/1/07, we determined the sequence of the NCRs for each vRNA segment for this virus strain. Viral RNA was self-ligated, then each of the NCRs amplified using a universal primer complementary to the opposite NCR and a seg- ment specific primer, as described by de Wit et al. [20]. Modifications were made to all but two of the published primer sequences to ensure that they were complemen- tary to equine influenza viruses sequenced previously (Table 3). The NCRs from each segment were amplified successfully, but with varying degrees of efficiency. In particular the 3′ of segment 8 was amplified to a high level, whereas products of the correct size for the 5′ of Discussion We and others have previously shown that reassortment has occurred between different EIV [4-8,15,16], but a lack of full genome sequences for EIV makes it difficult to ascertain the extent of reassortment between them, and whether reassortment has occurred between EIV and influenza A viruses from other species. We therefore developed a simple method for sequencing viral ge- nomes that included primers with M13 sequence tags to improve the sequencing efficiency. This was based upon the approach recommended by the WHO for sequen- cing swine influenza virus isolates in 2009 [21], however our method used only 26 PCR fragments to cover the segment-specific regions of EIV, rather than 46 frag- ments. Alternative methods have been employed for sequencing influenza A viruses, such as using universal primers to simultaneously amplify all eight genome seg- ments, or segment specific primers to amplify entire seg- ments; however, in our hands such protocols result in poor amplification of the three largest genome segments (data not shown). Other methods based on amplification Page 7 of 9 Page 7 of 9 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Table 3 Primer sequences used for sequencing 3′ and 5′ NCRs of H3N8 EIV Primer Primer sequence† Universal 3′ 5′-CCTTGTTTCTACTAGC-3′ Universal 5′ 5′-CCTGCTTTTGCTAGT-3′ PB2 3′ 5′-GGGTATTTCATTGCCATCATCC-3′ PB2 5′ 5′-GACTCTAGCATACTTACTGACAG-3′ PB1 3′ 5′-GACAGTATCCATGGTGTATCCTGT-3′ PB1 5′ 5′-GTATGGTTGAGGCCATGGTGTCC-3′ PA 3′ 5′-ATCCCTGTGATTTCAAATCTTTCTTC-3′ PA 5′ 5′-GAATGCCTGATTAATGATCCCTG-3′ HA 3′ 5′-AATGTTCCATTTGCTACTGCATG-3′ HA 5′ 5′-GGATTTCATTCGCCATATCATG-3′ NP 3′ 5′-TCAAATGCCGAAAGT-3′ NP 5′ 5′-CCGATCGTGCCTTCCTTTGACAT-3′ NA 3′ 5′-GTGGAGTAGATCATAAAATTGCC-3′ NA 5′ 5′-CAGACCTGTTTCATTGTTATTGAG-3′ M 3′ 5′-ATAAAGCGTCTACGCTGCAGTCC-3′ M 5′ 5′-AAAGAGGGCCTTCTACGGAAGG-3′ NS 3′ 5′-CCGTATTATCATTCCATTTAAG-3′ NS 5′ 5′-TGATAATACGGTTAGAATCTCT-3′ †Sequences from de Wit et al., [20] shown in italics with specific individual nucleotide changes in bold text, plus novel primer sequences for the N8 NA segment. Table 3 Primer sequences used for sequencing 3′ and 5′ NCRs of H3N8 EIV Primer Primer sequence† Universal 3′ 5′-CCTTGTTTCTACTAGC-3′ Universal 5′ 5′-CCTGCTTTTGCTAGT-3′ PB2 3′ 5′-GGGTATTTCATTGCCATCATCC-3′ PB2 5′ 5′-GACTCTAGCATACTTACTGACAG-3′ PB1 3′ 5′-GACAGTATCCATGGTGTATCCTGT-3′ PB1 5′ 5′-GTATGGTTGAGGCCATGGTGTCC-3′ PA 3′ 5′-ATCCCTGTGATTTCAAATCTTTCTTC-3′ PA 5′ 5′-GAATGCCTGATTAATGATCCCTG-3′ HA 3′ 5′-AATGTTCCATTTGCTACTGCATG-3′ HA 5′ 5′-GGATTTCATTCGCCATATCATG-3′ NP 3′ 5′-TCAAATGCCGAAAGT-3′ NP 5′ 5′-CCGATCGTGCCTTCCTTTGACAT-3′ NA 3′ 5′-GTGGAGTAGATCATAAAATTGCC-3′ NA 5′ 5′-CAGACCTGTTTCATTGTTATTGAG-3′ M 3′ 5′-ATAAAGCGTCTACGCTGCAGTCC-3′ M 5′ 5′-AAAGAGGGCCTTCTACGGAAGG-3′ NS 3′ 5′-CCGTATTATCATTCCATTTAAG-3′ NS 5′ 5′-TGATAATACGGTTAGAATCTCT-3′ †Sequences from de Wit et al., [20] shown in italics with specific individual nucleotide changes in bold text, plus novel primer sequences for the N8 NA segment. Table 3 Primer sequences used for sequencing 3′ and 5′ NCRs of H3N8 EIV differences between the two viruses. Discussion This was expected as these two proteins are under constant immune-driven selection pressure to undergo antigenic drift. Interest- ingly a high number of amino acid differences were found in PA, especially when compared to the other two polymerase subunits PB2 and PB1, and a similar finding was observed by Murcia et al. [5]. The other internal segments contained fewer changes, which is not surpris- ing as they are both smaller and may be under less im- mune pressure than the surface proteins. Interestingly, a mutation in the +1 reading frame of PA, causing a premature stop codon in the translated amino acid sequence of PA-X, was observed in A/ equine/Richmond/1/07. PA-X is a recently discovered protein containing the N-terminal 191 amino acids of PA and, in the majority of strains, a further 61 amino acids derived from a frameshift to the +1 reading frame of PA [24]. PA-X has been implicated in the modulation of influenza virus pathogenicity and virulence in a mouse model, whereby PA-X deficient viruses caused greater clinical signs and were less able to shut off host cell responses compared to wild-type viruses with full length PA-X [24]. The premature stop codon in A/ equine/Richmond/1/07 would lead to a truncation of the protein by 42 amino acids. Truncated forms of PA-X have been described previously, however the majority of these are due to a nonsense mutation at codon 42 in the +1 reading frame [25]. Sequencing of PA, as de- scribed here, revealed that several other virus isolates from different outbreaks in 2007 as well as from the same yard as A/equine/Richmond/1/07, had the same truncated form of PA-X, however the truncated form did not persist in the UK. †Sequences from de Wit et al., [20] shown in italics with specific individual nucleotide changes in bold text, plus novel primer sequences for the N8 NA segment. of small PCR fragments do not include the M13 se- quences in the primers, which makes the method de- scribed here simple and efficient. A method previously described for sequencing the NCRs of the influenza gene segments was also modified and successfully used for the first time on an equine influenza virus, with novel primers designed for an N8 subtype NA. Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Page 8 of 9 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 PCR amplification of gene segments polymerase segments, as found in other influenza viruses, and uracil at this position in the remaining 5 segments. This is the same pattern seen in the majority of other influ- enza A viruses for which the promoter sequences have been determined, including the prototype avian influenza virus, A/chicken/Rostock/34 (H7N1) [26]. Viral gene segments were amplified in 50 μl PCR reac- tions consisting of 2 μl cDNA (representing 10% of the reverse transcription reaction), dNTP mix (0.2 mM each final concentration) (Qiagen), 1 × Pfu buffer, 2.5U Native Pfu DNA polymerase (Stratagene), water and oligonucleo- tide pairs (final concentration each of 0.2 μM) as listed in Table 1. The cycling conditions were as follows: initial denaturation at 96°C for 1 minute, followed by 25 cycles of denaturation at 96°C for 15 seconds, primer annealing at 50-60°C (see Table 1) for 10 seconds and elongation at 60°C for 5 minutes. PCR reactions were analysed on a 1% agar- ose gel containing GelRed nucleic acid stain (Biotium) according to manufacturer’s directions. PCR products were purified using a QIAquick PCR purification kit (Qiagen) ac- cording to manufacturer’s directions. The methods outlined here can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. The techniques described here are affordable, and the equip- ment required is available in most research laboratories. The sequence assembly process is simple and does not require in depth bioinformatics, unlike next generation sequencing methodology. Due to the small genome size and small sample numbers usually associated with EIV, this method is therefore highly cost effective and straightfor- ward. Amplicon sequencing has also been shown to be less labour intensive and more affordable than plasmid cloning methods [27]. This method also permits the sequencing of individual gene segments with relative ease, as was the case with PA described here to investigate the frequency of the truncated form of PA-X. PCR amplification of non-coding regions The method described by de Wit et al. [20] was used with modifications, as detailed in Table 2. Novel primers were designed to amplify the N8 subtype NA segment. Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Briefly, following an initial denaturation at 65°C for 5 mi- nutes in the presence of T4 RNA ligase buffer and 20U RNAsin RNase inhibitor (Promega), 15 μl RNA was li- gated using 40U T4 RNA ligase (New England Biolabs) at 37°C for 1 hour. The ligation reaction was stopped by heat inactivation at 65°C for 10 minutes. cDNA was transcribed from 4 μl of the ligated RNA by incubating the RNA in a mixture consisting of 0.5 μg random pri- mer (Promega), dNTP mix (0.5 mM each final concen- tration) (Qiagen) and 20U RNasin at 65°C for 5 minutes, then cooling to 4°C, before adding 20U RNasin, 5 mM DTT, 200U Superscript II (Invitrogen) and 1 × First Strand buffer in a total reaction volume of 20 μl. The re- action mixture was subsequently incubated at 25°C for 5 minutes, followed by 50°C for 1 hour. 50 μl PCR reac- tions consisting of 4 μl cDNA (representing 20% of the reverse transcription reaction), dNTP mix (20 mM final concentration), universal primer (3′- or 5′- final concen- tration 0.2 μM) (Table 2), gene segment specific primer (3′- or 5′- final concentration 0.2 μM) (Table 2), 1 × Pfu buffer and 2.5U Native Pfu DNA polymerase (Strata- gene) were made. The cycling conditions were as fol- lows: initial denaturation at 96°C for 6 minutes, followed by 40 cycles of denaturation at 96°C for 30 seconds, pri- mer annealing at 37°C for 1 minute and elongation at 72°C for 2 minutes. PCR reactions were analysed on a 2.5% agarose gel containing GelRed nucleic acid stain (Biotium) according to manufacturer’s directions. PCR products were purified using a QIAquick PCR purifica- tion kit (Qiagen) according to manufacturer’s directions. Where multiple bands were present in the gel, bands of the correct size were excised and purified using a Methods Vi EIV A/equine/Richmond/1/07 and A/equine/Lincolnshire/ 1/07 had previously been isolated and passaged twice in embryonated chicken eggs [4]. RNA was isolated from 140 μl virus stocks containing ~107 EID50/ml using a QIAamp Viral RNA Mini Kit (Qiagen), accord- ing to manufacturer’s directions. RNA was eluted in 50 μl elution buffer. Conclusions We have developed a simple, efficient and affordable method for sequencing whole genomes of EIV that of- fers an improvement compared with previously pub- lished methods. The adoption of these methods should facilitate an increase in the number of full genome se- quences available for EIV. This will benefit surveillance programmes for EIV and improve understanding of the evolutionary paths taken by the virus, including the level of reassortment. Discussion Sequence analysis of the NCRs from each segment showed that EIV strain A/equine/Richmond 1/07 had cytosine at position 4 of the 3′ vRNA in the three The two segments encoding the surface glycoproteins, HA and NA, contained a large number of amino acid 200bp 100bp 400bp 600bp 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ Seg. 1 (PB2) Seg. 2 (PB1) Seg. 3 (PA) Seg. 4 (HA) 200bp 100bp 400bp 600bp 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ Seg. 5 (NP) Seg. 6 (NA) Seg. 7 (M) Seg. 8 (NS) Figure 3 Agarose gel electrophoresis analysis of non-coding region PCR products. Agarose gel (2.5%) showing PCR fragments for the non-coding regions of A/equine/Richmond/1/07 influenza virus gene segments. The positions of molecular weight markers are indicated by black arrows. Bands of the expected size were visible in all lanes except for NA 5′, M 3′ and 5′. Figure 3 Agarose gel electrophoresis analysis of non-coding region PCR products. Agarose gel (2.5%) showing PCR fragments for the non-coding regions of A/equine/Richmond/1/07 influenza virus gene segments. The positions of molecular weight markers are indicated by black arrows. Bands of the expected size were visible in all lanes except for NA 5′, M 3′ and 5′. Sequencing Sequencing reactions were performed using the BigDye terminator sequencing kit version 3.1 (Applied Biosys- tems). M13 forward and reverse primers were used for gene segment PCR products, whilst for the non-coding regions the primers used for the PCR stage were reused, both at a final concentration of 80nM. The sequencing reactions were run on a 3130xl genetic analyzer (Applied Biosystems), and the resulting nucleotide sequences were visualised, assembled and edited using SeqMan II ver- sion 5.03 (DNAStar, Inc) and BioEdit version 7.0.5.3 (Ibis Pharmaceuticals Inc.). p 8. Gorman OT, Bean WJ, Kawaoka Y, Webster RG: Evolution of the nucleoprotein gene of influenza A virus. J Virol 1990, 64:1487–1497. p 8. Gorman OT, Bean WJ, Kawaoka Y, Webster RG: Evolution of the nucleoprotein gene of influenza A virus. J Virol 1990, 64:1487–1497. 9. Kawaoka Y, Krauss S, Webster RG: Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 1989, 63:4603–4608. 9. Kawaoka Y, Krauss S, Webster RG: Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics. J Virol 1989, 63:4603–4608. 10. Okazaki K, Kawaoka Y, Webster RG: Evolutionary pathways of the PA genes of influenza A viruses. Virology 1989, 172:601–608. 11. 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Binns MM, Daly JM, Chirnside ED, Mumford JA, Wood JM, Richards CM, Daniels RS: Genetic and antigenic analysis of an equine influenza H3 isolate from the 1989 epidemic. Arch Virol 1993, 130:33–43. Competing interests The authors declare that they have no competing interests. 19. de Wit E, Spronken MI, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA: Efficient generation and growth of influenza A/PR/8/34 from eight cDNA fragments. Virus Res 2004, 103:155–161. from eight cDNA fragments. Virus Res 2004, 103:155–161. cDNA synthesis Using a UNI-12 primer, as described by Hoffmann et al. [28], cDNA was transcribed by denaturing 2 μl RNA in the presence of UNI-12 (1 μM final concentration) and 7 μl water at 70°C for 10 minutes and then cooling on ice. Following this, dNTP mix (final concentration each 0.5 μM), 1 × First Strand buffer, 200U Superscript II reverse transcriptase (Invitrogen) and water to a final volume of 20 μl were added. The reaction mixture was incubated at 42°C for 45 minutes. Page 9 of 9 Page 9 of 9 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 Rash et al. Virology Journal 2014, 11:159 http://www.virologyj.com/content/11/1/159 QIAquick gel extraction kit (Qiagen) according to manufacturer’s directions. 6. Gorman OT, Donis RO, Kawaoka Y, Webster RG: Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus. J Virol 1990, 64:4893–4902. 7. Ito T, Gorman OT, Kawaoka Y, Bean WT, Webster RG: Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins. J Virol 1991, 65:5491–5498. Acknowledgements h k This work was supported by the Horserace Betting Levy Board, EIP 2011–2012. 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Additional files Additional file 1: GISAID EpiFlu database [19] accession numbers for PA-X sequences. Additional file 2: GenBank accession numbers for A/equine/ Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu database [19] accession numbers for other strains. Additional file 3: Alignment of predicted amino acid sequences for PA-X from EIV isolated in the UK between 2005 and 2013. Amino acids of the C-terminal PA-X domain only, following the +1 frameshift, are shown. Additional file 1: GISAID EpiFlu database [19] accession numbers for PA-X sequences. 15. Adeyefa CAO, Quayle K, McCauley JW: A rapid method for the analysis of influenza virus genes: application to the reassortment of equine influenza virus genes. Virus Res 1994, 32:391–399. Additional file 2: GenBank accession numbers for A/equine/ Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu database [19] accession numbers for other strains. 16. Bean WJ: Correlation of influenza A virus nucleoprotein genes with host species. Virology 1984, 133:438–442. 17. Dong BB, Xu CL, Dong LB, Cheng HJ, Yang L, Zou SM, Chen M, Bai T, Zhang Y, Gao RB, Li XD, Shi JH, Yuan H, Chen T, Zhu Y, Xiong Y, Yang S, Shu YL: A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic farm in Poyang Lake area. Biomed Environ Sci 2013, 26:546–551. Additional file 3: Alignment of predicted amino acid sequences for PA-X from EIV isolated in the UK between 2005 and 2013. Amino acids of the C-terminal PA-X domain only, following the +1 frameshift, are shown. Additional file 3: Alignment of predicted amino acid sequences for PA-X from EIV isolated in the UK between 2005 and 2013. Amino acids of the C-terminal PA-X domain only, following the +1 frameshift, are shown. 18. Skehel JJ, Hay AJ: Nucleotide sequences at the 5′ termini of influenza virus RNAs and their transcripts. Nucleic Acids Res 1978, 5:1207–1219. Additional file 1: GISAID EpiFlu database [19] accession numbers for PA-X sequences. Additional file 2: GenBank accession numbers for A/equine/ Richmond/1/07 and A/equine/Lincolnshire/1/07, and GISAID EpiFlu database [19] accession numbers for other strains. Additional file 3: Alignment of predicted amino acid sequences for PA-X from EIV isolated in the UK between 2005 and 2013. Amino acids of the C-terminal PA-X domain only, following the +1 frameshift, are shown. References 1. McGeoch D, Fellner P, Newton C: Influenza virus genome consists of eight distinct RNA species. Proc Natl Acad Sci 1976, 73:3045–3049. 26. Robertson JS: 5′ and 3′ terminal nucleotide sequences of the RNA genome segments of influenza virus. Nucleic Acids Res 1979, 6:3745–3757. 27. Lee HK, Tang JW-T, Kong DH-L, Koay ES-C: Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus. PLoS One 2013, 8:e64785. 2. Waddell GH, Teigland MB, Sigel MM: A new influenza virus associated with equine respiratory disease. J Am Vet Med Assoc 1963, 143:587–590. 3. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA: Antigenic and genetic evolution of equine H3N8 influenza A viruses. J Gen Virol 1996, 77:661–671. g g 28. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR: Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 2001, 146:2275–2289. 4. Bryant NA, Rash AS, Russell CA, Ross J, Cooke A, Bowman S, MacRae S, Lewis NS, Paillot R, Zanoni R, Meier H, Griffiths LA, Daly JM, Tiwari A, Chambers TM, Newton JR, Elton DM: Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007. Vet Microbiol 2009, 138:41–52. doi:10.1186/1743-422X-11-159 Cite this article as: Rash et al.: An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein. Virology Journal 2014 11:159. 5. Murcia PR, Wood JL, Holmes EC: Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus. J Virol 2011, 85:5312–5322.
https://openalex.org/W3006657590	https://infectagentscancer.biomedcentral.com/track/pdf/10.1186/s13027-020-0272-3.pdf	English	null	HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer	Infectious agents and cancer	2,020	cc-by	6,109	RESEARCH ARTICLE Open Access Liu et al. Infectious Agents and Cancer (2020) 15:6 https://doi.org/10.1186/s13027-020-0272-3 Liu et al. Infectious Agents and Cancer (2020) 15:6 https://doi.org/10.1186/s13027-020-0272-3 Abstract Cervical cancer is the fourth most common malignant tumor in women worldwide. The persistent infection of high-risk Human Papillomavirus (hrHPV) is considered to be the primary cause of this disease. As an innate immune receptor, the nucleotide-binding oligomerization domain protein-1 (NOD1) recognizes the pathogen-associated molecular pattern (PAMP), subsequently initiating immune responses. NOD1 is also involved in the apoptotic signaling pathway and mutates in many cancer cells. In the study, we revealed that NOD1 expression decreased during the progression of cervical intraepithelial neoplasia to cervical cancer and that HPV16 E6/E7 oncoproteins induced down-regulation of NOD1. Moreover, the activation of NOD1 promoted the apoptosis of HPV16-positive cervical cancer cells. The data indicated that the dysregulation of NOD1-mediated inflammation and apoptosis may contribute to cervical intraepithelial neoplasia progression and cervical cancer. Keywords: NOD1, Cervical intraepithelial neoplasia, Cervical cancer, HPV16 E6/E7, Apoptosis receptors (PRRs) recognize pathogen-associated molecu- lar patterns (PAMPs), triggering the intracellular signaling cascades and inducing the transcriptional ex- pression of inflammatory mediators that can eliminate the invading pathogens [10, 11]. The nucleotide-binding oligomerization domain protein-1 (NOD1) is one of the most important members of the NOD-like receptor (NLR) family, a type of PRR [12]. Like other NLRs, NOD1 consists of multiple leucine-rich repeats (LRRs), a nucleotide-binding oligomerization domain, and a cas- pase activation and recruitment domain (CARD) [13]. NOD1 is a cytosolic protein that recognizes the PAMPs on bacterial cell walls, such as peptidoglycan (PGN) and lipopolysaccharide (LPS) [10, 14]. The recognition of PAMPs by NOD1 leads to the activation of the NF-κB pathway, which drives the proinflammatory and anti- microbial responses [14, 15]. Since epithelial cells are the first barrier against pathogens, NOD1 has functional expression in epithelial cells of human digestive and re- productive systems [16]. Several studies suggested that NOD1 plays crucial role in the development of cancers, such as gastric cancer colorectal cancer, and breast cancer [17, 18]. NOD1 was found involved in TNF- induced apoptosis and the overexpression of NOD1 could enhance sensitivity to TriDAP-induced apoptosis in breast cancer cells [18]. Moreover, NOD1 shows HPV-mediated down-regulation of NOD1 inhibits apoptosis in cervical cancer Xubin Liu†, Hanyu Ma†, Lingyan Fei, Mengjie Jiang, Meng Xia, Lihong Bai, Xufang Pi, Shangwu Chen and Li Yu* © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: yuli5@mail.sysu.edu.cn †Xubin Liu and Hanyu Ma contributed equally to this work. Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China Introduction Cervical cancer is the fourth most common cancer in women, with a high mortality rate [1]. Although cervical cancer can be prevented by screening tests and Human Papillomavirus (HPV) vaccines in most western coun- tries, it is still the third most common cancer in devel- oping countries [2]. High-risk human papillomavirus (hrHPV) persistent infection is considered the main risk factor for cervical cancer development [3]. E6 and E7 are the major oncoproteins of hrHPV. E6 can direct p53 ubiquitin-mediated degradation, while E7 binds to Rb protein and regulates the G1/S checkpoint. E6 co- operation with E7 promotes the development of cervical cancer [4]. About 90% of HPV infections are eliminated within 3 years [5]. Only a few patients may develop cervical squamous intraepithelial lesion (SIL), and less than 1% of patients will progress to cervical cancer [6]. Therefore, in addition to HPV infection, other factors such as smoking and oral contraceptives may also be re- lated to the occurrence of cervical cancer [7]. Inflammation is another important risk factor in cer- vical cancer development [8, 9]. The pattern-recognition * Correspondence: yuli5@mail.sysu.edu.cn †Xubin Liu and Hanyu Ma contributed equally to this work. Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China * Correspondence: yuli5@mail.sysu.edu.cn †Xubin Liu and Hanyu Ma contributed equally to this work. Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China Immunohistochemistry Sections (4 μm) were obtained from formalin-fixed, paraffin-embedded normal (n = 36), CIN I (n = 50), CIN II (n = 52), CIN III (n = 44), and ISCCs (n = 61) tissues. Immunohistochemistry (IHC) staining was conducted as described previously [20] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16INK4A (p16INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions. Color was developed with diaminobenzidine (Dako Corp, CA 95051, USA) incubated for 5–10 min at room temperature. The double immunostaining of NOD1 and p16INK4A was performed with DouSPTM double staining kit (MaxVision, Fuzhou, China). Slides were counter- stained with haematoxylin and photos were taken by light microscopy. Cell transfection The HPV-negative C33A cells were seeded in 6-well plates for 24 h before transfection. Cells grown to 50–70% confluency were transfected using Lipofecta- mine®2000 (Invitrogen, Shanghai, China) for HPV16 wild type E6 and E7 gene transfection. Cells were di- vided into four groups for treatment: the blank group (without plasmid), the negative control (empty vector) group, the E6 group (E6-expressing plasmid), and E7 group (E7-expressing plasmid). A volume of 20 μl Lipofectamine®2000 reagent was used per microgram of DNA. Plasmids were constructed with the pEGFP-N1 vector. All of these plasmids were kindly provided by Prof. Xudong Tang (Institute of Biochemistry and Molecular Biology, Guangdong Medical University). Page 2 of 10 Liu et al. Infectious Agents and Cancer (2020) 15:6 Liu et al. Infectious Agents and Cancer positive staining cells; (1+), less than 25% positive stain- ing cells with weak intensity and focal distribution; (2+), 26–50% positive staining cells with moderate intensity and focal distribution; and (3+), more than 50% positive staining cells with strong intensity and diffuse distribution. tumor suppressor properties in colon [19]. NOD1 could protect the intestinal epithelial cells from injury, bacter- ial translocation, and colitis by regulating cell survival [19]. NOD1 dysfunction resulted in inflammation, in- creasing the risk of colitis-associated colon cancer [19]. As the cervix is often in contact with pathogenic micro- organisms, we speculate that NOD1-related signal path- way may be involved in the occurrence of cervical cancer. In the current study, we investigated the expres- sion of NOD1 in different grade cervical lesions, the impact of HPV infection on NOD1 expression, and the role of NOD1-mediated signaling in cervical cancer. Cell culture The cervical squamous cell cancer-derived cell lines C33A, SiHa, and ME180 were purchased from Shanghai Institute for Biological Sciences (Shanghai, China). SiHa and C33A cells were cultured in Minimum Essential Medium (MEM) (GIBCO, Shanghai, China) and ME-180 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, Shanghai, China) in a humidified atmosphere contain- ing 5% CO2 at 37 °C, supplemented with 10% fetal bovine serum (GIBCO). Patients and tissue sampling A total of 243 formalin-fixed, paraffin-embedded tis- sue samples were used in this study, including 50 cer- vical intraepithelial neoplasia (CIN) I, 52 CIN II, 44 CIN III, 61 invasive squamous cell carcinomas (ISCCs) samples and 36 normal control cervical tis- sues obtained from surgically removed uteruses that were reported to be either hysteromyoma or adeno- myosis. The tissues were sectioned and histologically diagnosed by pathologists at the Department of Path- ology, the First Affiliated Hospital of Sun Yat-Sen University. ISCCs were diagnosed when the epithelial basement membrane was breached. In addition, 30 frozen samples including 20 normal cervical epithelia and 10 ISCCs tissues were included in the study. None of the patients has received radiotherapy or chemotherapy. Immunocytochemistry ll Cells were grown on sterile glass coverslips or 35 mm culture dishes. The adherent cells were rinsed briefly in phosphate-buffered saline (PBS) and incubated in 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. After washing 3 times with PBS, the cells were incubated with PBS containing 0.5% Triton X-100 for 20 min, then blocked in 1% BSA in PBST (PBS with 0.1% Tween 20) for 30 min, and incubated in the diluted antibodies against NOD1 and p16INK4A overnight at 4 °C. Color was developed as described in immunohisto- chemistry section. Result NOD1 expression decreased with CIN progression In order to understand the possible function of NOD1 in cervical cancer, we first profiled its expres- sion in CINs and ISCCs tissues by immunostaining. We found that 97.2% of the normal cervix (35/36) and 86.0% of CIN I samples (43/50) were stained positive for NOD1 (Table 1). The 47.9% of high-grade squamous intraepithelial lesion (HSIL) (46/96) con- sisting of CIN II and CIN III and 14.8% of ISCCs (9/ 61) were positive for NOD1. In term of immunostain- ing intensity, the strong NOD1 expression (2+ and 3+) was detected in 77.8% normal cervical, 30.0% CIN I, 9.6% CIN II, and 6.8% CIN III samples, respect- ively, while no strong staining was observed in ISCCs (Fig. 1, Table 1). Our results indicated that NOD1 positive rates, particularly immunostaining intensity, gradually decreased along with the progression of cer- vical lesions. When NOD1 protein was examined using Western blot in 3 paired tumor (T) and adja- cent normal (N) tissues from cervical cancer patients, NOD1 level in normal tissues was higher than that in cancer tissues (Fig. 2a). Compared with normal Real-time quantitative reverse transcription PCR Real-time quantitative reverse transcription PCR Total RNA was extracted from cultured cells or fresh cervical tissues using Trizol reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol. Total RNA (1 μg) was converted to cDNA with Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). The primers for NOD1, RIP2, and GAPDH were synthesized by Sangon (Shanghai, China). The primer sequences were 5′-CCACTTCACAGCTGGAGACA-3′ (forward) and 5′- TGAGTGGAAGCAGCATTTTG-3′ (reverse) for NOD1; 5′-ACGTCTGCAGCCTGGTATAGC-3′ (forward) and 5′-CATCTAGCGACTGGTTAAG-3′ (reverse) for RIP2; and 5′-AGAAGGCTGGGGCT CATTTG-3′ (forward) and 5′- AGGGGCCATC CACAGTCTTC-3′ (reverse) for GAPDH. qPCR was carried out in triplicate in a 20 μl reaction volume using FastStart Universal Probe Master (ROX) (Roche, Mannheim, Germany) and Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, California, USA). The relative quantity of the target mRNA was normalized to the level of GAPDH mRNA level. Relative gene expression was evaluated using the 2-ΔΔCt method [22]. Cell apoptosis assay Cell apoptosis was evaluated by flow cytometry with Annexin V-FITC assay kit (KeyGEN BioTECH, Nanjing, China). Approximate 3 × 105 typsin pre-treated cells were centrifuged at 2000 rpm for 5 min, rinsed twice Staining intensity was graded according to the follow- ing criteria described in previous studies [20, 21]: (−), no Liu et al. Infectious Agents and Cancer (2020) 15:6 Page 3 of 10 Liu et al. Infectious Agents and Cancer Immobilon Western Chemiluminescent HRP Sub- strate (Millipore, Burlington, Massachusetts, USA). Immobilon Western Chemiluminescent HRP Sub- strate (Millipore, Burlington, Massachusetts, USA). with ice-cold PBS, and resuspended in Annexin binding buffer containing Annexin V-FITC and propidium iod- ide. Cells were incubated at room temperature for 15 min and analyzed using a Becton Dickinson FACScan (BD Biosciences, San Jose, California, USA). Cyclohexi- mide (CHX) was obtained from Sigma (St. Louis, Missouri, USA). γ-D-Glu-mDAP (iE-DAP) was pur- chased from Invivogen (Carlsbad, California, USA). Statistical analysis Statistical analysis IBM SPSS 22.0 and GraphPad Prism 6.01 were used for statistical analysis of the data. The Kruskal–Wallis test was used to compare the differences of cumulative NOD1 and p16INK4A expression with CIN progression and ISCCs among the sample groups. The multiple com- parisons between every two groups were examined by Bonferroni test, and a p-value ≤0.05 was considered sta- tistically significant. The correlation of NOD1 and p16INK4A expression was examined by Spearman correl- ation test. Student’s t-test was used to compare the dif- ferences of NOD1 mRNA expression in normal cervical epithelia, CINs, and ISCCs. Western blot Diffuse and strong staining of p16INK4A was observed in tumor epithelial cells of carcinoma nests in ISCC (S). NOD1 staining was only detected in normal cervical squamous epithelium (d) with double staining of NOD1 (red) and p16INK4A (black), and NOD1 staining was not observed within atypical hyperplasia cells of CINs (H, L, P), where p16INK4A was positive. Strong p16INK4A staining but no NOD1 staining was observed in the tumor cells of carcinoma nests (t) Fig. 1 Expression of NOD1 and P16INK4A in normal cervix, CIN I, CIN II, CIN III and ISCC tissues detected by immunohistochemistry. Hematoxylin and eosin (HE) staining showed that in comparison to normal cervical squamous epithelium (a), atypical hyperplasia of squamous epithelium was restricted within lower third in CIN I (e), lower two-thirds in CIN II (i), or exceeded over lower two-thirds in CIN III (m), respectively. Carcinoma invaded into muscle tissue in ISCC (q). NOD1 showed the strongest immunoreactivity in normal cervical squamous epithelium (b), and the staining intensity of NOD1 gradually decreased from CIN I (f), CIN II (j) to CIN III (n). No NOD1 immunostaining was observed in cells within carcinoma nests (r). In contrast to NOD1, no p16INK4A staining was detected in the normal cervical tissue (c). The expression of p16INK4A gradually increased from CIN I (g), CIN II (K) to CIN III (o), and p16INK4A positive cells were diffusely distributed within the atypical hyperplasia tissue. Diffuse and strong staining of p16INK4A was observed in tumor epithelial cells of carcinoma nests in ISCC (S). NOD1 staining was only detected in normal cervical squamous epithelium (d) with double staining of NOD1 (red) and p16INK4A (black), and NOD1 staining was not observed within atypical hyperplasia cells of CINs (H, L, P), where p16INK4A was positive. Strong p16INK4A staining but no NOD1 staining was observed in the tumor cells of carcinoma nests (t) cervical tissues, the expression of NOD1 mRNA in cervical cancers decreased (Fig. 2b), although the dif- ference was not statistically significant. was stained positive (Table 2). An increased intensity of p16INK4A staining was also observed along with the increased histopathologic grade of cervical intraepithe- lial lesions (Fig. 1). The differences of positive stain- ing levels for p16INK4A among normal controls, CINs, and ISCCs were statistically significant, and so was CINs and ISCCs (P < 0.005) (Table 2). Western blot When double immunostaining of NOD1 and p16INK4A was per- formed, NOD1 showed high expression in normal cervical tissues and gradually decreased expression Western blot The proteins of cells or fresh tissues were extracted using Whole Cell Lysis Assay (KeyGEN BioTECH, Nanjing, China). Proteins concentration was deter- mined using BCA Protein Assay Kit (Beijing CoWin Biotech, Beijing, China). Proteins (30μg) were sepa- rated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then trans- ferred to polyvinylidene difluoride (PVDF) mem- branes (Millipore, Burlington, Massachusetts, USA). Membranes were blocked with 5% nonfat dried milk and incubated overnight with antibodies against NOD1 (MAB7090, 1:500; R&D Systems, Minneapolis, Minnesota, USA) or GAPDH (#2118, 1:8000; Cell Signaling Technology, Danvers, Massachusetts, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Immunoreactive bands were detected by using Table 1 Expression of NOD1 in CINs and ISCCs Tissues Degree of immunoreactivity(%) – 1+ 2+ 3+ Normala 1/36 (2.8) 7/36 (19.4) 15/36 (41.7) 13/36 (36.1) CIN Ib 7/50 (14.0) 28/50 (56.0) 14/50 (28.0) 1/50 (2.0) CIN IIc 25/52 (48.1) 22/52 (42.3) 5/52 (9.6) 0/52 (0.0) CIN IIId 25/44 (56.8) 16/44 (36.4) 3/44 (6.8) 0/44 (0.0) ISCCse 52/61 (85.2) 9/61 (14.8) 0/61 (0.0) 0/61 (0.0) CIN cervical intraepithelial neoplasia, ISCCs invasive squamous cell carcinomas a-bP < 0.005, a-cP < 0.005, a-dP < 0.005, a-eP < 0.005, b-cP < 0.005, b-dP < 0.005, b-eP < 0.005, c-dP > 0.05, c-eP < 0.005, d-eP < 0.05; Liu et al. Infectious Agents and Cancer Page 4 of 10 Liu et al. Infectious Agents and Cancer (2020) 15:6 Fig. 1 Expression of NOD1 and P16INK4A in normal cervix, CIN I, CIN II, CIN III and ISCC tissues detected by immunohistochemistry. Hematoxylin and eosin (HE) staining showed that in comparison to normal cervical squamous epithelium (a), atypical hyperplasia of squamous epithelium was restricted within lower third in CIN I (e), lower two-thirds in CIN II (i), or exceeded over lower two-thirds in CIN III (m), respectively. Carcinoma invaded into muscle tissue in ISCC (q). NOD1 showed the strongest immunoreactivity in normal cervical squamous epithelium (b), and the staining intensity of NOD1 gradually decreased from CIN I (f), CIN II (j) to CIN III (n). No NOD1 immunostaining was observed in cells within carcinoma nests (r). In contrast to NOD1, no p16INK4A staining was detected in the normal cervical tissue (c). The expression of p16INK4A gradually increased from CIN I (g), CIN II (K) to CIN III (o), and p16INK4A positive cells were diffusely distributed within the atypical hyperplasia tissue. NOD1 expression was inversely correlated to expression of p16INK4A We investigated whether NOD1 expression is related to p16INK4A expression. We found that more than 98% CINs and ISCCs samples were stained positive for p16INK4A, but none of the normal cervical tissues Page 5 of 10 Liu et al. Infectious Agents and Cancer Liu et al. Infectious Agents and Cancer (2020) 15:6 (2020) 15:6 B. Relative expression of NOD1 Normal (n=20) ISCC (n=10) 0 5 10 15 Normal ISCC A. Fig. 2 NOD1 expression in frozen fresh ISCC and normal cervical tissues. a. Expression of NOD1 protein in 3 paired tumor (T) and adjacent normal (N) tissues from 3 patients was detected by Western blot. b. Quantitative comparison of the NOD1 mRNA expression between ISCC and normal cervical tissues using RT-PCR with GAPDH as the control house keeping gene. The ratios of NOD1/GAPDH expression were calculated from triplicate. (P = 0.22) B. Fig. 2 NOD1 expression in frozen fresh ISCC and normal cervical tissues. a. Expression of NOD1 protein in 3 paired tumor (T) and adjacent normal (N) tissues from 3 patients was detected by Western blot. b. Quantitative comparison of the NOD1 mRNA expression between ISCC and normal cervical tissues using RT-PCR with GAPDH as the control house keeping gene. The ratios of NOD1/GAPDH expression were calculated from triplicate. (P = 0.22) with the increased histopathologic grade in CINs, while p16INK4A expression increased with the progres- sion from CINs to ISCCs. Interestingly, no NOD1 expression was detectable in p16INK4A positive cells (Fig. 1). The results indicated that the expression of NOD1 was inversely correlated to the expression of p16INK4A (rs = −0.382, P < 0.001) (Table 3). The expression of NOD1 was examined by immuno- cytochemistry in 3 cervical cancer cells: SiHa infected with type 16 HPV, the predominant HPV type (46– 63%) in cervical squamous cell carcinoma [23]; ME180 infected with type 68 HPV; and C33A without HPV infection. The NOD1 immunostaining intensity was stronger in HPV-negative C33A cells compared to HPV16-positive SiHa cells and HPV68-positive ME180 cells (Fig. 3a). Immunocytochemistry results were confirmed by RT-PCR and Western blot (Fig. 3 b, c), suggesting that HPV, especially hrHPV (type16), may be related to the down-regulation of NOD1 in cervical cancer cells. HPV16 E6/E7 down-regulated the expression of NOD1 Since the expression of NOD1 decreased with the progression of CINs, and was negatively correlated to the expression of p16INK4A, we analyzed whether the expression of NOD1 was related to hrHPV infection. Table 2 Expression of p16INK4A in CINs and ISCCs Tissues Degree of immunoreactivity(%) – 1+ 2+ 3+ Normala 36/36 (100.0) 0/36 (0.0) 0/36 (0.0) 0/36 (0.0) CIN Ib 1/50 (2.0) 31/50 (62.0) 15/50 (30.0) 3/50 (6.0) CIN IIc 1/52 (1.9) 5/52 (9.6) 27/52 (51.9) 19/52 (36.5) CIN IIId 0/44 (0) 6/44 (13.6) 25/44 (56.8) 13/44 (29.5) ISCCse 0/61 (0) 16/61 (26.2) 14/61 (23.0) 31/61 (50.8) CIN cervical intraepithelial neoplasia, ISCCs invasive squamous cell carcinomas a-bP < 0.005, a-cP < 0.005, a-dP < 0.005, a-eP < 0.005, b-cP < 0.005, b-dP < 0.005, b-eP < 0.005, c-dP > 0.05, c-eP > 0.05, d-eP > 0.05; Table 3 A cross tabulation of NOD1 and p16INK4A expression NOD1 expression level (cases) Total – 1+ 2+ 3+ p16INK4A expression level (cases) – 5 7 14 12 38 1+ 23 20 13 2 58 2+ 46 31 4 0 81 3+ 36 24 6 0 66 Total 110 82 37 14 243 243 specimens that include 146 CINs, 61 ISCCs and 36 controls Correlation of NOD1 and P16INK4A expression: rs = −0.382, p < 0.001 Table 3 A cross tabulation of NOD1 and p16INK4A expression NOD1 i l l ( ) T t l Liu et al. Infectious Agents and Cancer (2020) 15:6 Page 6 of 10 Liu et al. Infectious Agents and Cancer (2020) 15:6 ous Agents and Cancer (2020) 15:6 Fig. 3 Expression of NOD1 in 3 cervical squamous carcinoma cells was detected by immunocytochemistry (a), quantitative real-time PCR (b) and Western blot (c). NOD1 showed strongest expression in the HPV (−) C33A cells and was weakest in the HPV16 (+) SiHa cells. P < 0.05, **P < 0.0001 Fig. 3 Expression of NOD1 in 3 cervical squamous carcinoma cells was detected by immunocytochemistry (a), quantitative real-time PCR (b) and Western blot (c). NOD1 showed strongest expression in the HPV (−) C33A cells and was weakest in the HPV16 (+) SiHa cells. P < 0.05, ***P < 0.0001 Fig. 3 Expression of NOD1 in 3 cervical squamous carcinoma cells was detected by immunocytochemistry (a), quantitative real-time PCR (b) and Western blot (c). NOD1 showed strongest expression in the HPV (−) C33A cells and was weakest in the HPV16 (+) SiHa cells. HPV16 E6/E7 down-regulated the expression of NOD1 P < 0.05, **P < 0.0001 To explore the mechanism of down-regulation of NOD1 expression, HPV-negative C33A cells were trans- fected with E6 and E7 expressing plasmids or the empty vectors (VECT). When HPV16 E6 or E7 was ectopically expressed in C33A cells, the NOD1 expression de- creased significantly in both protein (Fig. 4a, b) and mRNA levels (Fig. 4c). RIP2 mRNA level was also sig- nificantly down-regulated in HPV16 E6/E7-expressing cells (Fig. 4d). significantly higher than that of iE-DAP or CHX alone (P < 0.005) (Fig. 5b). The results indicated that NOD1 can enhance the sensitivity of HPV16-positive cells to apoptosis induced by CHX and the decrease of NOD1 expression may contribute to the apoptosis resistance and the development of cervical cancer. Discussion Human papillomavirus infection is an essential causal factor of cervical cancer, but the role of other etiological factors is unclear [24]. Cervix is often exposed to bacteria, and Gram-negative bacteria- related inflammation has been reported [20]. NOD1, an innate immune receptor, recognizes specific mo- lecular patterns expressed on the cell walls of Gram- negative bacteria and initiates inflammatory response [12]. Our results demonstrated that HPV infection induced down-regulation of NOD1 in cervical intrae- pithelial lesions and ISCCs. The activation of NOD1 increased CHX-induced apoptosis The reduction of apoptosis and dysregulation of cell proliferation caused by hrHPV are the main cause of cervical cancer. We investigated the effect of NOD1 on apoptosis of hrHPV-infected cervical cancer cells. When HPV16-positive SiHa cells were stimulated with iE-DAP, the mRNA level of NOD1 was posi- tively correlated with the dose of iE-DAP (Fig. 5a). In the presence of CHX, the exposure of SiHa cells to iE-DAP induced about 13% cell death, which was hrHPV E6 and E7 oncoproteins have been found to inhibit the expression of Toll-like receptor 9 Liu et al. Infectious Agents and Cancer (2020) 15:6 Page 7 of 10 Liu et al. Infectious Agents and Cancer (2020) 15:6 Liu et al. Infectious Agents and Cancer Page 7 of 10 (2020) 15:6 Fig. 4 HPV oncoproteins down-regulated the expression of NOD1 and RIP2 in C33A cells. NOD1 expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by immunocytochemistry (a), Western blot (b), and RT-PCR (c). RIP2 mRNA expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by RT-PCR (d). BLANK, without plasmid, VECT, empty vector, E6, E6-expressing plasmids, and E7, E7-expressing plasmids Fig. 4 HPV oncoproteins down-regulated the expression of NOD1 and RIP2 in C33A cells. NOD1 expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by immunocytochemistry (a), Western blot (b), and RT-PCR (c). RIP2 mRNA expression was down-regulated in the cancer cells transfected with E6- or E7-expressing plasmids detected by RT-PCR (d). BLANK, without plasmid, VECT, empty vector, E6, E6-expressing plasmids, and E7, E7-expressing plasmids associated with cell apoptosis and development of breast cancer and oral cancer [18, 31]. Discussion The activa- tion of NOD1 by ligand up-regulates the RIP2 expression and turns on the downstream signaling pathways, which in turn initiates multiple cellular responses including the TAK1-mediated release of proinflammatory cytokines and NOD1-dependent apoptosis through caspase 8 [32]. iE-DAP is a dipep- tide in bacterial peptidoglycan (PGN), and recogni- tion of iE-DAP by NOD1 can induce a signaling cascade that promotes the activation of NF-κB and the production of inflammatory cytokines [29]. We found that NOD1 in cervical cancer cells can be activated by iE-DAP, consequently facilitating the CHX-induced apoptosis. Cycloheximide is a glutari- mide antibiotic that blocks the translation of mRNA and promotes apoptosis through inhibiting the expression of antiapoptotic molecules such as FLIP [33, 34]. Our results suggested that the down- regulation of NOD1 by HPV16 E6/E7 can reduce (TLR9), an innate immune receptor of TLRs family [25]. p16INK4A is a surrogate marker for the infection of hrHPV, and p16INK4A immunostaining has been suggested as an alternative approach to histology re- view, which is valuable for confirming the cervical intraepithelial lesions in clinicopathologic diagnosis [26–28]. We previously found that the expression of TLR4 was inversely correlated to p16INK4A in cer- vical intraepithelial lesions and ISCCs [20]. The HPV16 oncoproteins-mediated suppression of NOD1 may attenuate the pathogen-induced inflammation and subsequent elimination of pathogens. In addition to innate immune response, NOD1 is also involved in the activation of the apoptosis sig- naling pathway through its interaction with the CARD domain of RIP2. RIP2 is considered to be an important component of the multiprotein signaling complex that mediates innate immune responses in- duced by the activation of NOD1 [29, 30]. The ab- normal expression of NOD1 and RIP2 has been Liu et al. Infectious Agents and Cancer (2020) 15:6 Page 8 of 10 Liu et al. Infectious Agents and Cancer Fig. 5 The activation of NOD1 increased CHX-induced apoptosis. NOD1 expression increased in SiHa cells treated with iE-DAP (P < 0.005, *P < 0.0001) (a). The NOD1 activation enhanced CHX-induced apoptosis. HPV16-positive SiHa cells were treated with iE-DAP in the presence or absence of CHX. Cell viability was measured by flow cytometry. The apoptosis of SiHa cells increased significantly in the presence of CHX (P < 0.005) (b) Fig. 5 The activation of NOD1 increased CHX-induced apoptosis. NOD1 expression increased in SiHa cells treated with iE-DAP (P < 0.005, ***P < 0.0001) (a). The NOD1 activation enhanced CHX-induced apoptosis. Acknowledgements Declared none. 12. Mukherjee T, Hovingh ES, Foerster EG, Abdel-Nour M, Philpott DJ, Girardin SE. NOD1 and NOD2 in inflammation, immunity and disease. Arch Biochem Biophys. 2019;670(30):69–81. Funding Thi d This study was funded by the National Natural Science Foundation of China (No. 31670788 and No. 81172485), the Ph.D. Program Foundation of Ministry of Education of China (No. 20130171110007), and Open Fund of Guangdong Key Laboratory of Pharmaceutical Functional Genes (No.2014B030301028 and No.2017B030314021). 17. Velloso FJ, Trombetta-Lima M, Anschau V, Sogayar MC, Correa RG. NOD-like receptors: major players (and targets) in the interface between innate immunity and cancer. Biosci Rep. 2019;39(4):1–21. 18. da Silva CJ, Miranda Y, Austin-Brown N, Hsu J, Mathison J, Xiang R, Zhou H, Li Q, Han J, Ulevitch RJ. Nod1-dependent control of tumor growth. Proc Natl Acad Sci U S A. 2006;103(6):1840–5. Ethics approval and consent to participate 20. Yu L, Wang L, Li M, Zhong J, Wang Z, Chen S. Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia. Cancer Immunol Immunother. 2010;59(7):1021–8. All procedures performed in studies involving human participants were in accordance with the ethical standards of the First Affiliated Hospital of Sun Yat-sen University research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All procedures performed in studies involving human participants were in accordance with the ethical standards of the First Affiliated Hospital of Sun Yat-sen University research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 21. Kim WY, Lee JW, Choi JJ, Choi CH, Kim TJ, Kim BG, Song SY, Bae DS. Increased expression of toll-like receptor 5 during progression of cervical neoplasia. Int J Gynecol Cancer. 2008;18(2):300–5. Consent for publication not applicable. 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8. Availability of data and materials The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. 19. Chen GY, Shaw MH, Redondo G, Núñez G. The innate immune receptor Nod1 protects the intestine from inflammation-induced tumorigenesis. Cancer Res. 2008;68(24):10060–7. Received: 19 May 2019 Accepted: 12 January 2020 Received: 19 May 2019 Accepted: 12 January 2020 Received: 19 May 2019 Accepted: 12 January 2020 24. Cohen PA, Jhingran A, Oaknin A, Denny L. Cervical cancer. Lancet. 2019 Jan 12;393(10167):169–82. 25. Hasan UA, Bates E, Takeshita F, Biliato A, Accardi R, Bouvard V, Mansour M, Vincent I, Gissmann L, Iftner T, Sideri M, Stubenrauch F, Tommasino M. TLR9 expression and function is abolished by the cervical cancer-associated human papillomavirus type 16. J Immunol. 2007;178(5):3186–97. Discussion HPV16-positive SiHa cells were treated with iE-DAP in the presence or absence of CHX. Cell viability was measured by flow cytometry. The apoptosis of SiHa cells increased significantly in the presence of CHX (P < 0.005) (b) cell apoptosis, which may promote the development and progression of cervical cancer. cervicitis. It has been confirmed that the chronic inflammation of the cervix may be related to high- grade CIN development. Combined with the down- regulation of NOD1 expression in CINs and ISCCs, we suggest that the dysregulation of NOD1- mediated inflammation may be an important risk factor in cervical intraepithelial neoplasia and cer- vical cancer. Previous studies have shown that NOD1 signaling protects the gut against injury and inflammation- induced tumorigenesis by maintaining the integrity of the intestinal epithelium. Similar to the gut, the cervix is exposed to a bacterial environment, includ- ing symbionts and pathogens that can cause Page 9 of 10 Page 9 of 10 Page 9 of 10 Liu et al. Infectious Agents and Cancer (2020) 15:6 Liu et al. Infectious Agents and Cancer Authors’ contributions 13. Caruso R, Warner N, Inohara N, Núñez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014;41(6):898–908. LXB and MHY did experiments, interpreted the data, and wrote the manuscript. LYF and MJJ were involved in data analysis. MX collected some tissue samples. LHB performed cell culture and Western blotting assay. XFP read all cervical tissue slides and analyzed relevant data. SWC designed the study, provided advice, and critically read the manuscript. LY designed the study, provided advice, and read all cervical tissue slides. All authors read and approved the final Manuscript. 14. Inohara N, Ogura Y, Chen FF, Muto A, Nuñez G. Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem. 2001;276(4):2551–4. 15. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jéhanno M, Viala J, Tedin K, Taha MK, Labigne A, Zähringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ. Nod1 detects a unique muropeptide from gram- negative bacterial peptidoglycan. Science. 2003;300(5625):1584–7. 16. Uehara A, Fujimoto Y, Fukase K, Takada H. Various human epithelial cells express functional toll-like receptors, NOD1 and NOD2 to produce anti- microbial peptides, but not proinflammatory cytokines. Mol Immunol. 2007; 44(12):3100–11. Competing interests 23. Clifford GM, Smith JS, Plummer M, Muñoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. Br J Cancer. 2003;88(1):63–73. The authors declare that they have no competing interests. The authors declare that they have no competing interests. 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https://openalex.org/W2898381427	https://zenodo.org/records/3408738/files/020917021%20Inyang%20et%20al.pdf	English	null	Effect of Diazinon on Organosomatic Indices and Behavioural Responses of Clarias gariepinus (a Common Niger Delta Wetland Fish)	Greener Journal of Biological Science	2,017	cc-by	3,354	ABSTRACT Lethal and sublthal concentrations of toxicants affect on fish behaviour, reproduction, growth and general physiological processes in exposed organism. The purpose of this research was to determine the organosomatic indices and behavioural responses of Clarias gariepinus exposed to diazinon. Adult fish (mean length, 35.24±2.80cm) were acclimated to laboratory conditions for 7 days and then exposed to varying sublethal concentrations of diazinon (1.00, 2.50, 5.00, 7.50 and 10.0mg/l) in a semi-static bioassay for 30 days. Organosomatic index was determined in the liver, spleen, kidney and heart. Fish behaviour was also monitored for several hours on and before exposure to diazinon. The final condition factor values were not statistically significant (P>0.05). All organosomatic indices tested were significant, unveiling the effect of diazinon on the probe organism organs. Behavioural aberrations were also observed in experimental group/treatment that received the highest dose of the toxicant. Based on the results, diazinon used for agricultural are anthropogenic purposes could have a devastating effect on Clarias gariepinus (a common Niger Delta wet land fish) Keywords: Clarias gariepinus, organosomatic indices, diazinon, pesticides. Effect of Diazinon on Organosomatic Indices and Behavioural Responses of Clarias gariepinus (a Common Niger Delta Wetland Fish) Iniobong R. Inyang1, A.O. Ollor2, and Sylvester C. Izah1 1Environmental Toxicity Research Unit, Department of Biological Sciences, Niger Delta University, Wilberforce Island, Bayelsa State, Nigeria 2Department of Medical Laboratory Science, Rivers state University of Science and Technology, Port Harcourt, Rivers State, Nigeria Corresponding Author’s E-mail: dr.inyang2009 @gmail .com *Corresponding Author’s E-mail: dr.inyang2009 @gmail .com DOI: http://doi.org/10.15580/GJBS.2017.2.020917021 ISSN: 2276-7762 ICV: 5.99 Greener Journal of Biological Sciences Vol. 7 (2), pp. 015-019, February 2017 ISSN: 2276-7762 INTRODUCTION 7 (2), pp. 015-019, February 2017 ISSN: 2276-7762 ICV: 5.99 ISSN: 2276-7762 I ISSN: 2276-7762 Greener Journal of Biological Sciences ISSN: 2276-7762 ICV: 5.99 pesticides such as pyrethroids lindane and disulpoton. The mechanism of toxic effect of diazinon is the same as those of other organophosphate substances (Leudke and Bartley, 2006). pesticides such as pyrethroids lindane and disulpoton. The mechanism of toxic effect of diazinon is the same as those of other organophosphate substances (Leudke and Bartley, 2006). g p p ( y ) Lethal and sublethal concentrations of toxicants usually have adverse effect on fish behaviour, haematology, histopathology, growth, reproduction, feeding and general physiological developments of exposed organisms (Butler, 1971). This research work examines the suitability of somatic indices of Clarias gariepinus as yet effective biomarkers of diazinon induced stress in fish. Data analysis The data were subjected to analysis of variance (ANOVA). Where variation exist, Duncan multiple range test (DMRT) were used determine the source of the observed difference between treatments at P=0.05 (Wahua, 1999). MATERIALS AND METHODS Fish samples for this study were purchased from a private fish farm at Abuloma road, Port Harcourt, Rivers State of Nigeria. The fish samples were transported to the Department of Fisheries and Aquatic environment, Rivers State University of Science and Technology, Port Harcourt, Nigeria, were the assays were conducted. Fifty-eight adult Clarias gariepinus (mean weight 275±53.12g, mean length, 35.24±2.80cm) were acclimatized individually in rectangular aquaria for one week during which they were fed once a day (9.00-11.00 hours) with 35% crude protein diet at 1% biomass. p Sublethal concentrations of diazinon for the assay (1.0, 2.5, 5.0, and 10.0mg/l) were determined based on the range finding test (Inyang, 2008). These were prepared by transferring 0.02, 0.013, 0.25, 0.37 and 0.5ml, respectively of the original concentration of diazinon and making it up to 30L with borehole water in the aquaria, 30L of the diluent water was used as control. Four replications of each treatment level (concentrated) and control were set up by introducing fishes individually into each aquarium. Daily renewal bioassay was employed during the experiment which lasted for 30 days. The physico-chemical properties of the water used for the fish bioassay was carried out using standard methods (APHA, 1998); The result of the water quality were; temperature 26oC, pH 6.620 – 6.37, dissolved oxygen 5.38 – 7.21 mg/l, alkalinity 15.25 – 17.09mg/l, conductivity 99.50 – 136.12 µs/cm and turbidity 0.042 – 0.50NTU. y µ y After 30 days of exposure, fishes were weighed before sacrifice for collection of target organs (heart, liver, kidney, and spleen) for organosomatic index text (OIT). The OIT was determined by weighing each organ on digital weighing balance. Organosomatic index for all the organs was calculated using the expression: x 100 (Simeon, 2007) While the condition factor (k) of each specimen was calculated using foulton’s formular: K = x 100 W = weight of fish L = Length of fish K = x 100 W = weight of fish L = Length of fish K = x 100 W = weight of fish L = Length of fish Fishes were closely observed for abnormal or adverse behavioural change outside the normal range of variability, during and after exposure to diazinon. INTRODUCTION In spite of the immerse contribution of pesticides to agriculture and household uses, they have been reported to have negative ecological consequences on the environment (Far et al., 2012). The food chain/web is majorly affected. Pesticides applied in agricultural field or careless management of used pesticide containers find their way into the aquatic environment, either through water run-off and or as aerosols carried by wind (Inyang et al., 2016a-f, 2017). , ) Pesticides are classified according to their function including herbicides, insecticides, fungicides, acaricides, fumigants etc. They are also classified based on their chemical composition. Irrespective of their mode of classification, they affects target organisms, and in some cases non-target organisms as well. Organophosphate insecticides are widely used to control a variety of agricultural pests as well as ectoparasites in fish. The organophosphorus based insecticides are derived from the phosphoric or phosphorothioic acid (Díaz-Resendiz et al., 2015). Organophosphorus pesticides are group of highly toxic compounds and they are readily available commercially for domestic, agricultural and industrial purposes (Aardema, 2008; Kapka-Skrzypczak et al., 2011). They account for about 50% of all insecticides applied worldwide (Yang and Deng, 2007). Organophosphorus insecticides are toxic because of their inhibition of the enzyme acetylcholinesterase (Al-Ghanim, 2014; Muranli et al., 2015). These inhibition processes of the enzyme may results in the accumulation of acetylcholine in nerve tissue and effector organs, with the main site of action being the peripheral nervous system (PNS) (National Research Council, 1982; Hodgson et al., 2004). g p p y ( ) ( g ) Diazinon (an organophosphrate insecticide) is one of the most frequently used pesticides in the world (Muranli et al., 2015). According to Al-Ghanim (2014), diazinon is the most generally used pesticide in agricultural field and environmental health. In Nigeria, it is sold in an open market and it is well embraced by farmers. Diazinon is mostly used for the control flies, lice, insect pests (of ornamental plants and food crops, nematodes and soil insects in lawns and croplands (Vajargah et al., 2013), and other insect pest such as cuttworms, wireworms and maggots in soil and ectoparasites on sheep (Virtues and Clayton, 1997). Additionally diazinon uses in urban areas include dormant sprays on fruits trees, professional landscape and pest control services (Baily et al., 2 0 0 0 ) . Diazinon may be found in formulations with a variety of other 15 www.gjournals.org Greener Journal of Biological Sciences Vol. www.gjournals.org Organosomatic index Our data unveiled a statistically significant values in all somatic indices tested. A slight deviation in values was recorded in the cardiosomatic (Heart) and Spleenosomatic indices. Renosomatic (kidney) and spleenosomatic (Spleen) indices values reduced as the concentration of the toxicant increases. These fluctuation in values showed the effect of diazinon on these organs. The results were similar to those of Clarias gariepinus exposed to monoclotophos insecticide (an organophosphorus insecticide) for 30 days (Simeon, 2007). Table 1: Condition factor of Clarias gariepinus exposed to diazinon for 30 days (mean ±SD) Table 1: Condition factor of Clarias gariepinus exposed to diazinon for 30 days (mean ±SD) Conc of diazinon (mg/l Initial Condition (K1) % of Control Final Condition (K2) % of Control 0.00 0.68 ± 0.14a 100 0.69 ± 0.12a 100 1.00 0.88±0.14a 129 0.78 ±0.15a 113 2.50 0.72 ±0.15a 106 0.72 ±0.15a 104 5.00 0.76 ±0.12a 112 0.68 ±0.69a 99 7.50 0.67 ±0.08a 99 0.59 ±0.06a 86 10.0 0.70 ±0.07a 103 0.63 ±0.07a 91 Means with the same superscript in the row are not significantly different (p<0.05). Table 2: Condition factor of Clarias gariepinus exposed to diazinon for 30 days (mean ±SD) Conc of diazinon (mg/l Renasomatic (kidney) Cardiosomatic (Heart Hepetosomatic (Liver Spleenosomatic (Spleen) 0.00 0.01±0.02b 0.18±0.03a 1.29±0.32ab 0.23±0.03b 1.00 0.8±0.03a 0.15±0.02ab 1.28±0.45ab 0.9±0.03b 2.50 0.09±0.02a 0.14±0.02ab 1.27±0.35ab 0.11±0.04b 5.00 0.08±0.02a 0.13±0.03ab 1.25±0.39ab 0.07±0.01b 7.50 0.10±0.01a 0.16±0.03a 1.45±0.35a 0.08±0.04b 10.0 0.09±0.01a 0.14±0.05ab 1.44±0.23a 0.07±0.03b Means with the same superscript in the row are not significantly different (p<0.05). Table 2: Condition factor of Clarias gariepinus exposed to diazinon for 30 days (mean ±SD) Pesticides are metabolized in the liver through cytochrome P450 to hepatotoxic intermediates (Das and Gupta, 2013). Several field and laboratory studies have recorded an increase in hepatosomatic indices in fishes exposed to pesticides (Krurutap and Duran, 2001; Roche et al., 2000. Liver values increased slightly at the last two concentrations of diazinon (7.50 and 10.00mg/l). Liver enlargement suggests increased detoxification processes. p Spleen filters foreign substances from blood cells, release blood to sections in need, while kidney plays the major role in excretion of nitrogenous waste, control of water and electrolyte balance in the fish. A significant decline in size of these organs as recorded in this present study could impair these functions resulting in a more toxic internal environment which is detrimental to the fish. Condition factor The initial condition factor of the control and experimental group values were not statistically significant (P>.05). Albeit a slight progressive rise in values were observed as the concentration increased. The final condition values were not significantly different (P>0.05). Values fluctuate as the concentration increased. A slight increase in values at 1.00mg/l and 2.50mg/l were recorded and then a reduction in values at 5.00, 7.50 and 10.0mg/l (Table 1). A sudden drop in values is clear indication of effect of diazinon on general physiology of the probe organism. www.gjournals.org www.gjournals.org 16 ISSN: 2276-7762 ICV: 5.99 Vol. ISSN: 2276-7762 ICV: 5.99 Vol. 7 (2), pp. 015-019, February 2017 Vol. 7 (2), pp. 015-019, February 2017 Greener Journal of Biological Sciences There was no behavioural changes in the control and the first group that received the least dose (1.00mg/l) of the toxicant. The rest of the doses caused changes in behavioural patterns. Slight restlessness was observed REFERENCES Aardema, H., Meertens, J., Ligtenberg, J., Peter-Polman, J.E, Tulleken J.E. and Ijistra, G. 2008. Organophosphorus pesticide poisoning: cases and developments, Neth. J. Med., 66: 149 – 153. g p p p p g p Al-Ghanim, K.A. 2014. 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A study on acute toxicity, behaviour and growth in Indian flying barb, Esonus Dauricus (Hamilton – Buchanan) on exposure to organchlorine pesticide Endosulfan (EC35). Inter. J. Env. Sci. 3.2217-2223. Díaz-Resendiz, K. J. G., Toledo-Ibarra, G. A. and Girón-Pérez, M. I. 2015. Modulation of Immune Response by Organophosphorus Pesticides: Fishes as a Potential Model in Immunotoxicology. Journal of Immunology Research, http://dx.doi.org/10.1155/2015/213836 p g Far, M.S., Roodsari, H.V., Zamini, A., Mirrasooli, E. and Kazemi, R. (2012). Effects of Diazinon on Behavior and Some Hematological Parameters of Fry Rainbow Trout (Oncorhynchus mykiss). World Journal of Fish and Marine Sciences 4 (4): 369-375. ( ) Hodgson, E., Leidy, R., and Cope, V.2004. A textbook of modern toxicology. John Wiley and sons Pub: Hoboken New Jersey. y Inyang I.R, Thomas S and Izah, S.C. 2016a. Evaluation of activities of transfereases and phosphate in plasma and organs of Clarias gariepinus exposed to fluazifop-P-butyl. Journal of Environmental treatment techniques 4(3): 94 – 97 q ( ) Inyang, I.R., Kenobi, A. and Izah, S.C. 2016b. Effect of dimethoate on some selected metabolites in the brain, liver and muscle of Clarias lazera. Sky Journal of Biochemistry Research. 5(4): 63-68 Inyang, I.R., Akio, K. and Izah, S.C. 2016c. Behavioural observation There was no behavioural changes in the control and the first group that received the least dose (1.00mg/l) of the toxicant. The rest of the doses caused changes in behavioural patterns. Slight restlessness was observed 17 www.gjournals.org Greener Journal of Biological Sciences ISSN: 2276-7762 ICV: 5.99 Vol. 7 ISSN: 2276-7762 ICV: 5.99 Vol. 7 (2), pp. 015-019, February 2017 Greener Journal of Biological Sciences in the group/treatment that received the highest dose of the toxicant. Intermediately the fish turned on the flank and swam in half circles and weakening of jerks were observed. Also, sudden quick movement, settling at the bottom and excessive mucus secretion on the skin were observed. Additionally, fishes showed unusual movement as a result muscle spasm and lethargy. Similarly findings have been reported by several researchers in a number of fish species including Clarias gariepinus, Heterobrancus bidorsalis, Common Carp (Cyprinus carpio L) and Branchydario rerio (Omeregie and Okpanachi 1992; Svobodova et al., 2001; Avoajeh and Oti, 2002; Auta et al., 2004). These abnormal behavioural responses exhibited by Clarias gariepinus in this present study suggest that they suffered respiratory impairment due to the effect of the toxicant precisely on gills and general metabolism. CONCLUSION Our data and physical observation unveiled the effect of diazinon on Claria gariepinus. We also observed that the concentration of diazinon that caused negative effect (specially, the behavioural responses) was much lower than anticipated, hence anywhere this insecticide run-off is present; it is assumed that organism in that microenvironment will suffer negative effects from exposure to diazinon. Further research of the impact of diazinon on developing aquatic organisms should be done for better understanding of the effect of diazinon in aquatic ecosystem. REFERENCES Effect of dimethoate on lactate dehydrogenase, creatinine kinase and amylase in Clarias lazera. Biotechnological Research, 2(4): 155- 160 y g , ( ) Inyang, I.R., Obidiozo, OZ. and Izah, S.C. 2016d. Effects of Lambda cyhalothrin in protein and Albumin content in the kidney and liver of Parpohiocephalus obscurus. EC Pharmacology and Toxicology, 2(3): 148-153 Inyang, I.R., Okon, N.C. and Izah, S.C. 2016e. Effect of glyphosate on some enzymes and electrolytes in Heterobranchus bidosalis (a common African catfish) Biotechnological Research 2(4):161 165 y g ( ) Inyang, I.R., Obidiozo, OZ. and Izah, S.C. 2016d. Effects of Lambda cyhalothrin in protein and Albumin content in the kidney and liver of Parpohiocephalus obscurus. EC Pharmacology and Toxicology, 2(3): 148-153 in the kidney and liver of Parpohiocephalus obscurus. EC Pharmacology and Toxicology, 2(3): 148-153 nyang, I.R., Okon, N.C. and Izah, S.C. 2016e. Effect of glyphosate on some enzymes and electrolytes in Heterobranchus bidosalis (a common African catfish). Biotechnological Research, 2(4):161-165 y p p gy gy, ( ) Inyang, I.R., Okon, N.C. and Izah, S.C. 2016e. Effect of glyphosate on some enzymes and electrolytes in Heterobranchus bidosalis (a common African catfish). Biotechnological Research, 2(4):161-165 18 www.gjournals.org Greener Journal of Biological Sciences Vol. 7 (2), pp. 015-019, February 2017 ISSN: 2276-7762 ICV: 5.99 ISSN: 2276-7762 ISSN: 2276-7762 Inyang, I.R., Thomas, S. and Izah, S.C. 2016f. Activities of electrolytes in kidney and liver of Clarias gariepinus exposed to fluazifop-p-butyl. Journal of Biotechnology Research, 2(9): 68 – 72. p p p y gy , ( ) Inyang, I.R., Izah, S.C., Johnson, D.T. and Ejomarie, O.A. 2017. Effects of Lambda cyhalothrin on some electrolytes and metabolites in organs of Parpohiocephalus obscurus. Biotechnological Research, 3(1): 6- 10. Inyang, I.R. 2008. Haematological and biochemical responses of Clarias gariepinus to diazinon phD thesis, Rivers State University of Science and Technology, Port Harcourt. 138 – 153. Inyang, I.R. 2008. Haematological and biochemical responses of Clarias gariepinus to diazinon phD thesis, Rivers State University of Science and Technology, Port Harcourt. 138 – 153. Kapka-Skrzypczak, L., Cyrank, M., Skrzypczak, M. and Kruszewski, M. (2011). Biomonitoring and biomarkers of organophosphate pesticides exposure state of the art Annals of Agricultural and Environmental Rivers State University of Science and Technology, Port Harcourt. 138 – 153. Kapka-Skrzypczak, L., Cyrank, M., Skrzypczak, M. and Kruszewski, M. (2011). Biomonitoring and biomarkers of organophosphate pesticides exposure –state of the art. REFERENCES Annals of Agricultural and Environmental M di i 18(2) 294 303 Kapka-Skrzypczak, L., Cyrank, M., Skrzypczak, M. and Kruszewski, M. (2011). Biomonitoring and biomarkers of organophosphate pesticides exposure –state of the art. Annals of Agricultural and Environmental Medicine, 18(2): 294-303 , ( ) Kuruptap E.B., and Daoran F., 2001. The effects of endosulfan on activity and kinetic properties of Lactic dehydrogerase enzyme: A biochemical and Histopathological study, T. Klin. J. Med. Sci., 21:11-16 y g y p g y Leudke, K and Bartley, M. 2006. 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Accessed February 08th , 2017 Omeregie, E and Okpanachi, M.A. 1992. Growth of tilapia zilli exposures to sublethal concentration of crude extracts of Azadirachta indica. Acta Hydrobiologica, 34(2): 281-286. y g ( ) Roche H., Bult, A., Jonet, O., and Ramade, F. 2000. Organochlorine residues in European ed (Anguilla Anguilla), crucian carp (Carassius carassius) and catfish (Ictalurus nebulosus) from vaccares lagoon (French National Nature reserve of Camargue – effect of some physiological parameters. Aquatic Toxicology, 48:443-459 gy, Simeon, E.O 2007. Effect of monoclotophos (an organophosphate insecticide)on organosomatic indices and enzymes activity of a hybrid catfish. Msc thesis, Rivers State University of Sc and Tech. Port Harcourt. Svodovora, M., Luskova, V., Drastichora, J. and Zlabek, 2001. Effect if diazinon on haematological indices of common carp (Cyprinus carpio). Acta Vet. Brno. 70:457-465. Vajargah, M.F., Hossaini, S.A. and Hedayati, A. (2013). Acute toxicity test of two pesticides diazinon and deltamethrin on spirlin (Alburnoides bipunctatus) larvae and fingerling. Journal of Toxicology and Environmental Health Sciences, 5(6): 106-110 ( ) Virture, W.A and Voetorra, J.W. 1997. Sheep dip chemicals and water pollution. Environmental Toxicology, 22:194 -196. ( ) Virture, W.A and Voetorra, J.W. 1997. Cite this Article: Inyang IR, Ollor AO and Izah SC (2017). Effect of Diazinon on Organosomatic Indices and Behavioural Responses of Clarias gariepinus (a Common Niger Delta Wetland Fish). Greener Journal of Biological Sciences, 7(2):015-019, http://doi.org/10.15580/GJBS.2017.2.020917021 REFERENCES Sheep dip chemicals and water pollution. Environmental Toxicology, 22:194 -196. Wahua, T.A.T. 1999. Applied statistics for scientific studies, Africa link books, Ibadan. Wahua, T.A.T. 1999. Applied statistics for scientific studies, Africa link books, Ibadan. Yang, C. and Deng, J. 2007. Intermediate syndrome following organophosphate poisoning. J. Chin. Med. Assoc. 11:467 – 472. pp Yang, C. and Deng, J. 2007. Intermediate syndrome following organophosphate poisoning. J. Chin. Med. Assoc. 11:467 – 472. Cite this Article: Inyang IR, Ollor AO and Izah SC (2017). Effect of Diazinon on Organosomatic Indices and Behavioural Responses of Clarias gariepinus (a Common Niger Delta Wetland Fish). Greener Journal of Biological Sciences, 7(2):015-019, http://doi.org/10.15580/GJBS.2017.2.020917021 19 www.gjournals.org
https://openalex.org/W2805692389	https://europepmc.org/articles/pmc5977757?pdf=render	English	null	Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography	BMC biology	2,018	cc-by	14,551	Olmos et al. BMC Biology (2018) 16:59 https://doi.org/10.1186/s12915-018-0524-5 Olmos et al. BMC Biology (2018) 16:59 https://doi.org/10.1186/s12915-018-0524-5 Open Access RESEARCH ARTICLE Open Access Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography Jose L. Olmos Jr1†, Suraj Pandey2†, Jose M. Martin-Garcia3†, George Calvey4, Andrea Katz4, Juraj Knoska5,6, Christopher Kupitz2, Mark S. Hunter7, Mengning Liang7, Dominik Oberthuer5, Oleksandr Yefanov5, Max Wiedorn5,6, Michael Heyman8, Mark Holl3, Kanupriya Pande9, Anton Barty5, Mitchell D. Miller1, Stephan Stern5, Shatabdi Roy-Chowdhury3, Jesse Coe3, Nirupa Nagaratnam3, James Zook3, Jacob Verburgt2,10, Tyler Norwood2, Ishwor Poudyal2, David Xu1, Jason Koglin7, Matthew H. Seaberg7, Yun Zhao3, Saša Bajt11, Thomas Grant12, Valerio Mariani5, Garrett Nelson13, Ganesh Subramanian13, Euiyoung Bae14, Raimund Fromme3, Russell Fung2, Peter Schwander2, Matthias Frank15, Thomas A. White5, Uwe Weierstall13, Nadia Zatsepin13, John Spence13, Petra Fromme3, Henry N. Chapman5,6,16, Lois Pollack4, Lee Tremblay17,18, Abbas Ourmazd2, George N. Phillips Jr1 and Marius Schmidt2* Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography Jose L. Olmos Jr1†, Suraj Pandey2†, Jose M. Martin-Garcia3†, George Calvey4, Andrea Katz4, Juraj Knoska5,6, Christopher Kupitz2, Mark S. Hunter7, Mengning Liang7, Dominik Oberthuer5, Oleksandr Yefanov5, Max Wiedorn5,6, Michael Heyman8, Mark Holl3, Kanupriya Pande9, Anton Barty5, Mitchell D. Miller1, Stephan Stern5, Shatabdi Roy-Chowdhury3, Jesse Coe3, Nirupa Nagaratnam3, James Zook3, Jacob Verburgt2,10, Tyler Norwood2, Ishwor Poudyal2, David Xu1, Jason Koglin7, Matthew H. Seaberg7, Yun Zhao3, Saša Bajt11, Thomas Grant12, Valerio Mariani5, Garrett Nelson13, Ganesh Subramanian13, Euiyoung Bae14, Raimund Fromme3, Russell Fung2, Peter Schwander2, Matthias Frank15, Thomas A. White5, Uwe Weierstall13, Nadia Zatsepin13, John Spence13, Petra Fromme3, Henry N. Chapman5,6,16, Lois Pollack4, Lee Tremblay17,18, Abbas Ourmazd2, 1 2* George N. Phillips Jr1 and Marius Schmidt2* Abstract Background: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results: Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. Conclusions: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data. © Schmidt et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: m-schmidt@uwm.edu †Jose L. Olmos, Suraj Pandey and jose M. Martin-Garcia contributed equally to this work. 2Physics Department, University of Wisconsin-Milwaukee, 3135 N. Maryland Ave, Milwaukee, WI 53211, USA Full list of author information is available at the end of the article S h id l 20 8 O A Thi Background description of a reaction in real time [4, 5]. However, X-ray damage and the need for large single crystals made time-resolved crystallography very challenging. The advent of X-ray free-electron lasers (XFELs) has en- abled time-resolved serial femtosecond (fs) crystallog- raphy (SFX), where X-ray damage is outrun by ultrashort fs X-ray pulses [6, 7]. This approach has made it possible to follow and describe cyclic and non-cyclic reactions triggered by light. Examples include pioneering studies on the photoactive yellow protein [8, 9], myoglo- bin [10], bacteriorhodopsin [11], photoswitchable fluor- escent proteins [12, 13], and photosystem II [14–17]. However, structural investigations on one-pathway Observing the catalytic action of a biomolecule in atomic detail has been the dream of structural biologists since the first structure of an enzyme was solved [1, 2]. By exploiting X-ray radiation from powerful synchrotron sources, time-resolved crystallographic methods were developed [3] with the goal of achieving a complete Page 2 of 15 Page 2 of 15 Olmos et al. BMC Biology (2018) 16:59 enzymatic reactions present additional difficulties, be- cause diffusion of substrate(s) and products in and out of the crystals limit the accessible reaction times. Stand- ard crystallography can be used to track reaction inter- mediates of slow reactions by flash cooling [18–20], but the method is then unable to reveal enzymatic reactions at room temperature in real time. The problem is to start a reaction in large-sized crystals. Initiation by diffu- sion is far slower in these crystals than the typical milli- second turnover times of enzymes. It was proposed that one can trigger enzymatic reactions by light by soaking inactive (caged) substrates [21] into the crystals, which then can be activated by a laser pulse. The first proof of concept for time-resolved Laue crystallography triggered by a caged substrate was achieved in 1990 [22]. While this method has great potential, its application has so far been limited due to significant experimental challenges. Only a few time-resolved experiments have been re- ported where highly reactive, caged substrates are readily available [18, 22, 23], or the reactions are slow and allow the use of more conventional methods [24, 25]. It is therefore highly desirable to develop new methods that open the field of time-resolved crystallography to the study of biomolecular reactions at room temperature with the native enzyme and its natural substrate(s). Background residues to slow down (or stop) the reaction to the ex- tent that the binding of numerous antibiotics to BlaC could be studied [39]. In our experiments, however, car- ried out at the Linac Coherent Light Source (LCLS), mi- crocrystals of unmodified BlaC are mixed with CEF on the fly, and the cleavage and thereby inactivation of the antibiotics by the wild-type β-lactamase is followed at runtime. BlaC is a broad-spectrum β-lactamase which confers resistance to all classes of β-lactam antibiotics in tuberculosis [19, 40]. BlaC chemistry has rendered the frontline arsenal of antibacterial agents ineffective against this deadly disease, creating a global public health crisis. Beginning with the famous discovery of penicillin, β-lactam antibiotics were widely used to eliminate deadly bacterial infectious diseases [41]. More com- pounds with diverse chemical composition have been found through the years [42], the most prominent of them most likely the cephalosporins. The chemical structure of CEF is shown in Fig. 1. Unlike the penicil- lins, which feature a 5-membered thiazolidine ring, in the cephalosporins a 6-membered dihydrothiazine ring is fused to the β-lactam ring. However, rampant resist- ance against these antibiotics was observed shortly after their widespread use [41]. β-lactamases open the β-lactam ring, thereby rendering the antibiotic inactive. BlaC from M. tuberculosis, an Ambler class A β-lactamase [43], uses a conserved serine to attack the β-lactam ring (Fig. 1, blue arrow), thus inactivating the antibiotics. Because of the medical challenge that BlaC causes for the fight against infectious diseases, the process of catalysis has been studied by conventional biochemical methods in detail, leading to the hypothesis of a three-step model of the cleavage process. The first step is the formation of the enzyme-substrate (ES) com- plex (Fig. 1, species 1), and it has been proposed that the enzyme may use active site interactions to orient the β-lactam carbonyl carbon near the Ser-70 nucleophile [19, 40]. The next step proposed along the reaction co- ordinate is the nucleophilic attack of Ser-70, which re- sults in the opening of the β-lactam ring and the formation of the covalently bound active site acyl inter- mediate (species 3). For cephalosporins there is evidence that during the enzymatic reaction a leaving group (de- noted R in Fig. 1) is split off [44, 45]. In the third step, the open-ring β-lactam ligand is hydrolyzed and released by the enzyme (Fig. 1, species 4). Background Evidence for all four intermediate species is found in our experiments. (4) Species (3) is further hydrolyzed from Ser-70 and leaves the enzyme as product Fig. 1 Reaction of β-lactamase with ceftriaxone (CEF). (1) Formation of the enzyme substrate complex by non-covalently binding CEF. (2) Nucleophilic attack of the active site residue Ser-70 results in rearrangement of double bonds and ultimately leads to the opening of the β- lactam ring (blue arrow points to the bond to be cleaved) and the detachment of the leaving group (R). (3) Covalent bond formation between Ser-70 and a shortened species (E-CFO). Note the double bond Δ. The double bond may react with water to form an alcohol (OH). Evidence for all four intermediate species is found in our experiments. (4) Species (3) is further hydrolyzed from Ser-70 and leaves the enzyme as product Fig. 1 Reaction of β-lactamase with ceftriaxone (CEF). (1) Formation of the enzyme substrate complex by non-covalently binding CEF. (2) Nucleophilic attack of the active site residue Ser-70 results in rearrangement of double bonds and ultimately leads to the opening of the β- lactam ring (blue arrow points to the bond to be cleaved) and the detachment of the leaving group (R). (3) Covalent bond formation between Ser-70 and a shortened species (E-CFO). Note the double bond Δ. The double bond may react with water to form an alcohol (OH). Evidence for all four intermediate species is found in our experiments. (4) Species (3) is further hydrolyzed from Ser-70 and leaves the enzyme as product X-ray data. We base our interpretation on bias-free omit maps obtained by simulated annealing. Figure 2 and Additional file 1: Figures S2–S6 show details of these maps near the active site. As a complement, more con- ventional 2mFo-Fc maps are shown in Additional file 1: Figures S7–S9. the potential to directly visualize substrate chemical in- termediates and the accompanying active site interac- tions, with wide-ranging implications for all classes of β-lactams. Ultimately, knowledge of the molecular pro- cesses by which BlaC is able to bind and catalyze the breakdown of β-lactams will directly impact rational drug design against deadly human diseases. The critical questions in MISC concern whether the enzyme in the crystals is still catalytically active and whether the reaction is limited by constraints of crystal packing or the solvent/precipitant used for crystallization. We have therefore crystallized BlaC in two different crystal forms. Background Various rates have been reported for this step of the catalytic reaction across dif- ferent classes of β-lactams, followed by product release [40]. Static structures of some of the critical intermedi- ates have been determined and reported [19], including an initial enzyme substrate complex trapped by removal of catalytically important amino acid residues [39]. Obtaining time-resolved data on BlaC chemistry holds y Structural studies at XFELs offer the possibility of a breakthrough. The XFEL intensity is high enough to generate a diffraction pattern from an exposure to a sin- gle fs X-ray pulse even from micrometer- and submicrometer-sized crystals. These tiny crystals allow for fast (sub-millisecond to millisecond) diffusion times, which are not rate-limiting for many enzymatic reactions [26–32]. The microcrystals are mixed “on the fly” and injected into the XFEL beam, a method we call mix-an- d-inject serial crystallography (MISC) [28, 30]. In MISC, crystals react with their native substrate(s) at ambient temperature until they are probed by a single X-ray pulse that destroys them but not before a diffraction pat- tern has been recorded. The pulses are short enough to essentially outrun radiation damage by means of the “diffraction-before-destruction” principle [33–35]. Opti- mized injectors have been recently developed [36, 37] for MISC experiments with the potential to provide sub- millisecond time resolution [38]. The microcrystals may tolerate even larger conformational changes leading to unit cell or even space group changes [14, 31]. Here, we apply MISC to the study of a very important public health problem: bacterial antibiotic resistance. Specifically, we have obtained time-resolved crystallo- graphic data on the binding and cleavage of the third-generation antibiotic ceftriaxone (CEF) in micro- crystals of the enzyme β-lactamase from Mycobacterium tuberculosis (BlaC). Previous studies introduced muta- tions into BlaC by exchanging catalytically important Olmos et al. BMC Biology (2018) 16:59 Page 3 of 15 Fig. 1 Reaction of β-lactamase with ceftriaxone (CEF). (1) Formation of the enzyme substrate complex by non-covalently binding CEF. (2) Nucleophilic attack of the active site residue Ser-70 results in rearrangement of double bonds and ultimately leads to the opening of the β- lactam ring (blue arrow points to the bond to be cleaved) and the detachment of the leaving group (R). (3) Covalent bond formation between Ser-70 and a shortened species (E-CFO). Note the double bond Δ. The double bond may react with water to form an alcohol (OH). Background With phosphate as the pre- cipitant, the BlaC crystallizes in a shard-shaped crystal form with four copies in the asymmetric unit (Fig. 2a) as previously reported [30]. With polyethylene glycol (PEG) 1000 as the precipitant, needle-shaped crystals are ob- tained with one molecule in the asymmetric unit. The packing of BlaC in both crystal forms is shown in Add- itional file 1: Figure S11. Our previous results at 2 s after mixing showed that CEF can diffuse into the crystals and binds to the active site of the crystalline β-lactamase [30]. These first stud- ies showed that the catalytic reaction is heterogeneous, as the reactivity is specific to individual copies of the four β-lactamase chains in the asymmetric unit of the crystal. Only subunits B and D bind and process CEF, while subunits A and C do not directly contribute to ca- talysis, at least on the time scale of our experiments (Fig. 2a). This first proof-of-concept study was limited to a single time point about 2 s after reaction initiation [30]. Multiple time points covering the reaction are re- quired for any kinetic analysis. In our MISC experiment, the small microcrystals were rapidly mixed with CEF “on the fly” using optimized mixing devices (Additional file 1: Figure S1), and struc- tures of the reaction of BlaC with CEF were determined by scattering from femtosecond X-ray pulses at five time points (unmixed, and 30 ms, 100 ms, 500 ms, and 2 s after mixing, respectively) during the reaction in both crystal forms. Results are shown in Figs. 1 and 2. CEF Results Here we present a time series from 30 ms to 2 s after mixing with substrate in two different crystal forms, called shards and needles; this allows us to discover the conformational changes and to characterize the kinetics of this important class of enzymes directly from the Page 4 of 15 Olmos et al. BMC Biology (2018) 16:59 Fig. 2 Overview of BlaC as determined using 10 × 10 × 3 μm3 sized crystals in the shard form at 500 ms after mixing with 300 mM CEF at room temperature. The mFo-DFc SA-omit electron density is shown for the covalently bound intermediate E-CFO in green (contour level 2.5 σ). Electron density of an additional, stacked ceftriaxone molecule near the active site is shown in dark green (contour level 2 σ). a The BlaC subunits A–D displayed in blue, yellow, green and light yellow, respectively. Amino acid residues that interact with the stacked CEF are labeled. Panels b and c show enlarged views of the active sites of subunits B and D, respectively. Arg-126 and Tyr-127 with which the respective stacked CEF molecules interact are shown. Some important distances are also displayed (stacked molecules are also observed at the other time delays in the shard crystal form but not in the needles) Fig. 2 Overview of BlaC as determined using 10 × 10 × 3 μm3 sized crystals in the shard form at 500 ms after mixing with 300 mM CEF at room temperature. The mFo-DFc SA-omit electron density is shown for the covalently bound intermediate E-CFO* in green (contour level 2.5 σ). Electron density of an additional, stacked ceftriaxone molecule near the active site is shown in dark green (contour level 2 σ). a The BlaC subunits A–D displayed in blue, yellow, green and light yellow, respectively. Amino acid residues that interact with the stacked CEF are labeled. Panels b and c show enlarged views of the active sites of subunits B and D, respectively. Arg-126 and Tyr-127 with which the respective stacked CEF molecules interact are shown. Some important distances are also displayed (stacked molecules are also observed at the other time delays in the shard crystal form but not in the needles) component observed (~ 70%, see also Table 2). A minor fraction (~ 30%) has an open β-lactam ring (Fig. 3d, e, f). Results The open, covalently bound species E-CFO* can be identified more clearly at 500 ms, where it dominates the electron density (Fig. 3g, h, i). Only on a time scale longer than 100 ms does the nucleophilic attack of Ser-70 open the β-lactam ring. At 500 ms this results in high occupancy of an intermediate which is covalently bound to the enzyme called E-CFO* as shown in Fig. 3g, h, i. At the same time the leaving group R (Fig. 1) is split off, as witnessed by the vanishing density of the leaving group sulfur and some of the weak ring density features (compare Fig. 3d and g, or Fig. 3e and h; see also feature β in Additional file 1: Figure S10c). The covalently bound ligand is much shorter than CEF. The red arrow in Fig. 3g indicates that the double bond Δ (Fig. 1) may have reacted to an alcohol in subunit B, which does not occur in subunit D or in the needle form of the crystals. Additional file 1: Figure S10 shows the density in the un- mixed shard crystal form (Additional file 1: Figure S10a) binds to the active site of BlaC as shown in Fig. 2a. In Fig. 3 more details are shown for the substrate binding in the shard and needle crystal forms (see also Add- itional file 1: Figure S2 for details from another viewing direction, and Additional file 1: Figures S3–S10 for ste- reo representations of various viewing directions and time points). Strong electron density at 30 ms shows that substrate diffusion into the crystals was successful. At this time delay the formation of the non-covalently bound ES complex is observed (Fig. 3a, b, c). The ES complex can be identified by strong electron density of the leaving group sulfur (blue arrows in Fig. 3a, b), and somewhat stronger dioxo-triazine ring features (red ar- rows in Fig. 3a, b). Since the resolution of our X-ray data at the 30-ms time delay is limited to 2.75 Å, the distinc- tion between a non-covalently bound species and a cova- lently bound species (see below) is difficult. However, the non-covalently bound species dominates occupancy refinements (see Table 2a, and remarks therein). At 100 ms the ES complex still prevails and is the major binds to the active site of BlaC as shown in Fig. 2a. In Fig. Results j, k, l Mixture of the non-covalently bound, full-length CEF (blue arrow shows the leaving group sulfur feature) and covalently bound E-CFO* in the shard crystal form (both subunits) at 2 s. The electron density in the needle crystal form favors only the full- length CEF species Fig. 3 Ceftriaxone density in the active site in 10 × 10 × 3 μm3 shard and 5 × 2 × 2 μm3 needle crystal forms at various times after mixing with 200–300 mmol/L CEF. The main species is displayed in blue, the minor species in gray. First two columns: shard crystal form, mFo-DFc SA-omit density (green) contoured at 2.5 σ. Third column: needle crystal form. SA omit maps were calculated using extrapolated structure factors. Time delays are arranged from top (30 ms) to bottom (2 s). Black arrows show the electron density of the covalently bound acyl adduct (see also Additional file 1: Figure S2 for details). a, b, c The ES complex at 30 ms. The full-length CEF model (blue) is displayed. The ES complex can be observed in needles or shards (both subunits). Blue arrows: features of the leaving group sulfur, red arrows: dioxo-triaxine ring feature. d, e, f Early phases of the formation of a covalently bound CEF adduct at 100 ms. The full-length CEF model (blue) is displayed together with the minor E- CFO* species (gray), where the β-lactam ring is open and attached to Ser-70 in subunit-B (shard crystal form, panel d) and the needle crystal form (panel f). In the shard crystal form subunit D (panel e) the acyl adduct is not yet observed. g, h, i Covalently bound adduct (E-CFO* in blue) formation at 500 ms with a small contamination of full-length CEF (gray). The red arrow points to electron density that may favor the interpretation by an OH group. j, k, l Mixture of the non-covalently bound, full-length CEF (blue arrow shows the leaving group sulfur feature) and covalently bound E-CFO* in the shard crystal form (both subunits) at 2 s. The electron density in the needle crystal form favors only the full- length CEF species and a difference map between the 500 ms and the 100 ms time points (Additional file 1: Figure S10c), which displays changes in the region of the covalent at- tachment of the intermediate between 100 ms and 500 ms. Results 3 more details are shown for the substrate binding in the shard and needle crystal forms (see also Add- itional file 1: Figure S2 for details from another viewing direction, and Additional file 1: Figures S3–S10 for ste- reo representations of various viewing directions and time points). Strong electron density at 30 ms shows that substrate diffusion into the crystals was successful. At this time delay the formation of the non-covalently bound ES complex is observed (Fig. 3a, b, c). The ES complex can be identified by strong electron density of the leaving group sulfur (blue arrows in Fig. 3a, b), and somewhat stronger dioxo-triazine ring features (red ar- rows in Fig. 3a, b). Since the resolution of our X-ray data at the 30-ms time delay is limited to 2.75 Å, the distinc- tion between a non-covalently bound species and a cova- lently bound species (see below) is difficult. However, the non-covalently bound species dominates occupancy refinements (see Table 2a, and remarks therein). At 100 ms the ES complex still prevails and is the major Page 5 of 15 Olmos et al. BMC Biology (2018) 16:59 Fig. 3 Ceftriaxone density in the active site in 10 × 10 × 3 μm3 shard and 5 × 2 × 2 μm3 needle crystal forms at various times after mixing with Fig. 3 Ceftriaxone density in the active site in 10 × 10 × 3 μm3 shard and 5 × 2 × 2 μm3 needle crystal forms at various times after mixing with 200–300 mmol/L CEF. The main species is displayed in blue, the minor species in gray. First two columns: shard crystal form, mFo-DFc SA-omit density (green) contoured at 2.5 σ. Third column: needle crystal form. SA omit maps were calculated using extrapolated structure factors. Time delays are arranged from top (30 ms) to bottom (2 s). Black arrows show the electron density of the covalently bound acyl adduct (see also Additional file 1: Figure S2 for details). a, b, c The ES complex at 30 ms. The full-length CEF model (blue) is displayed. The ES complex can be observed in needles or shards (both subunits). Blue arrows: features of the leaving group sulfur, red arrows: dioxo-triaxine ring feature. d, e, f Early phases of the formation of a covalently bound CEF adduct at 100 ms. Results The full-length CEF model (blue) is displayed together with the minor E- CFO* species (gray), where the β-lactam ring is open and attached to Ser-70 in subunit-B (shard crystal form, panel d) and the needle crystal form (panel f). In the shard crystal form subunit D (panel e) the acyl adduct is not yet observed. g, h, i Covalently bound adduct (E-CFO* in blue) formation at 500 ms with a small contamination of full-length CEF (gray). The red arrow points to electron density that may favor the interpretation by an OH group. j, k, l Mixture of the non-covalently bound, full-length CEF (blue arrow shows the leaving group sulfur feature) and covalently bound E-CFO* in the shard crystal form (both subunits) at 2 s. The electron density in the needle crystal form favors only the full- length CEF species Fig. 3 Ceftriaxone density in the active site in 10 × 10 × 3 μm3 shard and 5 × 2 × 2 μm3 needle crystal forms at various times after mixing with 200–300 mmol/L CEF. The main species is displayed in blue, the minor species in gray. First two columns: shard crystal form, mFo-DFc SA-omit density (green) contoured at 2.5 σ. Third column: needle crystal form. SA omit maps were calculated using extrapolated structure factors. Time delays are arranged from top (30 ms) to bottom (2 s). Black arrows show the electron density of the covalently bound acyl adduct (see also Additional file 1: Figure S2 for details). a, b, c The ES complex at 30 ms. The full-length CEF model (blue) is displayed. The ES complex can be observed in needles or shards (both subunits). Blue arrows: features of the leaving group sulfur, red arrows: dioxo-triaxine ring feature. d, e, f Early phases of the formation of a covalently bound CEF adduct at 100 ms. The full-length CEF model (blue) is displayed together with the minor E- CFO* species (gray), where the β-lactam ring is open and attached to Ser-70 in subunit-B (shard crystal form, panel d) and the needle crystal form (panel f). In the shard crystal form subunit D (panel e) the acyl adduct is not yet observed. g, h, i Covalently bound adduct (E-CFO* in blue) formation at 500 ms with a small contamination of full-length CEF (gray). The red arrow points to electron density that may favor the interpretation by an OH group. Results Diffusion times and time scales of the ES formation (30 ms) are irrelevant compared to those for the E-CFO* intermediate formation (~ 500 ms). As a consequence, the ability to observe the E-CFO* inter- mediate does not critically depend on CEF diffusion times into the crystals (compare the solid and dashed lines in Fig. 4). Accordingly, the reaction dynamics of the catalytic reaction in the needle and the shard crystal forms appear to be similar despite the different crystal morphologies and packing (see further explanation in the Methods section). Subtle differences between the re- sults from the two crystal forms, and between the sub- units in different crystal environments, confirm previous preliminary results [30] and previous predictions from biochemical results for other cephalosporin species [44]. An additional CEF molecule (CEFstack) can be identi- fied near the catalytic clefts of subunits B and D, each in the shard crystal form (Fig. 2a, b, c, and Additional file 1: Figure S9). This molecule stacks to the CEF species that occupy the active sites on all time scales. CEFstack is non-covalently interacting with Arg-126 and Tyr-127 of subunit A or C, which are adjacent to the active catalytic clefts of subunit B or D, respectively. For more details see Additional file 1: Figure S9 for a stereo view. This way CEFstack is quite close, pre-oriented, and can rapidly access the active site after the initial CFO has been hy- drolyzed and has left the enzyme. Stacking of multiple cephalosporin (cefamandole) molecules has also been observed in orthorhombic crystals of the BlaC K73A mutant [39]. In these crystals the stacked molecules interact with Asp-192 and Arg-194 of a symmetrically equivalent BlaC molecule; this is different from the in- teractions seen here. As with most proteins, BlaC can crystallize in crystal forms with different numbers of copies in the asymmetric unit [39, 48, 49]. Since stacking is not observed in crystals that pack in the same way as our needle crystal form [19], it might be argued that it represents a non-physiological, non-specifically bound substrate that occurs only in the environment in the shard crystals. However, the binding of the additional CEF molecule could be a mechanism to steer the sub- strate towards, and orient it with respect to, the active site under certain conditions. Results At 2 s, the binding sites are occupied mainly by the full-length CEF with a minor contribution from E-CFO* (Table 2a, b). substoichiometric occupancy ranging from 20% to 40%. The reason for this might be that the enzyme is more tightly packed in the needle crystal form (Additional file 1: Figure S11). To reach full occupancy in the needles, at least 30 mmol/L of CEF (one CEF molecule per asym- metric unit) is initially required, which needs to be deliv- ered by diffusion from the solution to the side of the crystal. While the outside CEF concentration is on the order of 200 mmol/L in both experiments (Table 3c), the ratio of CEF to enzyme varies in the shard and nee- dle crystals. Additional file 1: Figure S11 shows how the solvent volume that contains CEF surrounding the BlaC molecules in the crystals varies. The solvent volume as In the multi-copy shard crystal form, subunits A and C do not directly participate in catalysis, at least not in the first 2 s. In the monomeric needle crystal form, it ap- pears that the reaction proceeds similarly to that ob- served in subunit D in the shards. However, substrate occupancy is lower than that in the shards, with Olmos et al. BMC Biology (2018) 16:59 Page 6 of 15 Page 6 of 15 estimated by the CCP4 program “truncate” [46] is on the order of 59% for the shard-shaped crystal, and it is sub- stantially lower (28%) in the needles. Additional file 1: Figure S11 also shows that there are substantial differ- ences in the solvent channel sizes in the two crystal forms. When measured by the program Coot [47], cav- ities with diameters as wide as 90 Å can be identified in the shards, and only 20-Å voids exist in the needles. Both may significantly impact diffusion of the CEF sub- strate (which is about 20 Å long and 10 Å wide) into the crystals. However, compared to other antibiotics such as ampicillin [40], CEF binds relatively slowly to BlaC, so it may first build up by diffusion (see also the discussion below and estimates in the Methods section). Strong electron density in our maps shows that diffusion and formation of the ES complex is near completion on a 30-ms time scale. Results It appears, however, that at the very high concentrations of CEF applied here (around 200 mmol/L), stacking is not required for effect- ive catalysis, as the kinetics in the monomeric needles, where stacking does not occur, is similar to that in the shard crystal form. When only low CEF concentrations are present, stacking might well be essential to recruit antibiotic substrate molecules to promote effective BlaC function. Fig. 4 Concentration profile of the catalytic BlaC reaction with CEF as simulated with realistic parameters and a kinetic mechanism as discussed. The solid lines are calculated with τD = 15 ms, the dashed lines with τD = 1.5 ms. Black lines: free enzyme (E). Green lines: enzyme substrate complex (ES). Blue lines: enzyme acyl intermediate complex (E-CFO). Red lines: product P (CFO, inactive CEF without leaving group, lactam ring open), released from the enzyme. Black dots: time delays are shown together with the approximate expected ratio of CEF to E-CFO Discussion Since only five time delays are available, the parameters in the mechanism cannot be independently determined, but we do show that our in- terpretation is consistent with known kinetic parameters in solution. After initial formation of the ES complex represented by a non-covalently bound full-length CEF, the intermediate E-CFO* has its peak concentration at 500 ms. It has been previously suggested [19] that the hydrolytic cleavage of an acyl adduct from Ser-70 (hy- drolysis of species 3 in Fig. 1) should be the rate-limiting step in BlaC catalysis. Then the E-CFO* species should be the dominant species in the steady state. However, this is not the case, as the ES complex with the non-covalently bound, full-length CEF is prevalent (> 70%) in our MISC data at 2 s (Table 2). The simulation can explain this, if the nucleophilic attack of Ser-70 on species 2 in Fig. 1 is inhibited or slowed down. High product concentrations of > 10 mmol/L are already reached after one catalytic cycle due to the very high en- zyme and substrate concentrations (Table 3). In initial velocity solution studies, the enzyme concentration is kept in the low micromolar range. Even under a saturat- ing substrate (vmax) condition, only micromolar concen- trations of product can be produced per turnover. With a turnover rate of 0.8 s−1 of BlaC, it may take hours to reach millimolar concentrations of product. In BlaC crystals and with stoichiometric CEF concentrations (of 16 mmol/L for the shard crystal form, and 30 mmol/L for the needles), however, these concentrations are already reached after one turnover. Consequently, prod- uct inhibition is not only plausible but likely, as previous studies have shown that lactamases do show product in- hibition by similar lactams with inhibitory constants in the millimolar range [52]. In this scenario, after an initial burst during the first second, the nucleophilic attack on the lactam ring by Ser-70 represented by rate coefficient k2 likely becomes the rate-limiting process (the E-CFO* formation slows down), and the ES complex accumu- lates later, as observed in our X-ray data. Discussion One of the major questions addressed here is whether the structural data obtained by MISC can be interpreted in accordance with previous investigations on BlaC ca- talysis. Ideally, a compatible chemical kinetic mechanism can be developed and expressed in the terminology of enzyme kinetics [50, 51]. Accordingly, we set up a kin- etic mechanism (see Methods, Scheme 1) which allows for diffusion of substrate and which features a suffi- ciently large number of intermediate states to explain our observations. Initially, we simulated the catalytic cycle employing literature values of the Michaelis con- stant Km (~ 500 μmol/L) and kcat (0.8 s−1) [40] (Table 3). Our simulations describe the change from the transient state kinetics regime at 30 ms to 2 s, covering a large Fig. 4 Concentration profile of the catalytic BlaC reaction with CEF as simulated with realistic parameters and a kinetic mechanism as discussed. The solid lines are calculated with τD = 15 ms, the dashed lines with τD = 1.5 ms. Black lines: free enzyme (E). Green lines: enzyme substrate complex (ES). Blue lines: enzyme acyl intermediate complex (E-CFO). Red lines: product P (CFO, inactive CEF without leaving group, lactam ring open), released from the enzyme. Black dots: time delays are shown together with the approximate expected ratio of CEF to E-CFO Page 7 of 15 Page 7 of 15 Olmos et al. BMC Biology (2018) 16:59 range of substrate and product concentrations. We then vary the rate coefficients in the mechanism to explain our MISC experiment. Since only five time delays are available, the parameters in the mechanism cannot be independently determined, but we do show that our in- terpretation is consistent with known kinetic parameters in solution. After initial formation of the ES complex represented by a non-covalently bound full-length CEF, the intermediate E-CFO* has its peak concentration at 500 ms. It has been previously suggested [19] that the hydrolytic cleavage of an acyl adduct from Ser-70 (hy- drolysis of species 3 in Fig. 1) should be the rate-limiting step in BlaC catalysis. Then the E-CFO* species should be the dominant species in the steady state. However, this is not the case, as the ES complex with the non-covalently bound, full-length CEF is prevalent (> 70%) in our MISC data at 2 s (Table 2). The simulation can explain this, if the nucleophilic attack of Ser-70 on species 2 in Fig. Discussion 1 is inhibited or slowed down. High product concentrations of > 10 mmol/L are already reached after one catalytic cycle due to the very high en- zyme and substrate concentrations (Table 3). In initial velocity solution studies, the enzyme concentration is kept in the low micromolar range. Even under a saturat- ing substrate (vmax) condition, only micromolar concen- trations of product can be produced per turnover. With a turnover rate of 0.8 s−1 of BlaC, it may take hours to reach millimolar concentrations of product. In BlaC crystals and with stoichiometric CEF concentrations (of 16 mmol/L for the shard crystal form, and 30 mmol/L for the needles), however, these concentrations are already reached after one turnover. Consequently, prod- uct inhibition is not only plausible but likely, as previous studies have shown that lactamases do show product in- hibition by similar lactams with inhibitory constants in the millimolar range [52]. In this scenario, after an initial burst during the first second, the nucleophilic attack on the lactam ring by Ser-70 represented by rate coefficient k2 likely becomes the rate-limiting process (the E-CFO* formation slows down), and the ES complex accumu- lates later, as observed in our X-ray data. l d d d l h b l f needles (Table 3c). Diffusion may also be further en- abled, and facilitated, by protein dynamics [53] at ambi- ent temperatures. Although the reaction kinetics in crystals might be different compared to that in solution [54], structures of intermediate states that are occupied along the catalytic pathway are highly relevant as long as the enzyme is active in the crystal. With more conven- tional X-ray sources, radiation damage might impede the collection of even a single diffraction pattern [55] from these microcrystals. The ultrashort, brilliant hard X-ray pulses available at XFELs circumvent these difficulties. With high X-ray pulse-repetition rates expected at LCLS-II [56] and the European XFEL [57], a large num- ber of finely spaced time delays may be collected rapidly to allow for a comprehensive description of the reaction in terms of structure and kinetics. Then the extraction of a more accurate kinetic mechanism and the analytical separation of mixtures into pure constituents become possible [4, 58]. range of substrate and product concentrations. We then vary the rate coefficients in the mechanism to explain our MISC experiment. Conclusions As we demonstrate here, the structural characterization of enzyme-catalyzed reactions on the millisecond time scale is possible by making use of very small crystals. MISC can be employed to investigate a large number of non-cyclic (single-pass) reactions in proteins and en- zymes, some of which are of immense biological signifi- cance and might be important targets for structure-based drug design. MISC may become a major tool to address fundamental questions on biomolecular reactions at existing and new pulsed X-ray sources. General overview Using a continuous-flow mixing apparatus (Add- itional file 1: Figure S1), we injected active microcrystals of BlaC simultaneously with the β-lactam substrate cef- triaxone (CEF) into a liquid jet for delivery to the beam as a stream of randomly oriented hydrated nanocrystals undergoing catalysis. The catalytic reaction is initiated by solution mixing at the junction of two capillaries [38] and the nanocrystals intersected by the X-ray pulse at specific time points during the reaction. The use of nanocrystals is essential for observation at short times and for effective and uniform reaction initiation [28]. The 120-Hz repetition rate of LCLS allowed for the rapid collection of diffraction snapshots at a number of delay times (time points) after reaction initiation. Accur- ate reflection intensities were extracted from the snap- shots at each time point by indexing and Monte Carlo-type integration [59, 60]. The data were phased using the structural model for BlaC reported by Kupitz et al. [30]. This model is based on the BlaC Protein Data Bank (PDB) entry 2GDN [48]. The sequence convention y Our results depend decisively on the ability of CEF to penetrate the crystals. Although pore sizes for shard and needle crystal forms largely differ (see above), CEF sub- strate swiftly arrives at BlaC molecules in the crystals. This is primarily due to the large substrate concentra- tions that facilitate diffusion (see Methods and Table 3). The osmotic pressure π of the outside CEF into the crystals can be estimated as π = MRT, where M is the molarity of the outside CEF concentration, R is the gas constant, and T is the temperature in kelvins. This pres- sure can be as high as 0.5 MPa (5 bar) with 300 mmol/L CEF, which promotes rapid and uniform diffusion, even in crystals with tight cavities such as those in our Olmos et al. BMC Biology (2018) 16:59 Page 8 of 15 ranging from 30 ms to 2000 ms. Two high-performance liquid chromatography (HPLC) pumps (Shimadzu LC-20 AD) drove the flow. Crystals (shards: 10 × 10 × 3 μm3, needles: 5 × 2 × 2 μm3) were held in a custom reservoir built by Coherent X-ray Imaging (CXI) staff, while the buffer was held in a larger reservoir (KNAUER VariLoop), allowing water flow through the HPLC pump without diluting either sample or buffer. A pressure con- troller (Proportion-Air GP1) was used to regulate helium pressure in the device. Crystal forms l y Cloning, overexpression, and purification of M. tubercu- losis BlaC was performed as described previously [30]. BlaC was crystallized in the shard crystal form as de- scribed earlier [30]. The slurry was stirred overnight at 30 °C to avoid the growth of larger crystals that other- wise need to be crushed to be suitable for MISC experi- ments. Crystals grown this way were of dimensions 10 × 10 × 3 μm3. An additional crystal form was obtained from a different crystallization condition using the free interface diffusion (FID) method [61]. In a 1.5-mL Eppendorf tube, 250 μL of a precipitant solution (35% PEG 1000, sodium acetate pH 5.0), was slowly added dropwise through 250 μL of a protein solution at 10 mg/ mL. Needle-shaped crystals of dimensions 5 × 2 × 2 μm3 grew at room temperature in about 48 h. The microcrys- talline sample was highly monodisperse as demonstrated by dynamic light scattering (Additional file 1: Figure S12). The suspension showed an intense second order, non-linear imaging of chiral crystals (SONICC) signal demonstrating the crystallinity of the sample. X-ray powder diffraction was used as a quality test to verify the existence of diffracting crystals. A very high-density pellet of microcrystals was transferred to a transparent plastic capillary (MiTiGen, Ithaca, NY, USA). A small amount of precipitant solution was kept to prevent the crystals from drying out. The capillary was mounted onto a regular goniometer base, and data were collected for 3 min on a Rigaku Micro Focus 007 high-flux X-ray generator. Intense powder rings were observed up to 7 Å. Weaker rings were also observed to extend up to approximately 4 Å. General overview For each condition, the solution is considered mixed when the CEF concentration ex- ceeds 40 mM, which is sufficiently high to cause rapid binding. The reported mixing times are the time for the concentration around the average crystal to reach this threshold, with upper and lower bounds given for the first and third quartiles. In these calculations, the crys- tals are assumed to be much smaller than the focused jet, and fluctuations in flow rate are neglected. The mix- ing times for each time point are reported in Table 1. The delay time is defined as the time that the reaction is allowed to proceed after mixing. During this time, the crystals traverse the device before being probed by the X-ray beam. Uncertainty in the delay time results from errors in the sample and buffer flow rates (which come from the factory specifications for the Shimadzu LC-20 AD HPLC pumps that we used to drive the flows) and from small variations in the diameters and lengths of the capillaries used to make the mixing injectors. Mixing injectors were designed so that the delay time slightly exceeded the nominal time point to allow for additional time for the ceftriaxone to diffuse into the crystals. Table 1 lists the delay times and flow parame- ters for different time points. reported in PDB entry 2GDN has also been used by others [19], and we use it here throughout for homogen- eity. Accordingly, we obtained, as a function of time, in- formation on distinct chemical intermediates of β-lactam substrates within the active site of BlaC. The BlaC enzyme requires limited conformational changes to execute catalysis, allowing us to observe the full enzym- atic reaction within a crystal. Data collection, data analysis, and structure determination Serial femtosecond crystallography (SFX) experiments were performed at the CXI instrument [62]. Microcrys- tals (10 × 10 × 3 μm3 shard-shaped crystals or 5 × 2 × 2 μm3 needles) were mixed with the antibiotic ceftriax- one (200–300 mmol/L) before injection into a vacuum using a mixing jet injector (described above) that allowed millisecond time resolution. Diffraction patterns were recorded on a Cornell-Stanford Linear Accelerator Center (SLAC) pixel array detector (CSPAD) [63] oper- ating at 120 Hz to match the X-ray pulse frequency. Data for shards and needles were analyzed in an identi- cal fashion. Cheetah [64] was used to filter out diffrac- tion patterns containing Bragg reflections. These patterns were indexed and integrated using the CrystFEL (version 0.6.2) program suite [60, 65]. Partial intensities were scaled and merged using linear and Debye-Waller factor scale factors. Data statistics are listed in Table 2. The BlaC structures were solved for the needles and Injectors 1) were placed in the positive Δρomit t and initially real-space refined in Coot using ad- equate crystallographic information files (CIFs), which define the geometry and provide restraints. CIFs were generated for the full-length ceftriaxone (CEF) as well as an open form with the leaving group split off (E-CFO) as previously described [30]; compare also Fig. 1. One oxygen of the open lactam carboxyl in E-CFO was re- moved, and the carboxyl carbon was connected to the Ser70-Og with a weak distance restraint of 1.6 Å. At all time points, either CEF, E-CFO* (bound to Ser-70), or a mixture of both was observed. Their structures were first refined in real space in Coot. shards using molecular replacement by Phaser [66]. For the shards, the structure determined by Kupitz et al. [30] with four subunits (A–D) in the asymmetric unit was used as the initial model. For the monomeric structure in the needles, subunit D from this structure was ex- tracted and used as a search model. Reference structures Sref,n and Sref,s were determined for the needles and shards, respectively, using the respective “unmixed” data for both crystal forms. To determine structural changes after mixing, difference maps were determined. For the shards, unit cell changes on the order of 2 Å and larger were observed after mixing. This prevents the calcula- tion of isomorphous difference maps. With the needles, however, unit cell changes were not observed (Table 2), and isomorphous difference maps can be calculated. Ac- cordingly, two different strategies were followed to analyze the two types of data. 1. Structures for the shard crystal form. Since isomorphous difference maps could not be calculated, structural interpretation has been based on omit difference maps. The reference model was refined using simulated annealing (SA) in PHENIX against the observed jFobs t j. For this refinement, water and phosphate molecules residing in the active sites of all subunits were removed. In addition Ser-70 was replaced by a glycine (Gly-70) in subunits B and D. The structure was heated to 5000 K (default) and slowly cooled to 300 K. As a result, a model of the apo-protein without any li- gands in the active site was obtained. Injectors The mixing injectors used in this experiment were based on the design by Calvey et al. [36] shown in Add- itional file 1: Figure S1. In these devices, a crystal sus- pension and a buffer (either 1 mol/L sodium phosphate or sodium acetate, pH 5) containing 200–300 mmol/L CEF are flowing in coaxial capillaries. The flows are combined and forced into a constriction, thinning the crystal flow to a narrow jet and allowing rapid CEF dif- fusion. By varying the length of the device, the sample and buffer flow rates, or by placing an expanded region after the constriction, we were able to probe time scales Page 9 of 15 Olmos et al. BMC Biology (2018) 16:59 Page 9 of 15 Table 1 Mixing parameters for each time point. The buffer contained 200–300 mmol/L CEF in either 1.0 mol/L sodium phosphate (shard crystal form), pH 5, or in 100 mmol/L sodium acetate, pH 5 (needle crystal form). The superscript and subscript numbers in the second column indicate that deviations to shorter times are different from those to longer times Table 1 Mixing parameters for each time point. The buffer contained 200–300 mmol/L CEF in either 1.0 mol/L sodium phosphate (shard crystal form), pH 5, or in 100 mmol/L sodium acetate, pH 5 (needle crystal form). The superscript and subscript numbers in the second column indicate that deviations to shorter times are different from those to longer times g Nominal time point (ms) Mixing time (ms) Delay time (ms) Sample flow (μL/min) Buffer flow (μL/min) Constriction diameter (μm) 30 5þ6 −3 42 ± 2 4.0 ± 0.5 66.0 ± 0.6 75 ± 1 100 10þ13 −8 114 ± 4 10.0 ± 0.5 70.0 ± 0.7 75 ± 1 500 7þ10 −5 510 ± 20 8.0 ± 0.5 32.0 ± 0.5 50 ± 1 2000 15þ20 −11 2300 ± 50 10.0 ± 0.5 45.0 ± 0.5 75 ± 1 the water molecules re-appeared, a result that was also previously reported [30]. Hence, the structures of the catalytic clefts in these subunits A and C were restored back to the reference. The Δρomit t in the catalytic clefts of subunits B and D was exceptionally strong at all time delays (Fig. 3, Additional file 1: Figures S2–S4, S6). Ap- propriate CEF species (Fig. Injectors After the re- finement, mFo-DFc omit difference maps Δρomit t were calculated for each time point t, where the Fo correspond to the jFobs t j and the Fc are determined from the refined (partial) model, m is the figure of merit, and D is a coordinate error-dependent weighting term [67, 68]. The resulting omit map is essentially free of phase bias towards the ligand-free “unmixed” structure. Mixtures of full-length, non-covalently bound CEF configurations and Ser-70 bound, open forms (CFO) were refined together in PHENIX. Note that E-CFO* was replaced at 500 ms in subunit B with a species dis- playing an alcohol (Figs. 1d, 3g and Additional file 1: Figure S6b) instead of the double bond Δ, the structure of which was refined as described. Further refinement including occupancy refinement of the two species was performed with PHENIX [69] against the jFobs t j. Since a large volume of electron density is shared by CEF and the shorter E-CFO, occupancy refinement is not reli- able. Numbers obtained reflect the fact that the two molecules are present. Essentially complete ligand occu- pancy is reached at all time delays. Therefore, a potential presence of an unmixed BlaC species was not taken into account during the refinement. An additional CEF mol- ecule, which can be identified near, but not bound to, the active site (CEFstack) has been added to the last phase of the refinement. The leaving group (the large dioxo-triazine ring) is π-π stacking with the small amino-thiazol ring of the CEF species in the active site, resulting in an antiparallel alignment. Distances between the rings are on the order of 3.5 Å. However, as men- tioned later, the main interactions are with Tyr-127 Strong electron density appeared in subunits B and D that was reminiscent of CEF molecules. In subunits A and C, the electron density of only the phosphate and Olmos et al. Injectors BMC Biology (2018) 16:59 Page 10 of 15 Table 2 Data collection and refinement statistics Reference 30 ms 100 ms 500 ms 2 s (a) Shard-shaped crystals Hits 98,895 35,065 88,413 158,620 39,140 Indexed images 73,170 24,397 79,328 134,583 32,201 Resolution (Å) 2.45 2.75 2.15 2.20 2.30 Space group P21 P21 P21 P21 P21 Unit cell (Å,o) (a, b, c, and β) 79.0 97.2 110.6 108.7 78.7 96.8 112.6 109.7 79.2 96.5 113.7 109.9 78.8 96.3 113.5 110.0 78.2 95.6 112.3109.9 Volume (Å3) 804,442 807,597 817,098 809,346 789,415 BlaC/unit cell 8 8 8 8 8 Completeness 100(100) 100(100) 100(100) 100(100) 100(100) Multiplicity 1221 (103.3) 526 (142.0) 895 (58.8) 1363 (81.3) 330 (59.0) SNR 8.9(2.4) 6.4(0.9) 7.1(1.0) 8.3(0.9) 5.4(1.1) Rsplit (%) 9.8(209.4) 14.2(121.1) 11.18(111.0) 9.7(126.3) 11.9(104.1) CC-half (%) 99.4(41.1) 98.6(34.5) 99.4(37.5) 99.6(31.0) 96.8(35.4) Refinement Rcryst/Rfree (%) 19.2/24.4 19.3/25.0 20.9/23.9 21.9/25.0 23.5/26.6 BCEF/E-CFOa 0/0 91 (23b) 57/32 40/36c 58/25 DCEF/E-CFOa 0/0 89 (24b) 54/40 38/44 51/31 Stacking no yes yes yes yes H2O 315 143 499 431 399 Average B value (Å2) 48.2 51.7 42.3 37.3 36.2 Protein amino acid residues in asym. unit 265 × 4 265 × 4 265 × 4 265 × 4 265 × 4 Ligands 0 2 + 2 (stacking) 2 + 2 (stacking) 2 + 2 (stacking) 2 + 2 (stacking) RMSD bond lengths (Å) 0.008 0.010 0.008 0.008 0.008 RMSD bond angles (degrees) 1.10 1.72 1.66 1.67 1.74 PO4 4 2 2 2 2 (b) Needle-shaped crystals Hits 171,314 64,507 115,223 141,935 36,606 Indexed images 111,466 34,590 87,580 87,058 23,278 Resolution (Å) 1.8 1.9 1.8 1.9 2.05 Space group P21 P21 P21 P21 P21 Unit cell (Å,o) (a, b, c, and β) 39.6 41.6 69.3 104.8 39.5 41.6 69.3 104.8 39.6 41.6 69.3 104.9 39.6 41.7 69.5 104.9 39.6 41.7 69.5 104.9 Volume (Å3) 110,375 110,096 110,323 110,908 110,908 Completeness (%) 100(100) 100(100) 100(100) 100(100) 100(100) Multiplicity 985 (54.5) 330 (26.8) 831 (89.0) 806 (36.5) 238 (27.3) Signal-to-noise ratio 9.6(1.2) 5.8(0.8) 9.6(1.6) 8.6(0.9) 5.1(1.1) Rsplit (%) 6.6(97.0) 12.2(136.3) 6.6(72.5) 8.8(129.1) 14.0(105.9) CC (%) 99.9(75.0) 99.9(76.1) 99.9(84.3) 99.9(68.1) 99.8(74.8) CC-half (%) 99.7(39.1) 99.4(40.4) 99.7(55.1) 99.7(30.2) 99.13(38.8) Refinement Rcryst/Rfree (%) 21.5/24.5 20.7/26.2 23.0/26.7 21.7/26.4 20.0/25.0 Nd na 9 9 6 5 CEF/E-CFOa 0/0 59/0 51/35 43/53 71/0 Stacking no no no no no H2O 167 203 154 104 175 Average B value (Å2) 34.7 16.9 10.5 15.7 18.3 Protein amino acid residues in asym. Injectors The CEF concentration is much higher than the con- centration of BlaC molecules in the crystals (16 mmol/L in the shard crystal form, 30 mmol/L in the needles). The stoichiometric concentration of CEF is reached at a time t, much faster than τD. This time t can be estimated as t = τD ∙f where f = −lnð1−s outÞ, s is the concentration of BlaC in the crystals, and out is the outside CEF concentra- tion (Table 3c). f is ~ 0.1 for the shards and ~ 0.2 for the needles (see Table 3c). The experiment becomes robust to- wards variations in diffusion times caused by crystal size, crystal morphology, and crystal packing. Pore sizes in the shards (up to 90 Å) are up to four times larger than those in the needles (see discussion above, and also Add- itional file 1: Figure S11), which could severely impede dif- fusion, especially of a molecule as large as CEF (554.6 g/ mol). As shown in Table 3c, diffusion times may be slower by two orders of magnitude compared to those in solu- tion, and still the substrate would diffuse sufficiently fast to reach stoichiometric concentrations. (between Tyr-127Oη and OI of the CEFstack dihydrothia- zine carboxyl) and Arg-126 (between Arg-126Nε and OI of CEFstack-OI) of the adjacent (non-reactive) dimer sub- unit (see Fig. 2b, c and Additional file 1: Figure S9). CEFstack is pre-oriented this way very close to the active site. In order to access the active site, CEFstack only has to flip in, which may be initiated when the CFO species leaves the active site. B-factors of the various CEF spe- cies in the shard crystal form are shown in Additional file 1: Table S1. (between Tyr-127Oη and OI of the CEFstack dihydrothia- zine carboxyl) and Arg-126 (between Arg-126Nε and OI of CEFstack-OI) of the adjacent (non-reactive) dimer sub- unit (see Fig. 2b, c and Additional file 1: Figure S9). CEFstack is pre-oriented this way very close to the active site. In order to access the active site, CEFstack only has to flip in, which may be initiated when the CFO species leaves the active site. B-factors of the various CEF spe- cies in the shard crystal form are shown in Additional file 1: Table S1. 2. Structures for the needle crystal form. Injectors unit 265 265 265 265 265 Page 11 of 15 Olmos et al. BMC Biology (2018) 16:59 Table 2 Data collection and refinement statistics (Continued) Reference 30 ms 100 ms 500 ms 2 s Ligands 0 1 1 1 1 RMSD bond lengths (Å) 0.008 0.007 0.007 0.003 0.008 RMSD bond angles (degrees) 1.06 1.57 1.74 1.49 1.57 Bfor subunit B Dfor subunit D aoccupancy of full-length, intact CEF to covalently bound, open E-CFO, which has lost R. Numbers are rough estimates and should represent only trends (the error is on the order of 25%, see note b) bomit maps show only CEF in the active site. If E-CFO‘s occupancy is refined at the same time, values around 24% are obtained. We consider this the error of our occupancy refinement cAddition of OH instead of the double bond Δ dIf N does not extrapolate to 100% occupancy, a fraction of reference structure is present. This is ignored in the refinement na not applicable RMSD root mean square deviation Table 2 Data collection and refinement statistics (Continued) the active site as well as the diffusion coefficient of CEF in the crystals are unknown. Accordingly, we need to assume values that yield plausible results. When the koff rate coef- ficient is assumed to be equal to the kcat rate coefficient in solution, the kon rate coefficient for the binding of CEF is kon ¼ 2xkcat Km ¼ 2x0:8 500 x 10−6 L mol−1s−1 ¼ 3200 L mol−1s−1 . As- suming a diffusion coefficient of 2.3 × 10−6 cm2/s for CEF in water [70], characteristic diffusion times τD into the centers of the 10 × 10 × 3 μm3 shards and the 5 × 2 × 2 μm3 needles would be a few milliseconds (Table 3c) [28]. It should be mentioned here that diffusion times in crystals may be very different from, and much slower than, those in solution. At the characteristic diffusion time, ð1−1 eÞ or 63% of the outside CEF concentration is reached in the crystal center. Crystal suspensions are typ- ically mixed 1:4 with large concentrations of CEF (be- tween 200 mmol/L and 300 mmol/L). Mixing ratios can be up to 1:17 for the fastest mixing times (Table 1). As a result, the CEF solution is only slightly diluted after mix- ing. Injectors Difference structure factor amplitudes ΔFiso t were calculated for each time point t by subtracting the observed reference structure factor amplitudes jFobs ref jcollected with no CEF present from the time-dependent structure factor amplitudes jFobs t j. From the ΔFiso t and the phases derived from Sref,n, isomorphous dif- ference maps were calculated. In order to model the BlaC structure including a (potentially modified) CEF ligand, conventional electron density maps ρext t were calculated where the ligand occupancy was ex- trapolated to 1.0. Extrapolated structure factors Fext t were calculated by adding the ΔFiso t N times (see Table 2) to the calculated structure factors derived from Sref,n. The extrapolated electron density ρext t was derived from the Fext t . The structures of appro- priate CEF derivatives (see above and Fig. 1) were inserted using Coot [47]. At all time points, either CEF, E-CFO (bound to Ser-70), or a mixture of both was observed (Fig. 3c, f, i, l). Their structures were first refined in real space against the ρext t in Coot. Further occupancy refinement was performed as described above (1). Enzyme kinetics The Michaelis constant Km is on the order of 500 μmol/ L for BlaC with CEF, and kcat in solution is 0.8 s−1 [40]. The koff rate coefficient of dissociation of substrate from The time-dependent concentrations of species along the enzymatic pathway were simulated by Page 12 of 15 Page 12 of 15 Page 12 of 15 Olmos et al. BMC Biology (2018) 16:59 Table 3 Results from the simulations and estimates of diffusion times. (a) Parameters used in the simulation: apparent diffusion time τD based on crystal size, initial enzyme concentration E0, outside substrate concentration S0, rate coefficients k1 … k3, and product inhibition Ip (Scheme 1). (b) Occupancy of the various enzyme species as obtained by the simulation. They can be compared to occupancy values listed in Table 2. E free enzyme, ES non-covalently bound ceftriaxone in the active site, with leaving group present, E-CFO* enzyme intermediate with CFO bound covalently, P free product (CFO). (c) Diffusion times τD, and times t to reach stoichiometric concentration in the shard and needle crystals. As an example, estimates for the 1:4 (crystal:CEF) mixing ratio are listed. Time variations between 200 mmol/L and 300 mmol/L CEF are negligible. Times are lower limits, since they are estimated from diffusion in water. Even if they are allowed to vary by orders of magnitude, sufficient occupancy would be achieved after 30 ms. They also imply that the time resolution may be given by the mixing times (Table 1) in some crystal forms, and not by the diffusion times, since the former are longer than the latter (a) τD (ms) E0 (mmol/L) S0 (mmol/L) k1 (L mmol−1 s−1) k−1 (s−1) k2 (s−1) k3 (s−1) IP (mmol/L) 15/1.5 25 200 3.2 0.01 7 0.8 6 (b) 0 ms 30 ms* 100 ms 500 ms 2 s E (%) 100 10/2 2 1 1 ES (%) 0 84 58 19 59 E-CFO* (%) 0 6/14 40 80 40 P ([mmol/L) 0 0.05 0.5 5.8 23.4 (c) Shard-shaped crystals Needle-shaped crystals BlaC concentration in crystal 16 mmol/L 30 mmol/L Average size([μm3) 10 × 10 × 3 5 × 2 × 2 τD 3.5 ms 0.8 ms Mixing ratio crystal:CEF 1:4 1:4 CEF = 200 mmol/L CEF after mixing 160 mmol/L 160 mmol/L Time to reach stoichiometric conc. 0.36 ms 0.17 ms CEF = 300 mmol/L CEF after mixing 240 mmol/L 240 mmol/L Time to reach stoichiometric conc. Enzyme kinetics 0.24 ms 0.10 ms For 15-ms and 1.5-ms diffusion times, respectively For 15-ms and 1.5-ms diffusion times, respectively The substrate concentration S in the crystal is deter- mined by the outside substrate concentration S0 (50 mmol/L in the simulation) and the characteristic diffu- sion time τD. S is fed to the free enzyme E and bound to the enzyme with k1. The total enzyme concentration was set to 25 mmol/L. Results are shown in Fig. 4 and Table 3b for τD = 15 ms (solid lines) and for τD = 1.5 ms (dashed lines). Sufficient accumulation (occupancy) of the enzyme substrate complex (ES, green) is achieved after 30 ms even with the longer diffusion time (Fig. 4), which agrees with our observations by MISC (Fig. 3a, b, c). Initially, k3 was assumed to account for the rate-limiting process and set to kcat = 0.8 s−1. As the low k3/Km ratio found in the litera- ture [40] (~ 1.5 × 103 L mol−1 s−1) suggests, CEF binds slowly to the enzyme. In Fig. 4, we show results for 1.5-ms as well as 15-ms diffusion times. As the kinetics are very similar, the MISC experiment is robust against crystal size heterogeneities as well as against mixing time jitter (Table 1). The ES complex accumulates slightly faster with numerically integrating the coupled differential equa- tions of the simple kinetic mechanism shown in Scheme 1 using the above rate coefficients which re- produce the known Km. Note that formation of the covalent E-CFO* complex (acyl intermediate) is irre- versible due to the cleavage of the leaving group R from CEF. Table 3a lists the parameters that enter the calculation. numerically integrating the coupled differential equa- tions of the simple kinetic mechanism shown in Scheme 1 using the above rate coefficients which re- produce the known Km. Note that formation of the covalent E-CFO* complex (acyl intermediate) is irre- versible due to the cleavage of the leaving group R from CEF. Table 3a lists the parameters that enter the calculation. Scheme 1 Page 13 of 15 Olmos et al. BMC Biology (2018) 16:59 the fast diffusion time, but the kinetics are essentially the same for both simulated diffusion times. The acyl inter- mediate (E-CFO, blue) forms essentially on the same time scale (500 ms) for both crystal forms (Fig. 3 g, h, i). Authors’ contributions JLO, JMM-G, CK, DO, MDM, NN, JZ, JV, TN, DX, EB, RF, LT, GNP, MS prepared and crystallized samples. MSH, ML, JaK, M.H.S. operated the CXI beamline. A.K., G.C., Ju.K., D.O., M.W.,JLO, JMM-G, CK, DO, MDM, NN, JZ, JV, TN, DX, EB, RF, LT, GNP, and MS prepared and crystallized the samples. MSH, ML, JaK, and MHS operated the CXI beamline. AK, GC, JuK, DO, MW, MiH, MaH, SS, SR- C, JC, NN, JZ, YZ, GN, SB, UW, HNC, and LP provided the injector systems, and operated injection at the CXI beamline. SP, DO, OY, KP, AB, TN, IP, TG, VM, GS, RaF, PS, MF, TW, and NZ RF, LT, GNP, and MS prepared and crystallized the samples. MSH, ML, JaK, and MHS operated the CXI beamline. AK, GC, JuK, DO, MW, MiH, MaH, SS, SR- C, JC, NN, JZ, YZ, GN, SB, UW, HNC, and LP provided the injector systems, and operated injection at the CXI beamline. SP, DO, OY, KP, AB, TN, IP, TG, VM, GS, RaF, PS, MF, TW, and NZ Funding This work was supported by the National Science Foundation (NSF)-Science and Technology Center (STC) “BioXFEL” through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-SC0002164 (to A.O., algorithm design and development) and by the NSF under contract number 1551489 (to A.O., underlying analytical models). Portions of this research were performed at the Linac Coherent Light Source (LCLS). Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-AC02-76SF00515. This material is based upon work supported by the NSF Graduate Research Fellowship Program to J.L.O. under grant no. 1450681. The work was also supported by funds from the National Institutes of Health grants R01 GM117342-01 and R01 GM095583, by funds from the Biodesign Center for Applied Structural Discovery at Arizona State University, and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. Part of this work was also supported by program-oriented funds of the Helmholtz Association. Availability of data and materials The structures and diffraction data of BlaC, unmixed and mixed with ceftriaxone at 0 s, 30 ms, 100 ms, 500 ms, and 2 s for shard and needle crystal forms are deposited to the Protein Data Bank (PDB) with the following access codes: Shards 6B5X (unmixed), 6B5Y (30 ms), 6B68 (100 ms), 6B69 (500 ms), 6B6A (2 s); needles 6B6B (unmixed), 6B6C (30 ms), 6B6D (100 ms), 6B6E (500 ms), 6B6F (2 s). None of the authors declare competing interests. Clones and other material such as computer code are available on request from the corresponding author. Enzyme kinetics The catalytic cleft of BlaC, further details, including a difference map between the 500 ms and 100 ms time points. Figure S11. Crystal packing in shards and needles. Figure S12. Dynamic light scattering results. Table S1. B- factors for CEF species observed in the shard crystals at different time delays. (PDF 1646 kb) 2 s). Figure S8. 2mFo-DFc electron density and structural details in the catalytic clefts of BlaC in the needle crystal form (stereo representation from 30 ms to 2 s). Figure S9. Details in the catalytic cleft of subunit B in the shard crystal form at 500 ms including the stacked CEF, 2FoFc maps, and simulated annealing omit maps (stereo representation). Figure S10. The catalytic cleft of BlaC, further details, including a difference map between the 500 ms and 100 ms time points. Figure S11. Crystal packing in shards and needles. Figure S12. Dynamic light scattering results. Table S1. B- factors for CEF species observed in the shard crystals at different time delays. (PDF 1646 kb) Abbreviations BlaC: Mycobacterium tuberculosis β-lactamase; CEF: Ceftriaxone; CFO: Ceftriaxone with lactam ring open and the leaving group split off; CSPAD: Cornell SLAC pixel area detector; CXI: Coherent X-ray imaging; E- CFO: CFO species covalently bound to the enzyme; ES: Enzyme-substrate (complex); FID: Free interface diffusion; Km: Michaelis constant; LCLS: Linac Coherent Light Source; MISC: Mix-and-inject serial crystallography; SLAC: Stanford Linear Accelerator Center; SFX: Serial femtosecond crystallography; XFEL: X-ray free-electron laser Additional file Additional file 1: Figure S1. Schematics of the short-time-point mixing injector. Figure S2. Selected views of the CEF binding site in the BlaC shard crystals including simulated annealing omit maps. Figure S3. Structural details, and simulated annealing omit maps, shard crystal form, subunit B (stereo representation, from 30 ms to 2 s). Figure S4. Structural details and simulated annealing omit maps, shard crystal form, subunit D (stereo represen- tation, from 30 ms to 2 s). Figure S5. Structural details, and simulated annealing omit maps, needle crystal form (stereo representation, from 30 ms to 2 s). Figure S6. Backside view of the catalytic cleft of BlaC in the shard crystal form, structural details and simulated annealing omit maps (stereo representation, selected time points). Figure S7. 2mFo-DFc electron density in the catalytic clefts of BlaC in the shard crystal form (stereo representation, from 30 ms to collected and processed the data. JLO, SP, JMM-G, CK, DO, MiH, MDM, TN, IP, DX, RaF, RuF, PF, GNP, and MS analyzed and interpreted the data. LT, AO, and MS conceived the project. JS, PF, AO, GNP, and MS wrote the manuscript with contributions from all authors. All authors read and approved the final manuscript. Enzyme kinetics In our MISC X-ray data we do not see clear evidence of a prod- uct complex (EP) where the CFO has been hydrolyzed (detached) from Ser-70 and is non-covalently bound to the enzyme. It appears as if this product state is depopu- lated faster than it is populated, and it therefore does not accumulate sufficiently to be detected. Importantly, the ES complex reappears in our MISC data at 2 s (Fig. 3j, k, l). That means the E-CFO* cannot be the rate-limiting species (with the hydrolysis of the covalent bond the rate-limiting step); otherwise, E-CFO* would be the dominant species in the steady state. However, if ES were initially rate-limiting (and the nucleophilic attack of Ser-70 the rate-limiting step), E-CFO* would not accumulate sufficiently to be de- tected so clearly at 500 ms. To solve this dilemma, we as- sume that rate coefficient k2 (the Ser-70 nucleophilic attack) decreases with product concentration. Unlike in so- lution, in the crystal enzyme and substrate concentrations are so high that already after one turnover more than 10 mmol/L of substrate is converted to product. Accord- ingly, on time scales > 1 s, product inhibition was assumed by lowering k2: k2 ¼ k0 2ð1−e−Pn=IpÞ, where Pn is the concen- tration of the released product P divided by an characteristic inhibitory concentration Ip in mmol/L (Table 3a). BlaC in- hibition by penicilloic acids was also reported previously [52]. This detail of the BlaC reaction awaits further investi- gations which are outside the scope of this paper. By no means do we suggest that this mechanism is unique. There are only four time points (plus the unmixed, free enzyme species). The rate coefficients in the mechanism may vary widely and still reproduce the observations. Within a large number of plausible mechanisms, our mechanism is the simplest that explains our experimental observations at lim- ited time points. If a more complex mechanism is to be jus- tified, the collection of additional, more finely spaced time points is necessary. 2 s). Figure S8. 2mFo-DFc electron density and structural details in the catalytic clefts of BlaC in the needle crystal form (stereo representation from 30 ms to 2 s). Figure S9. Details in the catalytic cleft of subunit B in the shard crystal form at 500 ms including the stacked CEF, 2FoFc maps, and simulated annealing omit maps (stereo representation). Figure S10. Author details 1 1Department of BioSciences, Rice University, 6100 Main Street, Houston, TX 77005, USA. 2Physics Department, University of Wisconsin-Milwaukee, 3135 N. 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https://openalex.org/W4296838021	https://www.frontiersin.org/articles/10.3389/fonc.2022.1017026/pdf	English	null	MET alterations in advanced pulmonary sarcomatoid carcinoma	Frontiers in oncology	2,022	cc-by	6,314	OPEN ACCESS EDITED BY Maoshan Chen, Army Medical University, China REVIEWED BY Jiao Liu, Tulane University, United States Yuwen Li, Tulane University, United States Murat Kara, Istanbul University, Turkey EDITED BY Maoshan Chen, Army Medical University, China Chen Gong, Huihua Xiong, Kai Qin, Jianhua Wang, Yi Cheng, Jing Zhao and Jing Zhang* Chen Gong, Huihua Xiong, Kai Qin, Jianhua Wang, Yi Cheng, Jing Zhao and Jing Zhang* Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Pulmonary sarcomatoid carcinoma (PSC) is a rare subset of NSCLC that accounts for about 0.5-1% of all primary lung carcinoma, and its malignant biological behavior is more aggressive than other pathological types of lung cancer. Recent studies have reported a variety of gene mutations associated with the occurrence, development and treatment of PSC, especially the mesenchymal-epithelial transition (MET) proto-oncogene alterations, including the exon 14 (METex14) skipping mutations as well as the ampliﬁcation and overexpression of MET gene, which are associated with molecularly targeted therapy for PSC. METex14 skipping mutation is the most common and well-studied mutation type, occurring in about 22-31.8% of PSC patients, while the prevalence of MET ampliﬁcation is reported as 4.8-13.6% and MET ovexpression is about 20.2%. Molecular pathology tests, including IHC and NGS, are valuable in determining the prognosis of patients with PSC and helping to determine the treatment. The existing clinical data have conﬁrmed the efﬁcacy of MET-TKI in PSC patients with MET alteration, among which the clinical study of Savolitinib has enrolled the largest proportion of PSC patients and achieved relatively good efﬁcacy, but more clinical researches are still needed. The multi-disciplinary team may maximize the optimal treatment options for patients with the advanced PSC. Gong C, Xiong H, Qin K, Wang J, Cheng Y, Zhao J and Zhang J (2022) MET alterations in advanced pulmonary sarcomatoid carcinoma. Front. Oncol. 12:1017026. doi: 10.3389/fonc.2022.1017026 COPYRIGHT © 2022 Gong, Xiong, Qin, Wang, Cheng, Zhao and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. MET, pulmonary sarcomatoid carcinoma, skipping mutation, ampliﬁcation, overexpression TYPE Review PUBLISHED 23 September 2022 DOI 10.3389/fonc.2022.1017026 TYPE Review PUBLISHED 23 September 2022 DOI 10.3389/fonc.2022.1017026 TYPE Review PUBLISHED 23 September 2022 DOI 10.3389/fonc.2022.1017026 TYPE Review PUBLISHED 23 September 2022 DOI 10.3389/fonc.2022.1017026 Wild type MET structure and function The MET gene is located on the long arm of human chromosome 7q21-q31 (125kb long) and contains 21 exons. The MET proto-oncogene encodes a membrane MET tyrosine kinase receptor that mainly expressed in epithelial cells, also known as hepatocyte growth factor (HGF) receptors, which have been identiﬁed as a tumor driver gene and potential target of NSCLC (9). MET can bind with HGF with high afﬁnity and induce a series of biological effects, and it is frequently associated with and functionally supports the Epithelial-to-Mesenchymal Transition, which is mainly manifested to stimulate cell proliferation, survival, invasion and migration in tumors (8). In other words, the ectopic activation of MET pathways can drive the development, growth, and metastasis of various malignancies, including lung cancer, breast cancer, cervical cancer, gastric cancer and colon cancer (10). Pulmonary sarcomatoid carcinoma (PSC) is a rare subset of NSCLC that accounts for about 0.5-1% of all primary lung carcinoma, and its malignant biological behavior is more aggressive than other pathological types of lung cancer (5–7). According to the World Health Organization (WHO) Classiﬁcation of Lung Tumors (2015 version), PSC is deﬁned as a group of poorly differentiated NSCLC containing a component of sarcoma-like elements or true sarcomatous areas that is currently solely based on morphological characteristics, and is divided into ﬁve subtypes such as spindle cell carcinoma (a carcinoma almost completely composed of epithelial spindle cells), giant cell carcinoma (a carcinoma almost entirely composed of tumor giant cells), pleomorphic carcinoma (a poorly differentiated NSCLC that contains at least 10% spindle and/or giant cells or a carcinoma consisting only of spindle and giant cells), carcinosarcoma (a mix of NSCLC and true sarcoma) and biphasic pulmonary blastoma (a tumor composed of embryonal-type epithelial elements and primitive mesenchymal stroma) (6, 8). Being structured with a 50kD a chain and a 145kD b chain linked by disulﬁde bonds, MET protein also contain an N- terminal extracellular binding domain, a transmembrane helical domain, and an intracellular C-terminal domain with tyrosine kinase activity (11). The extracellular domain contains three distinct functional regions, including the Semaphorin (SEMA) domain covering the whole a chain and the N-terminal of some b chain, cystine-rich MET- related domain (Plexins-semaphorins-Integrins, PSI) containing four disulphide bonds and four immunoglobulin- plexin transcription regions (IPT) (3). Introduction Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for approximately 85% of all lung cancer with signiﬁcant heterogeneity and it may be associated with some known and/or unknown driver gene changes (1). According to the Non-Small Cell Lung Cancer, Version 3.2022, NCCN Clinical Practice Guidelines Frontiers in Oncology 01 frontiersin.org Gong et al. 10.3389/fonc.2022.1017026 10.3389/fonc.2022.1017026 in Oncology, NSCLC can be further classiﬁed into adenocarcinoma, squamous carcinoma, adenosquamous carcinoma, large cell carcinoma, and sarcomatoid carcinoma (2). Twenty years ago, there were limited treatments, such as surgery, radiotherapy and chemotherapy for advanced NSCLC, and the overall prognosis was dismal (3). However, the prognosis of advanced NSCLC has improved dramatically. And one of the important reasons is the discovery of the driver genes, their somatic genomic alterations (also known as molecular biomarkers) includes gene mutations and fusions, and the emergence of molecular targeted drugs (2–4). In the NCCN guidelines, the recommended genes for genetic testing in most patients with advanced NSCLC include ALK, BRAF, EGFR, KRAS, MET, NTRK1/2/3, RET and ROS1. The broad genomic testing can be used to assess mechanisms of drug resistance in patients who have relapsed after the targeted therapies, to distinguish primary lung cancer from intrapulmonary metastasis, and to help determining suitability for certain molecular-driven clinical trials (2). Although the precise molecular characterization of PSC is largely unexplored, many studies have found that a variety of gene mutations may be associated with the occurrence, development, and treatment of PSC. Several recent studies have conﬁrmed the mesenchymal-epithelial transition (MET) proto-oncogene alterations in PSC, including MET exon 14 (METex14) skipping mutations and MET ampliﬁcation, with an incidence of approximately 22% and association with molecular-targeted therapy for PSC (6). This review aims to update the molecular pathology and clinical features of MET gene alteration in PSC. Frontiers in Oncology frontiersin.org Wild type MET structure and function The intracellular domain (957-1390 amino acid residues) after the transmembrane helical domain also consists of three regulatory regions, including the juxtamembrane (JM) domain containing Tyr1003 and Ser985 phosphorylation sites, the catalytic domain containing Y1234 and Y1235 phosphorylation sites, and the C-terminal multifunctional binding region containing Y1349 and Y1356 acting as a docking site for adaptor proteins, which leads to downstream signaling via phosphoinositide 3-kinase (PI3K)/ AKT (protein kinase B), signal transducer and activator of PSC usually occurs in older men with a history of smoking, and its clinical symptoms are non-speciﬁc compared with other types of NSCLC, such as cough, chest pain, hemoptysis, and dyspnea, which makes early detection and diagnosis difﬁcult to some extent (5, 6). PSC has a very poor prognosis, with overall survival (OS) and 5-year survival rate signiﬁcantly lower than other types of lung cancer. In different studies, 5-year survival for sarcomatoid carcinoma has been reported ranging from 20.1% to 36.1%, with less than 5% survival and less than 7 months survival in advanced stage (5, 6). Due to the rapid progression of PSC and its insensitivity to conventional chemotherapy agents, previously, many patients with PSC do not have the opportunity to receive a second-line chemotherapy or to try other novel antitumor agents. Frontiers in Oncology 02 frontiersin.org Gong et al. 10.3389/fonc.2022.1017026 transcription protegins (STAT), mitogen-activated protein kinase (MAPK), Wnt/beta-catenin, extracellular signal regulated kinase (ERK), mammalian target of rapamycin (mTOR)and nuclear factor-kB (NF-kB) (3, 11, 12). ALK and ROS1, and is also associated with poor prognosis of lung cancer, including sarcomatoid cancer (19). Therefore, some studies recognized the lung cancer with METex14 skipping mutation as an independent molecular subtype and carried out individualized treatment (20). ALK and ROS1, and is also associated with poor prognosis of lung cancer, including sarcomatoid cancer (19). Therefore, some studies recognized the lung cancer with METex14 skipping mutation as an independent molecular subtype and carried out individualized treatment (20). HGF is the only known natural ligand of MET, and its binding to MET leads to receptor dimerization and phosphorylation of Y1234 and Y1235 tyrosine residues in the kinase domain’s catalytic loop and autophosphorylation of the carboxy-terminal bidentate substrate-binding sites 1349 and 1356, which can activates RTK-mediated downstream signaling pathways mentioned above (3, 11). These signaling transduction pathways are widely involved in cell proliferation, survival, cell motility, embryogenesis, organogenesis, angiogenesis, epithelial-mesenchymal transition and many other important biological behaviors in normal cells (13). Wild type MET structure and function Compared with the common type of non-small cell lung cancer, the incidence of METex14 skipping mutation is signiﬁcantly higher in PSC (7). It is important to improve the sensitivity and speciﬁcity of the genetic testing for METex14 skipping mutation, which is an independent driver mutation of NSCLC without exception for PSC and its clinical signiﬁcance is more prominent. Reverse transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing have both been used to detect MET mutations, but these targeted methods are rarely used because of the efﬁciency of detection (21). Compared with testing other lung cancer driver genes, such as EGFR and KRAS, the application of NGS in the diagnosis of MET alterations is not very efﬁcient, especially for METex14 skipping mutation. The main reason is that DNA sequencing can only detect the genomic sequence alterations to predict or postulate a possible splicing result, which will not conﬁrm the actual METex14 skipping event. MET dysfunction is considered to be one of the driver event of lung cancer, which is often caused by gene copy number ampliﬁcation, receptor protein overexpression, genetic sequence variations; exon 14 JM skipping mutations cause alternative splicing variant, and MET gene fusions (14). Many studies have reported that the frequency of MET alteration in PSC is higher than that in other types of lung cancer. At present, the most important MET alteration in the ﬁeld of PCS is MET overexpression, MET ampliﬁcation, and METex14 skipping mutations. At the same time, routine application of genetic testing in clinical practice can identify potential genetic biomarkers for developing targeted treatments and provide treatment options in addition to surgery, radiation, and chemotherapy. There are two DNA-based NGS technologies commonly used for genetic diagnosis of tumors currently. The ﬁrst method is amplicon-based methods, which uses primers to capture the sequences of target genes in genomic regions by multiplex polymerase chain reaction (PCR) ampliﬁcation (22). This method has a relatively shorter detection time and can better capture some regions that are difﬁcult to sequence, but it is prone to sequencing distortion for regions with small insertion/ deletions (indels), homopolymers, and allele loss. Amplicon- based method may fail to identify all METex14 skipping mutation, mainly because ampliﬁcation primers are not designed for high-quality sequencing and fail to capture key mutation sites. Wild type MET structure and function In the case of single-nucleotide variant or small indels in the primer region, the primers may not bind due to mismatches, eventually leading to allele dropout (20). In addition, the binding site of the primer may also be lost if there is an entire deletion in the genomic region. In other words, the location and size of the genetic alterations that cause the METex14 skipping mutation may lead to allelic dropout and false-negative results, which ultimately lead to a low detection rate of METex14 skipping mutation using the Amplicon-based method (23). Another approach is hybrid capture-based method, which uses long biotinylated oligonucleotides to hybridize target regions in the genome and enable ﬂanking regions to be sequenced (22). These probes are signiﬁcantly longer than the PCR primers used in the amplicon-based method and thus can tolerate binding site mismatches without interfering with target hybridization, which can avoid the problem of allelic deletions seen in amplicon based methods (22). Chen et al. (24) reported that hybrid capture-based method is the preferred method to avoid common allele deletions caused by amplicon-based assays. Frontiers in Oncology frontiersin.org MET mutations MET mutations can be detected in 3% to 5% of patients with non-small cell lung cancer (mainly adenocarcinoma) and occur more frequently in PSC, and METex14 skipping mutation is the most common and well-studied mutation type, occurring in about 3%-4% of adenocarcinoma patients and 22% of PSC patients (15, 16). Therefore, we will focus on the MET14 exon skipping mutation, including the biology, genetic testing, diagnosis and related molecular targeted therapy strategies of METex14 skipping mutation in this section. The molecular mechanism of METex14 skipping mutation in NSCLCs was reported by Kong Beltran et al. in 2006 (17). Exon 14 encodes the 47-amino acid JM domain of the MET receptor, a key regulatory region that prevents MET hyperactivation. When MET mutation occurs, the binding sites for Y1003 and c-CBL are lost and the process of CBL- mediated MET protein degradation is impaired, MET receptors are aggregated, and MET oncogenic signals are overactivated (18). METex14 skipping mutation is considered to be an independent lung cancer driver, which is usually mutually exclusive with other lung cancer driver genes such as EGFR, 03 frontiersin.org Gong et al. 10.3389/fonc.2022.1017026 10.3389/fonc.2022.1017026 Unlike the detection mechanism of DNA NGS, RNA NGS can detect fusion of METex13 to 15, which is a common consequence of any altered splicing mechanism or deletion (13). Thus, it has the advantage of theoretically detecting all genomic events that lead to METex14 skipping mutation (15). Subramanian et al. (22) and Socinski et al. (21) reported that the accuracy and detection rate of RNA NGS were higher than that of DNA NGS in the detection of MET14 mutations. Davies et al. (15) found that the detection rate of RNA NGS was 4.2% (17/ 404) for METex14 skipping mutation, which was signiﬁcantly higher than DNA NGS (1.3%, 11/856). But there are also problems with RNA NGS. RNA is less stable than DNA, which limits the shelf life of the tissue. In addition, interpretation of RNA NGS results poses challenges due to the high variability of mRNA expression between nonmalignant and tumor tissues (25). Teishikata et al. (26) also reported that the differences of METex14 skipping mutation detection not only exist between different sequencing technologies, but also between different NGS platforms. MET ampliﬁcation MET ampliﬁcation is also known as MET gene replication, mainly acquired by chromosomal 7 or local regions duplication. Aberrant chromosomal 7 replication, containing MET gene, always results in segmental chromosomal polysomy. The presence of polysomy can lead to the contiguous gene ampliﬁcation, including MET and other genes on the affected chromosomal region. However, local ampliﬁcation is usually caused by regionally cryptic copy number gain, but not microscopic chromosomal duplication. Now there is no uniﬁed diagnostic criterion for MET ampliﬁcation, with different cut-offs for different detection methods, such as immunohistochemistry (IHC), ﬂuorescence in situ hybridisation (FISH), quantitative Real-Time reverse transcriptase-PCR (qRT-PCR), or NGS (Next Generation Sequencing). However, IHC appears to be a poor screen for MET ampliﬁcation since MET IHC-positive cases may be MET ampliﬁcation negative and vice versa (32). qRT-PCR has been used to detect MET ampliﬁcation, although it isn’t well characterized compared to FISH and NGS (33). As mentioned above, the incidence of METex14 skipping mutation ranged from 22% to 31.8% in PSC, a higher mutation rate than in other types of NSCLC (16, 27). Li et al. (28) reported that in a study of 77 PSC patients, patients with advanced PSC and a positive METex14 skipping mutation had a faster rate of disease progression than patients without any driver gene mutation, and they have a median PFS of not yet reached vs. 3.97 months during follow-up (P =0.017). Therefore, the efﬁcacy of molecular targeted therapy for MET is a hot topic at present, and many clinical studies of drugs are being carried out. Crizotinib, a multi-target TKI covering MET, has been reported in some small retrospective studies to treat advanced PSC with METex14 mutation. Unfortunately, these data have not been analyzed independently from other non-small cells (5). Capmatinib is an oral, potent, and selective MET inhibitor that has been approved by the FDA for the treatment of NSCLC patients with the METex14 mutation. A phase I single-arm trial (NCT01324479) enrolled four PSC patients with METex14 mutations, including one with stable disease and three with partial response (29). Tepotinib, the ﬁrst highly selective TKI for METex14 mutation approved for marketing in the world, enrolled 2 patients with PSC out of 152 patients in the VISION study (open-label, phase 2), but the trial data were not analyzed by pathological type (30). MET mutations In short, the detection of METex14 skipping mutation by DNA NGS and RNA NGS has its own advantages and disadvantages, and it is possible to obtain more comprehensive and accurate information about METex14 skipping mutation by using dual-omics detection for some lung sarcomatoid carcinomas. PSC subgroup was 5.5 months, mOS was 10.6 months, ORR was 50%, and disease control rate (DCR) was 90% (31). It is the only MET inhibitor with PSC population data, which has brought a breakthrough for the treatment of PSC patients. MET ampliﬁcation The frequency of MET ampliﬁcation in PSC has rarely been reported. Mignard et al. (40) reported MET ampliﬁcation in about 8.5% of PSC patients, Tong et al. (41) about 13.6%, and Liu et al. (42) about 4.8%. These variations may depend on the sample size and different methods used in each study. and acquired MET ampliﬁcation (secondary MET ampliﬁcation) are typically identiﬁed in about 5-20% patients with oncogene- positive NSCLC following resistance to tyrosine kinase inhibitors (TKIs), such as EGFR TKIs (38, 39). Until now, there was no large-scale prospective or retrospective studies of PSC with MET ampliﬁcation. The frequency of MET ampliﬁcation in PSC has rarely been reported. Mignard et al. (40) reported MET ampliﬁcation in about 8.5% of PSC patients, Tong et al. (41) about 13.6%, and Liu et al. (42) about 4.8%. These variations may depend on the sample size and different methods used in each study. The most commonly used detection method of MET expression is IHC with kinds of antibodies, consisted of monoclonal antibodies, polyclonal antibodies and antibodies to phosphorylated MET (46). Generally, the IHC staining of MET were always assessed by the pathologists, providing the basis for various semiquantitative scoring systems of MET protein expression and overexpression. The degree of MET expression is usually quantiﬁed as a staining score from 0 to 3+ (47). IHC 1+ indicates MET expression, while MET overexpression is deﬁned as IHC 2+ and 3+ (47). The H-score is another typical scoring system calculated by multiplying the percentages of cells of MET expression with their staining intensity score, and ranging from 0 to 300 (43). The score ≥200 usually indicates MET overexpression, but different thresholds vary between different clinical studies (43). MET ampliﬁcation is a type of conﬁrmed mechanisms of acquired resistance to EGFR-TKIs and ALK inhibitors in NSCLC (43). Several case reports of MET ampliﬁcation with PSC have been published. Wang et al. reported a 74-year-old female PSC patient with co-existing mutation in exon 21 L858R of EGFR and MET ampliﬁcation at diagnosis (44). Combination of EGFR and MET inhibitors, geﬁtinib and crizotinib, respectively, were used. The patient acquired a partial response and remained stable for 9.7 months after terminated treatment. This observation highlights the importance of genetic testing and paves the way for combined targeting strategies in PSC. MET ampliﬁcation Savolitinib is a highly selective MET TKI, which is the ﬁrst and currently the only approved selective MET inhibitor in China. A multicenter, single-arm, open-label, phase 2 study of savolitinib enrolled a total of 70 patients with NSCLC who had METex14 skipping mutation (25 of whom were patients with PSC). The mPFS of Traditionally, MET ampliﬁcation was detected by FISH with MET gene copy number (GCN) or the ratio of MET to chromosome enumerating probe against chromosome 7 (CEP7). Generally, MET GCN≥5 or MET : CEP7≥2.0 is used as the FISH criteria for MET ampliﬁcation (3, 11). MET : CEP7 is more accurate than GCN, for MET : CEP7 is able to distinguish the true MET ampliﬁcations and MET polysomy. The degree of ampliﬁcation were categorized into three groups by MET : CEP7, low (≥1.8 to ≤2.2), intermediate (>2.2 to <5) and high (≥5) or low (≥1.8 to ≤2.2), intermediate (>2.2 to <4) and high (≥4) (34, 35). In general, the MET : CEP7 ≥5 was identiﬁed as an appropriate cut-off with no overlap with other oncogenes compared with low and intermediate groups and seems to be the strongest predictor of MET-driven tumors (13, 36). Additionally, the targeted therapies, like crizotinib, showed more effective in patients with high MET ampliﬁcation than low and intermediate categories (35). Now, NGS is widely used in detecting MET ampliﬁcation. MET ampliﬁcation was commonly deﬁned by copy number fold change of 1.8x or more by NGS. Similar to FISH, the cut-offs may vary signiﬁcantly between different assays. The main limitation of NGS is that the result is highly dependent on the quality of the sample and, more importantly, the amount of non-tumor DNA from the non-tumor cells in the sample (37). De novo MET ampliﬁcation (primary MET ampliﬁcation) occurs in about 1-5% of non-small-cell lung cancers (NSCLC), 04 Frontiers in Oncology frontiersin.org frontiersin.org 10.3389/fonc.2022.1017026 Gong et al. overexpression partially due to discrepancies in its causes (1). Fortunately, new therapeutic targets for MET are being explored, including biparatopic antibodies (targeting two different epitopes on the same target protein), antibodies and ADCs combinations. and acquired MET ampliﬁcation (secondary MET ampliﬁcation) are typically identiﬁed in about 5-20% patients with oncogene- positive NSCLC following resistance to tyrosine kinase inhibitors (TKIs), such as EGFR TKIs (38, 39). Until now, there was no large-scale prospective or retrospective studies of PSC with MET ampliﬁcation. MET ampliﬁcation This was the ﬁrst reported case of PSC patient with concurrent EGFR mutation and MET ampliﬁcation prior to treatment, who may beneﬁt from combination EGFR and MET inhibitors. MET overexpression has been reported with high frequencies in NSCLC, ranging from 22.2-74.5%, and seems to portend poorer prognosis (4). In contrast, studies on MET overexpression in PSCs are limited. Liu et al. (42) found MET overexpression in 20.2% PSCs and carried survival analysis of MET gene alterations and protein expression in Chinese PSCs with only surgery but no MET TKI treatment. They found that MET ampliﬁcation suffered shorter mOS, while METex14 skipping mutation and overexpression didn’t affect patients’ survival. The studies showed different prognostic value of MET overexpression between PSCs and NSCLCs. He et al. (44) Reported that a 62-year-old male patient carrying two rare EGFR mutations, exon 18 L719S and exon 19 L797S. After application of afatinib for 6 months, the patient experience disease progression. NGS found new acquired MET ampliﬁcation with original mutations of EGFR exon 18 L719S and exon 19 L797S. Then the patient was treated with afatinib combined with crizotinib, with a result of a partial response of the disease. Combined therapies may be efﬁcient for the MET ampliﬁcation PSC patients with concurrent mutation of other oncogenes or secondary to resistance of previous treatment of TKIs. More large-scale clinical trials are required to conﬁrm the ﬁndings. Xavier Mignard et al. (40) found MET overexpression as a poor predictor of MET ampliﬁcations or exon 14 mutations in PSCs. They reported that MET exon 14 mutations could cause loss of ubiquitination and improve the presence of c-MET membrane, while lack of association between them and MET overexpression remains to be understood. These results may be induced by the different genomic backgrounds between PSCs and NSCLCs, or other mechanisms in the oncogenesis of MET exon 14 mutations. The lack of clinical trials about PSCs results in the limited understanding of the biological mechanisms, development, diagnosis, prognosis and treatment of the tumors (48). We are looking forward to more relevant clinical studies. MET overexpression MET ovexpression is thought to be one of the earliest MET dysregulation event in oncogenic process. MET can be found transcriptionally overexpressed in the presence of hypoxia and inﬂammation, thereby activating proliferation, reducing apoptosis, promoting migration, these all contributing to tumorigenesis (45). MET overexpression is present in many types of cancers, such as epithelial, mesenchymal and hematological malignancies. In addition, MET can be overexpressed in cancers with activated genomic signature, including those with primary and/or secondary MET ampliﬁcations or METex14 skipping mutation. Until now, MET TKIs showed little effect on patients with MET Frontiers in Oncology Author contributions CG wrote the section of introduction and MET mutation. HX and JZhang designed the review and revised the manuscript. KQ wrote the section of wild-type MET. JW wrote the section of Conclusion PSC is a subtype of NSCLC with unique malignant biological behavior. The symptoms are not speciﬁc, and PSC is often found in the advanced stage. It is not sensitive to traditional chemotherapy and radiotherapy, and the expected survival time is short, and the prognosis is poor. It is most likely that Frontiers in Oncology 05 frontiersin.org Gong et al. 10.3389/fonc.2022.1017026 MET ampliﬁcation. JZhao wrote the section of MET overexpression. YC wrote the section of conclusion. All authors contributed to the article and approved the submitted version. the treatment of PSC should continue to follow the treatment guidelines for advanced NSCLC. Molecular pathology tests, including IHC and NGS, are valuable in determining the prognosis of patients with PSC and helping to determine the treatment of NSCLC patients. MET alterations occur more frequently in PSC than in other types of NSCLC, and METex14 skipping mutation is the most common type. 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https://openalex.org/W2971065807	https://bmcneurol.biomedcentral.com/track/pdf/10.1186/s12883-019-1444-x	English	null	Anti-NMDAR encephalitis with simultaneous hypertrophic pachymeningitis in a 68-year-old male: a rare case report	BMC neurology	2,019	cc-by	3,066	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is one of the most frequent types of autoimmune encephalitis. However, the instigating mechanisms are as yet not fully ascertained. Cardinal clinical manifestations of anti-NMDAR encephalitis include acute behavioural change, psychosis, and catatonia. As the level of diagnosis increases, encephalitis becomes more common, but there are never been published in patients with anti-NMDAR encephalitis and simultaneous hypertrophic pachymeningitis. Case presentation: A sixty-eight-year-old man who presented with mental, behavioral abnormalities, unstable walking, headaches, and erratic hand movements. The neuropsychiatric symptoms and cerebrospinal fluid examination was consistent with the diagnosis criteria of anti-NMDAR encephalitis. Magnetic resonance imaging of the brain showed a thickening of dura mater localized at the left tentorium cerebelli, left cerebral hemisphere, and cerebral falx; the thickening dura mater was characterized by an intense contrast enhancement after the administration of gadolinium. High doses of intravenous methylprednisolone were administrated during his hospitalization. After 5 days, the patient’s condition improved. Conclusions: We herein describe a rare case of a 68-year-old man with anti-NMDAR encephalitis presenting with concomitant hypertrophic pachymeningitis. We systematically expounded anti-NMDAR encephalitis and hypertrophic pachymeningitis, and made bold conjectures on the etiology and pathogenesis of these two diseases, hoping to stimulate new ideas from clinicians and basic medical researchers. MDA receptor encephalitis, Autoimmune encephalitis, Hypertrophic pachymeningitis (HP), Dural AR Keywords: Anti-NMDA receptor encephalitis, Autoimmune encephalitis, Hypertrophic pachymeningitis (HP), Dural hypertrophy, NMDAR Anti-NMDAR encephalitis with simultaneous hypertrophic pachymeningitis in a 68-year-old male: a rare case report Huafang Jia1†, Xiaoli Xie2†, Faying Qi1, Long Wang2, Lijuan Wang2† and Fengyuan Che1,2† Background rapid surge in the number of cases reported. Hypertrophic pachymeningitis (HP) is a relatively rare condition that causes localized or diffused thickening of the dura mate [2]. The dominant clinical symptoms are chronic headaches with or without neurological manifestations [3]. Anti-N-methyl-D-aspartate receptor (NMDAR) encephal- itis is an autoimmune nervous system disease mediated by NMDAR antibodies. It is one of the most frequent types of autoimmune encephalitis. Patients often present with sub- acute psychiatric symptoms, memory loss, movement dis- orders, and seizures [1]. Since its recognition, clinicians have inreasingly identified the disease and there has been a Several cases of anti-NMDAR encephalitis and those of HP have previously been published. In fact, to our knowledge, simultaneous occurrence of both conditions has never been published before. We herein describe the case of a 68-year-old man with anti-NMDAR encephal- itis presenting with concomitant HP. We discuss the causes, presentation, and treatment in patients with anti- NMDAR encephalitis and HP. * Correspondence: wanglj730@163.com; che1971@126.com Huafang Jia and Xiaoli Xie devoted equally to this manuscript. Lijuan Wang and Fengyuan Che devoted equally to this manuscript. 2Central Laboratory, Linyi Key Laboratory of Neurophysiology, Linyi People’s Hospital, 27 East Section of Jiefang Road Lanshan District, Linyi 276000, Shandong, China 1 1Department of Neurology, The Eleventh Clinical Medical College of Qingdao University, Linyi People’s Hospital, 27 East Section of Jiefang Road Lanshan District, Linyi 276000, Shandong, China Jia et al. BMC Neurology (2019) 19:215 https://doi.org/10.1186/s12883-019-1444-x Jia et al. BMC Neurology (2019) 19:215 https://doi.org/10.1186/s12883-019-1444-x Open Access Case presentation serum IgG4 was 60.5 mg/dL. whereas serum values were negative for tumor markers (AFP, CEA, CA125, CA199, FPSA, NSE, CYFRA21-1, CA72–4), rheumatoid factor (RF), antineutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), MPO antibodies and PR3 antibodies. serum IgG4 was 60.5 mg/dL. whereas serum values were negative for tumor markers (AFP, CEA, CA125, CA199, FPSA, NSE, CYFRA21-1, CA72–4), rheumatoid factor (RF), antineutrophil cytoplasmic antibodies (ANCA), antinuclear antibodies (ANA), MPO antibodies and PR3 antibodies. p A 68-year-old man was admitted to the Department of Neurology, Linyi People’s Hospital in 15 May of 2018 with mental and behavioral abnormalities, unstable walking, head- aches, and erratic hand movements. Family members com- plained that over the past 2 months, the patient had been showing impulsiveness and irritability, wastefulness, particu- larly with food, and using foul language. These behaviours commonly lasted a half an hour each time, and the patient would often be conscious of his actions afterwards. He re- ported that he frequently suffered from mild headaches in the left frontal occipital region, numbness in the left facial face, and urinary incontinence at night. He had no known al- lergies. He was raised and lived in the area. He has been smoking for 30 years but but did not drink alcohol or use any illicit drugs. He had not traveled recently, and reported no exposures to patient with similar symptoms, farm or live- stock, or bitter insect bites. There was no family history of genetic diseases and autoimmune diseases. Lumbar puncture and subsequent cerebrospinal fluid (CSF) examination results showed a raised protein level (831 mg/L), white blood cell count was 8000 with mainly lympho- cytes, and no abnormal performance from infection. An im- munological examination of the CFS results also tested positive for anti-NMDAR antibodies, whereas IgG4 and anti- CASPR2, anti-AMPA1, anti-AMPA2, anti-LGI1, anti- GABAB antibodies were normal. There was no lesion sug- gestive of lung disease in Chest High Resolution CT. Mag- netic resonance imaging (MRI) of the brain showed a thickening of dura mater localized at the left tentorium cere- belli, left cerebral hemisphere, and cerebral falx; the thicken- ing dura mater was characterized by an intense contrast enhancement after the administration of gadolinium (Fig. 1a, b). Case presentation Magnetic resonance venography (MRV) showed no obvi- ous indication of the left internal jugular vein, and both of the left transverse sinus and the sigmoid sinus was slim with local stenosis; there was also increased cortical drainage in the left cerebral hemisphere (Fig. 1c). Video electroenceph- alogram (EEG) monitoring showed that each electrode de- tected a small amount of low-amplitude slow wave. On examination, his temperature was 37 °C, blood pressure was 90/53 mmHg, heart pulse was 99 beats per minute, respiratory rate was 21 breaths per minute. The patient was fully awake and communicated with the doc- tor in a normal way, but was slow to respond, had poor memory and computational power. He had a stiff face, nuchal rigidity, and mild ptosis on the left side. The bi- lateral pupils were normal. Sensory examination revealed hypoesthesia on the left side of the face. The strength of the limbs was normal. No pathological reflexes were de- tected. Cerebellar testing was not carried out because the patient was not cooperative. The patient underwent a composite evaluation and a diagnosis of anti-NMDAR encephalitis with dural hyper- trophy was proposed. Following recent research findings and expert advice, the patient received high doses of intra- venous methylprednisolone during his hospitalization. No adverse or unexpected events occurred during the treat- ment. After 5 days, the patient’s condition improved and he was asked to leave the hospital. The results of the MRI res- onance examination showed almost the same as before the treatment. The patient is very satisfied with the treatment and treatment results received. After hospital discharge, 30 Routine blood, No abnormalities in liver, kidney and thy- roid function tests. The levels of albumin, globulin, electro- lytes and glucose in the blood are also normal. Plasma C- reactive protein (CRP) was 98 mg/l, Erythrocyte sedimenta- tion rate (ESR) was 30 mm/h. Autoimmunity exams revealed that anti-NMDAR antibodies were positive and levels of Fig. 1 Magnetic resonance imaging of the brain: a thickening of dura mater localized at the left tentorium cerebelli, left cerebral hemisphere, and cerebral falx; the thickening dura mater was characterized by an intense contrast enhancement after the administration of gadolinium (a, b). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 4 Jia et al. BMC Neurology (2019) 19:215 Jia et al. BMC Neurology (2019) 19:215 Case presentation Magnetic resonance venography: no obvious indication of the left internal jugular vein, and both of the left transverse sinus and the sigmoid sinus was slim with local stenosis; there was also increased cortical drainage in the left cerebral hemisphere (c) Fig. 1 Magnetic resonance imaging of the brain: a thickening of dura mater localized at the left tentorium cerebelli, left cerebral hemisphere, and cerebral falx; the thickening dura mater was characterized by an intense contrast enhancement after the administration of gadolinium (a, b). Magnetic resonance venography: no obvious indication of the left internal jugular vein, and both of the left transverse sinus and the sigmoid sinus was slim with local stenosis; there was also increased cortical drainage in the left cerebral hemisphere (c) Page 3 of 4 Jia et al. BMC Neurology (2019) 19:215 Page 3 of 4 thrombosis due to HP, which shows no development of the left internal jugular vein and local stenosis of the left transverse sinus and sigmoid sinus and increased cortical drainage of the left cerebral hemisphere. While the over- lap may have been occasional, one could raise the con- sideration of a congenerous pathogenic mechanism, owing to an underlying disturbances of immune mecha- nisms. Our next step is to conduct an in-depth study of the relevance of these two diseases from multiple perspectives. mg/die of prednisone, per os was recommended. The pa- tient was referred to us again at 10 months after discharge. There had been no symptoms include mental and behav- ioral abnormalities. Nevertheless, slight chronic headache persisted. A follow-up MRI did not find Significant changes. Prednisone was followed by maintenance treatment. Fol- low-up information through telephone call showd partial regression of headaches in 1 August of 2019. Ethics approval and consent to participate This case report has been approved by the Linyi People’s Hospital and Qingdao University. Acknowledgements We are very grateful to our patients for allowing the publication of this case report. Consent for publication We have obtained written informed consent from the patient for the publication of this case report and image, The Editor of this journal can review the copy of written informed consent at any stage. Funding We receive no funding support. Availability of data and materials All data generated or analysed during this study are included in this published article. All data generated or analysed during this study are included in this published article. Abbreviations CSF b i CSF: cerebrospinal fluid; EEG: electroencephalogram; FLAIR: fluid-attenuated inversion recovery; HP: Hypertrophic pachymeningitis; MRA: Magnetic resonance angiography; MRI: Magnetic resonance imaging; MRV: Magnetic resonance venography; NMDAR: N-methyl-D-aspartate receptor Discussion and conclusions We report the case with anti-N-methyl-D-aspartate re- ceptor (NMDAR) encephalitis and simultaneous HP. Our patient who presented with episodic abnormal men- tal behavior with unstable walking, the CSF anti- NMDAR antibodies is positive. Magnetic resonance im- aging (MRI) of the brain showed a thickening of dura mater localized at the left tentorium cerebelli, left cere- bral hemisphere, and cerebral falx; the thickening dura mater was characterized by an intense contrast enhance- ment after the administration of gadolinium. Dural hypertrophy is a clinical manifestation of HP. In actual- ity, HP is a potential manifestation of numerous clinico- pathological entities including infectious diseases, auto- immune disorders, malignant neoplasms, and no obvious cause of idiopathic hypertrophic pachymeningitis [4]. The pathogenic mechanism of HP is obscure, but more and more evidence suggests that abnormal autoimmun- ity may play an important role [5]. Hypertrophic pachy- meningitis has been reported to be associated with a variety of autoimmune disease, including rheumatoid arthritis, sarcoidosis, IgG4-related disorder, Sjögren’s syndrome, Rosai-Dorfman disease, antiphospholipid syn- drome and anti-centromere antibody-positive status [3, 6–10]. In our patient, the serum and CSF IgG4 level were within the normal range. Serum values were nega- tive for RF, ANCA, ANA, MPO antibodies and PR3 anti- bodies. We are very regret that the patient did not have a histological proof of HP because of various restrictions and his personal wishes, so we can only diagnose HP from the imaging level. According to Dalmau J et al. MRI showed that 14 of the 100 patients had an en- hanced contrast of the overlaying meninges [11]. Irani et al. speculated that the dura mater lacking the blood- brain barrier may be one of the first sites of inflamma- tion in anti-NMDAR encephalitis through clinical obser- vation of disease progression [12]. Suzuki et al. reported a case of anti-NMDAR encephalitis preceded by dura mater lesions and predicted that the dura mater lesions on MRI may be available diagnosis and give insight into the the etiology and pathogenesis of anti-NMDAR en- cephalitis [13]. In our experience, it is unclear whether dural hypertrophy was a manifestation of anti-NMDAR encephalitis or merely coincidental. Unexpected discov- ery, the performance of MRV does not rule out venous Authors’ contributions Th d h The corresponding authors are LW and FC, who dominated this article. HJ and XX completed the collection of clinical data and the writing. FQ guided the completion of this paper. LW had provided lots of help in literature retrieval and article revision. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 17 May 2019 Accepted: 25 August 2019 Received: 17 May 2019 Accepted: 25 August 2019 References 1. Titulaer MJ, McCracken L, Gabilondo I, Armangue T, Glaser C, Iizuka T, et al. Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study. Lancet Neurol. 2013;12(2):157–65. https://doi.org/10.1016/S1474-4422(12)70310-1. 1. 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https://openalex.org/W2098001636	https://irishvetjournal.biomedcentral.com/counter/pdf/10.1186/2046-0481-59-5-278	English	null	Comparison of the arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal carbon dioxide tension during sevoflurane or isoflurane anaesthesia in rabbits	Irish veterinary journal	2,006	cc-by	3,616	Introduction Sevoflurane is a rapid-acting, inhaled anaesthetic used frequently in human beings and increasingly in animal patients in clinical practice. It has emerged as the most promising agent for inhalation induction in human patients (Smith et al., 1992; Ebert et al., 1998). Due to its low blood solubility (its blood/gas partition coefficient is 0.68), induction and emergence are rapid (Strum and Eger, 1987; Young and Apfelbaum, 1995; Ebert et al., 1998; Flecknell et al., 1999). Sevoflurane has a pleasant, non-pungent odour which permits a smooth and rapid induction by inhalation; it has made mask induction more attractive (Smith et al., 1992; Ebert et al., 1998). The cardiovascular effects of sevoflurane appear to be comparable to those of isoflurane. However, the effects of this agent on blood gas, arterial oxyhaemoglobin saturation and end-tidal CO2 tension in rabbits have not been well studied. Continuous monitoring of end-tidal CO2 (ETCO2) tension and arterial oxyhaemoglobin saturation by pulse oximetry (SpO2) has been considered the minimal standard of care during anaesthesia. The technology to objectively and non-invasively assess gas exchange in anaesthetised animals has recently become readily available. Because of the potential problems associated with hypoventilation, it is important to closely monitor the ventilation status of animals during inhalation The purposes of this study were to determine the effects of sevoflurane on arterial blood gas, arterial oxyhaemoglobin saturation, end-tidal CO2 tension and physiological parameters in rabbits and to compare these effects with those induced by isoflurane. Comparison of the arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal carbon dioxide tension during sevoflurane or isoflurane anaesthesia in rabbits Because of the potential problems associated with hypoventilation, it is important to closely monitor the ventilation status of animals during inhalation anaesthesia by assessing PaCO2 (Mutoh et al., 1995). Capnography and pulse oximetry allow for continuous and non-invasive monitoring of ventilation and oxygenation, respectively, in anaesthetised animals (Grosenbaugh and Muir, 1998). Arterial blood gas analysis is an invasive monitoring procedure. However, analysis of carbon dioxide and oxygen in an arterial blood sample defines sufficiency of gaseous exchange. The cardiovascular effects of sevoflurane appear to be comparable to those of isoflurane. However, the effects of this agent on blood gas, arterial oxyhaemoglobin saturation and end-tidal CO2 tension in rabbits have not been well studied. The purposes of this study were to determine the effects of sevoflurane on arterial blood gas, arterial oxyhaemoglobin saturation, end-tidal CO2 tension and physiological parameters in rabbits and to compare these effects with those induced by isoflurane. Materials and methods Twenty (10 male, 10 female) young New Zealand White rabbits, aged six to 12 months and weighing 4.2±0.6 kg (mean ± 1 SD) were obtained from a research centre for breeding test animals in Uludag University (Turkey) and were free from recognised respiratory pathogens (Pasteurella and Bordetella spp). The rabbits were floor- housed in groups, on dust-free shavings and autoclaved hay. They were fed a commercial pelleted diet (Ankara Feedstuff Industry, Turkey) and water ad libitum. No animal was used more than once. All experiments were conducted in accordance with the Animal Research Ethics Committee at the Uludag University of Turkey. Author for Correspondence: Ayse Topal Uludag Üniversitesi Veteriner Fakültesi Klinikleri Mudanya Cad. No: 2 16190 Bursa Turkey Tel: + 90 224 2347655 Fax: + 90 224 2346395 Email: atopal@uludag.edu.tr Irish Veterinary Journal Volume 59 (5) 278-281 2006 Irish Veterinary Journal Volume 59 (5) 278-281 2006 Keywords: Rabbit, Anaesthesia, Inhalation, Sevoflurane, Isoflurane anaesthesia by assessing PaCO2 (Mutoh et al., 1995). Capnography and pulse oximetry allow for continuous and non-invasive monitoring of ventilation and oxygenation, respectively, in anaesthetised animals (Grosenbaugh and Muir, 1998). Arterial blood gas analysis is an invasive monitoring procedure. However, analysis of carbon dioxide and oxygen in an arterial blood sample defines sufficiency of gaseous exchange. Volume 59 (5) : May, 2006 Peer reviewed Irish Veterinary Journal Volume 59 (5) : May, 2006 Peer reviewed Irish Veterinary Journal Volume 59 (5) : May, 2006 Irish Veterinary Journal Volume 59 (5) : May, 2006 Irish Veterinary Journal Comparison of the arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal carbon dioxide tension during sevoflurane or isoflurane anaesthesia in rabbits Ayse Topal and Nihal Gül Department of Surgery, Faculty of Veterinary Medicine, Uludag University, 16190 Bursa, Turkey Department of Surgery, Faculty of Veterinary Medicine, Uludag University, 16190 Bursa, Turkey The effects of sevoflurane or isoflurane on arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal CO2 tension were monitored during induction and maintenance of anaesthesia in 10 premedicated New Zealand White (NZW) rabbits. The effects of sevoflurane or isoflurane on arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal CO2 tension were monitored during induction and maintenance of anaesthesia in 10 premedicated New Zealand White (NZW) rabbits. For induction, the anaesthetic agents were delivered via a face-mask. After induction was completed, an endotracheal tube was introduced for maintenance of anaesthesia for a period of 90 minutes. Changes in heart rate, respiratory rate, arterial blood gas, arterial oxyhaemoglobin saturation, blood pH and end-tidal CO2 tension were recorded. Although sevoflurane and isoflurane produce similar cardiopulmonary effects in premedicated rabbits, sevoflurane provides a smoother and faster induction because of its lower blood/gas partition coefficient. Thus sevoflurane is probably a more suitable agent than isoflurane for mask induction and maintenance. Its lower blood solubility also makes sevoflurane more satisfactory than isoflurane for maintenance of anaesthesia because it allows the anaesthetist to change the depth of anaesthesia more rapidly. 278 Irish Veterinary Journal Volume 59 (5) 278-281 2006 Keywords: Rabbit, Anaesthesia, Inhalation, Sevoflurane, Isoflurane Introduction Sevoflurane is a rapid-acting, inhaled anaesthetic used frequently in human beings and increasingly in animal patients in clinical practice. It has emerged as the most promising agent for inhalation induction in human patients (Smith et al., 1992; Ebert et al., 1998). Due to its low blood solubility (its blood/gas partition coefficient is 0.68), induction and emergence are rapid (Strum and Eger, 1987; Young and Apfelbaum, 1995; Ebert et al., 1998; Flecknell et al., 1999). Sevoflurane has a pleasant, non-pungent odour which permits a smooth and rapid induction by inhalation; it has made mask induction more attractive (Smith et al., 1992; Ebert et al., 1998). Continuous monitoring of end-tidal CO2 (ETCO2) tension and arterial oxyhaemoglobin saturation by pulse oximetry (SpO2) has been considered the minimal standard of care during anaesthesia. The technology to objectively and non-invasively assess gas exchange in anaesthetised animals has recently become readily available. Apparatus pp A small plastic face-mask (Kruuse [Veterinary Equipment] Ltd, Marslev, Denmark) and a Jackson-Rees modification of the Ayre’s T-piece were used for induction. Isoflurane was delivered from an Isoflurane A-3 vaporiser (Kimura Medical Co., Tokyo, Japan) and sevoflurane from a temperature and flow compensated vaporiser (Blease, Genuine Part, England). Both agents were delivered in 100% oxygen at 4L/ minute. Electrodes were placed on the rabbit’s skin, on the medial aspects of the upper forelegs and of the upper left hindleg, and the electrocardiogram was recorded by connecting the electrodes to an electronic monitoring apparatus (Burdick Medic-5, Burdick Inc., USA). Pulse oximetry (SpO2) readings and pulse rate were obtained with a pulse oximeter (Vet/OXTM 4403, SDI, USA) with the probe placed on the tongue. End-tidal CO2 (ETCO2) tension and respiratory rate were monitored continuously with a sidestream-sampling capnograph (Normocap 200, Datex-Ohmeda). During the induction period, the sampling adapter (AMS CO2 absorber for veterinary purpose, AMS Company, Ankara, Turkey) was connected between the face-mask and the Jackson-Rees modification of the Ayre’s T-piece. During the maintenance period, the sampling adapter was connected between the endotracheal tube and the Jackson-Rees modification of the Ayre’s T-piece in a similar manner. A catheter (22g, IV cannula, Transplastics, Germany) was inserted into the ear artery for blood gas analysis. Arterial blood samples were collected in heparinised capillary tubes, and blood pH, PaO2 and PaCO2 were measured by blood gas analyser (RapidLab 248, Chiron Diagnostic Ltd, Halstead, Essex, UK). Lactated Ringer’s solution (10 ml/kg per hour) was administered into the other ear vein. All data were expressed as mean ± 1 SD. The cardiopulmonary variables and values for time to event were compared between groups using one-way analysis of variance (ANOVA) for repeated measures. If statistically significant effects were found, Tukey’s post hoc test was applied to the differences between groups. The changes within each anaesthetic group were compared using one-way ANOVA for repeated measures, followed by Tukey’s post hoc test when appropriate. Values of P <0.05 were considered statistically significant. Experimental protocol The animals were prepared as described above. After a five to 10 minute period, baseline measurements were recorded. Xylazine (3mg/kg: Rompun®, Bayer, Turkey) was injected intramuscularly, for premedication. The animals were restrained manually by gently positioning the operator’s arms on either side of the animal’s body. After placement of the face-mask, 100% oxygen was provided for two to three minutes at a fresh gas flow rate of 4L/minute. Any struggling was noted by an observer, and this was used to assess the quality of induction. After this initial period, sevoflurane (Sevorane®, Abbott, UK) at a vaporiser setting of 6%, or isoflurane (Forane®, Abbott, UK) at a vaporiser setting of 4%, was delivered in the oxygen stream in a similar manner. The anaesthetic gas concentration was maintained at this level until induction was completed. When induction was complete, an endotracheal tube was inserted using a premature baby laryngoscope and the non-rebreathing system was attached to it. The anaesthetic vaporiser was then turned to a setting of 3% (sevoflurane) or 2% (isoflurane) for maintenance of anaesthesia for a 90 minute period. A decrease in respiratory rate compared with the pre-induction rate was observed during induction and maintenance with both agents. An associated respiratory acidosis, characterised by an increase in PaCO2, was observed in both groups. The respiratory depressant effects, characterised by increased PaCO2 and decreased blood pH values, were associated with decreases in respiratory rate. Arterial PaCO2 rose from 30.5mmHg (±7.0) to a peak of 50.6mmHg (±10.2) in the sevoflurane group and from 33.6mmHg (±5.4) to a peak of 51.5mmHg (±11.7) in the isoflurane group. Blood pH values were normal in all animals prior to induction and fell to 7.25 ± 0.11 during sevoflurane anaesthesia and to 7.16 ± 0.09 during isoflurane anaesthesia. These changes were significant (P<0.05). Time to recovery of righting reflex also differed significantly between treatment groups: 219 seconds (±103) in the sevoflurane group versus 615 seconds (±326) in the isoflurane group. The recovery was smooth and uneventful in all rabbits. Cardiopulmonary responses (heart rate, respiratory rate, rectal temperature, arterial oxyhaemoglobin saturation and end-tidal CO2 tension) and blood gas values were recorded every 15 minutes from the time of induction to the beginning of recovery. After anaesthesia, all rabbits were extubated and observed in the post-anaesthesia care unit for a further hour. Results Cardiopulmonary data recorded during mask induction and maintenance of anaesthesia with sevoflurane and isoflurane are summarised in Table 1. Pre-induction values for each variable in conscious and premedicated animals were not significantly different between the sevoflurane group and the isoflurane group. Intramuscular administration of xylazine induced moderate sedation in both groups. Placement of the face-mask and administration of oxygen did not cause any change in respiratory rate. Exposure to sevoflurane caused very little aversion and relatively few bouts of struggling behaviour. In contrast, isoflurane induced excessive struggling in all animals, necessitating very firm restraint, in spite of sedation. Times to loss of the palpebral reflex and to successful tracheal intubation were significantly shorter with sevoflurane (126 seconds ±36) than with isoflurane (150 seconds ±31). None of the rabbits given sevoflurane had episodes of induction–related complications (struggling, ataxia or excitement). Cardiopulmonary effects of sevoflurane and isoflurane, during the induction and maintenance periods, were similar with the exception of a slightly decreased heart rate in the sevoflurane group. The heart rate values during anaesthesia with isoflurane were slightly higher than pre-induction rates, although they were still within the physiological reference range. Materials and methods Twenty (10 male, 10 female) young New Zealand White rabbits, aged six to 12 months and weighing 4.2±0.6 kg (mean ± 1 SD) were obtained from a research centre for breeding test animals in Uludag University (Turkey) and were free from recognised respiratory pathogens (Pasteurella and Bordetella spp). The rabbits were floor- housed in groups, on dust-free shavings and autoclaved hay. They were fed a commercial pelleted diet (Ankara Feedstuff Industry, Turkey) and water ad libitum. No animal was used more than once. All experiments were conducted in accordance with the Animal Research Ethics Committee at the Uludag University of Turkey Author for Correspondence: Ayse Topal Uludag Üniversitesi Veteriner Fakültesi Klinikleri Mudanya Cad. No: 2 16190 Bursa Turkey Tel: + 90 224 2347655 Fax: + 90 224 2346395 Email: atopal@uludag.edu.tr 278 Peer reviewed Peer reviewed Volume 59 (5) : May, 2006 Irish Veterinary Journal Volume 59 (5) : May, 2006 Irish Veterinary Journal Volume 59 (5) : May, 2006 Irish Veterinary Journal Discussion This study was designed to monitor arterial blood gas, arterial oxyhaemoglobin saturation and end-tidal CO2 tension in rabbits during mask induction and maintenance of anaesthesia with sevoflurane and isoflurane. The adverse effects of halogenated volatile anaesthetics associated with mask induction, as experienced clinically 279 Volume 59 (5) : May, 2006 PEER REVIEWED Irish Veterinary Journal PEER REVIEWED Volume 59 (5) : May, 2006 Irish Veterinary Journal Volume 59 (5) : May, 2006 Irish Veterinary Journal Table 1. Table 1. Cardiorespiratory parameters (heart rate, respiratory rate, arterial oxyhaemoglobin saturation and end-tidal CO2), blood gas values and rectal temperatures of rabbits anaesthetised with sevoflurane (SEVO) and isoflurane (ISO). Cardiorespiratory parameters (heart rate, respiratory rate, arterial oxyhaemoglobin saturation and end-tidal CO2), blood gas values and rectal temperatures of rabbits anaesthetised with sevoflurane (SEVO) and isoflurane (ISO). B I 15 30 45 60 75 90 HR ISO 218±70 188±43ab 200±31ab 209±25b 201±14ab 196±23ab 209±31b 198±39ab (beats/min) SEVO 234±58 54±27a 233±35 244±39 240±42 255±41a 260±42a 266±29a RR SEVO 100±30 38±7ab 37±8a 36±5a 34±5a 36±11a 35±9a 34±9a (beats/min) ISO 78±24 43±13a 36±12a 34±10a 37±10a 34±10a 32±7a 35±5a SpO2 SEVO 91±5 91±4 91±4 90±4 94±2 94±2 92±4 89±6 (%) ISO 94±3 94±4 90±4 91±5 93±4 93±3 94±4 94±3 ETCO2 SEVO ND 32±9 36±6 37±6 35±7 36±4 36±6 37±5 (mmHg) ISO ND 28±10 29±8 30±6 30±5 33±7 34±7 32±8 pH SEVO 7.40±0.04 7.25±0.11a 7.32±0.06 7.39±0.08 7.41±0.04 7.38±0.05 7.41±0.05 7.39±0.05 ISO 7.38±0.05 7.16±0.09a 7.34±0.08 7.40±0.06 7.41±0.04 7.41±0.05 7.41±0.05 7.42±0.05 PaCO2 SEVO 30.5±7.0 48.1±11.5a 46.1±6.9a 45.5±9.7a 43.3±9.0a 48.3±8.6a 48.7±8.8a 50.6±10.2a (mmHg) ISO 33.6±5.4 56.0±9.6a 52.1±9.7a 47.1±8.6a 8.9±9.2a 51.3±10.0a 52.3±7.2a 51.5±11.7a PaO2 SEVO 67.70±21.37 248.16±177.47a 227.53±181.16a 397.88±166.10a 306.52±172.38a 308.60±107.28a 361.28±137.38a 344.25±126.63a (mmHg) ISO 72.89±13.37 240,58±191.60a 302.71±173.45a 300.78±137.90a 295.11±139.80a 317.45±131.76a 370.55±164.92a 373.39±114.11a RT SEVO 39.02±0.35 38.70±0.46 38.42±0.76 38.17±0.94 37.80±1.11 37.50±1.23 37.28±1.35 37.05±1.45 (OC) ISO 38.52±0.68 39.20±0.57 39.09±0.59 38.74±0.70 38.27±0.88 38.00±0.92 37.67±0.95 37.45±1.13 Data are expressed as mean ±1SD. B: basal values; I: induction values; 15, 30, 45, 60, 75, 90: stage of anaesthesia (expressed in minutes); HR: heart rate; RR: respiration rate; SpO2 (%): arterial oxyhaemoglobin saturation; ETCO2 (mmHg): end-tidal CO2; RT (OC): rectal temperature; ND: not done. Arterial blood gas values: PaCO2 (mmHg), PaO2(mmHg) and pH. aSignificantly different from baseline value (P <0.05); bSignificantly different from ISO group (P <0.05). Discussion g p ( ) ( ) B: basal values; I: induction values; 15, 30, 45, 60, 75, 90: stage of anaesthesia (expressed in minutes); HR: heart rate; RR: respiration rate; SpO2 (%): arterial oxyhaemoglobin saturation; ETCO2 (mmHg): end-tidal CO2; RT (OC): rectal temperature; ND: not done. Arterial blood gas values: PaCO2 (mmHg), PaO2(mmHg) and pH. aSignificantly different from baseline value (P <0.05); bSignificantly different from ISO group (P <0.05). methodology. Because of the occurrence of periods of apnoea in the study by Flecknell et al. (1999), inhalation of a gradually rising concentration of anaesthetic was not used here. It was thought that a technique that gives a rapid increase in the concentration of the inhaled agent during mask induction may be more appropriate than a technique that delivers a slowly increasing concentration of the agent. By rapid mask induction, it was possible to avoid long induction periods and violent struggling efforts. Surgical anaesthesia was reached faster with sevoflurane than with isoflurane because of the lower blood/gas partition coefficient. in animals, are characterised by apnoea, breath-holding, laryngospasm, bronchoconstriction or body movements (Mitsuhata et al., 1994; Mutoh et al., 2001a,b). Some of these induction-related complications (e.g., struggling) also accompany mask induction in rabbits (Flecknell et al., 1995,1999) and dogs (Mutoh et al., 1995). In this experiment, induction-related complications induced by isoflurane necessitated very firm restraint in spite of sedation whereas these complications were not evident when sevoflurane was used. This was unlike the study reported by Flecknell et al. (1999). However, none of their rabbits were premedicated. The protocol followed for face-mask induction was identical to that used in the study by Flecknell et al. (1999), which had been derived from standard veterinary anaesthetic induction and maintenance In this study, xylazine was selected for premedication because it produced light to moderate sedation with small changes in cardiopulmonary functions in rabbits. It was clearly demonstrated that 280 Peer reviewed Peer reviewed Volume 59 (5) : May, 2006 Irish Veterinary Journal \References Ebert, T.J., Robinson, B.J., Uhrich, T.D., Mackenthun, A. and Pichotta, P.J. (1998). Recovery from sevoflurane anaesthesia: A comparison of isoflurane and propofol anesthesia. Anaesthesiology 89: 1524-1531. Flecknell, P.A., Cruz, I.J., Liles, J.H. and Whelan, G. (1995). Induction of anaesthesia with halothane and isoflurane in the rabbit: a comparison of the use of a face-mask or an anaesthetic chamber. Laboratory Animals 30: 67-74. Flecknell, P.A. and Liles, J.H. (1996). Halothane anaesthesia in the rabbit: a comparison of the effects of medetomidine, acepromazine and midazolam on breath-holding during induction. Journal of the Association of Veterinary Anaesthetists 23: 11-14. g y pp y Both inhalants had similar cardiopulmonary effects during the induction and the maintenance periods, with the exception of a decreased heart rate in the sevoflurane group. The heart rate values during anaesthesia with isoflurane were higher than preinduction rates, although they were still within the physiological reference range for maintenance under isoflurane anesthesia. The difference in heart rates between two groups may have been related to more stress in rabbits anaesthetised with isoflurane because it is more irritating than sevoflurane (Mutoh et al., 1995; Flecknell et al., 1999; Hedenqvist et al., 2001). Okamoto et al. (1996) reported that inhalation of 1% to 4% isoflurane in anaesthetised rabbits has been shown to increase heart rate as a result of direct sympathetic stimulation, in addition to stimulation of the baroreflex elicited by a decrease in arterial blood pressure. Flecknell, P.A., Roughan, J.V. and Hedenqvist, P. (1999). Induction of anaesthesia with sevoflurane and isoflurane in the rabbit. Laboratory Animals 33: 41-46. Grosenbaugh, D.A. and Muir, W.W. (1998). Accuracy of non- invasive oxyhaemoglobin saturation, end-tidal carbon dioxide concentration, and blood pressure monitoring during experimentally induced hypoxemia, hypotension, or hypertension in anesthetised dogs. American Journal of Veterinary Research 59: 205-212. Hedenqvist, P., Roughan, J.V., Antunes, L., Orr, H. and Flecknell, P.A. (2001). Induction of anaesthesia with desflurane and isoflurane in the rabbit. Laboratory Animals 35: 172-179. Mitsuhata, H., Saitoh, J., Shimizu, R., Takeuchi, H., Hasome, N. and Horiguchi,Y. (1994). Sevoflurane and isoflurane protect against bronchospasm in dogs. Anaesthesiology 81: 1230-1234. All volatile anaesthetic agents have been reported to cause a dose- related respiratory depression in rabbits (Flecknell et al., 1995, 1996, 1999; Okamoto et al., 1996; Hedenqvist et al., 2001). In this study, decreased respiratory rates, which were reflected in higher end-tidal CO2 tensions, were recorded in both anaesthetic groups. \References The mild respiratory depression was qualitatively similar to those of previous studies in anaesthetised rabbits (Flecknell et al., 1999; Hedenqvist et al., 2001). It is possible that slowing of breathing during induction and maintenance of anaesthesia is caused by both respiratory reflexes and anaesthetic effects of the inhalants. Mutoh, T., Nıshimura, R., Kim, H.Y., Matsunaga, S., Kadosawa, T., Mochizuki, M. and Sasaki, N. (1995). Rapid inhalation induction of anaesthesia by halothane, enflurane, isoflurane and sevoflurane and their cardiopulmonary effects in dogs. Journal of Veterinary Medicinal Science 57: 1007-1013. Mutoh, T., Kojima, K., Takao, K., Nıshimura, R. and Sasaki, N. (2001a). Comparison of sevoflurane with isoflurane for rapid mask induction in midazolam and butorphanol sedated dogs. Journal of Veterinary Medicine Series- A 48: 223-230. Mutoh, T., Kanamaru, A., Tsubone, H., Nıshimura, R. and Sasaki, N. (2001b). Respiratory reflexes in response to upper airway administration of sevoflurane and isoflurane in anaesthetised spontaneously breathing dogs. Veterinary Surgery 30: 87-96. As in previous studies (Flecknell et al., 1999; Hedenqvist et al., 2001), respiratory depression due to volatile anaesthetic agents produced elevations of arterial PaCO2 and end-tidal CO2 tension. However, reduction in arterial PaO2 and hypoxaemia did not occur. Arterial oxyhaemoglobin saturation was constant during the anaesthetic period with both agents predictable because of the high inspired oxygen fraction. Okamoto, H., Hoka, S., Kawasaki, T., Okuyama, T. and Takashashi, S. (1996). Dose-dependent increases in the renal sympathetic nerve activity during rapid increase in isoflurane concentration in intact, lower airway-deafferented, and baroreceptor- deafferented rabbits. Anaesthesiology 84: 1196-1204. In conclusion, both sevoflurane and isoflurane produced similar cardiopulmonary effects in premedicated rabbits. However, sevoflurane was considered a more suitable agent for mask induction since it provided a smoother and more rapid induction, due to its lower blood/gas partition coefficient. Its lower blood solubility also makes sevoflurane more desirable for maintenance of anaesthesia than isoflurane because of the facility to change the depth of anaesthesia more rapidly and to hasten recovery. Smith, I., Ding, Y. and White, P.F. (1992). Comparison of induction, maintenance, and recovery characteristics of sevoflurane-N2O and propofol-sevoflurane-N2O with propofol-isoflurane-N2O anaesthesia. Anaesthesia and Analgesia 74: 253-259. Strum, D.P. and Eger, E.I. (1987). Partition coefficients for sevoflurane in human blood, saline, and olive oil. Anaesthesia and Analgesia 66: 654-656. Young, C.J. and Apfelbaum, J.L. (1995). Inhalation anaesthetics: Desflurane and sevoflurane. Journal of Clinical Anaesthesiology 7: 564- 577. 281
https://openalex.org/W4361011406	https://hess.copernicus.org/preprints/hess-2023-12/hess-2023-12.pdf	English	null	Reply on RC2	null	2,023	cc-by	14,738	ERROR: type should be string, got "https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 \n1College of Water Resources and Architectural Engineering, Northwest A&F University, 712100 Yangling, Shannxi Province, \n5 \nChina \n2Institute of Soil and Water Conservation, Chinese Academy of Science & Ministry of Water Resources, 712100, Yangling, \nShaanxi Province, China \n3Institute of Soil and Water Conservation, Northwest A&F University, 712100, Yangling, Shaanxi Province, China \n4National Engineering Research Center of Water Saving and Irrigation Technology at Yangling, 712100, Shaanxi Province, \n10 \nChina \n5Institute of Green Development Yellow River Drainage Basin, Lanzhou University, 730000, Lanzhou, Gansu Province, \nChina \nCorrespondence to: Xiaodong Gao (gao_xiaodong@nwafu.edu.cn) 1 \nIntroduction The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 na He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nao2,3,4 1College of Water Resources and Architectural Engineering, Northwest A&F University, 712100 Yangling, Shannxi Province, \n5 \nChina \n2Institute of Soil and Water Conservation, Chinese Academy of Science & Ministry of Water Resources, 712100, Yangling, \nShaanxi Province, China \n3Institute of Soil and Water Conservation, Northwest A&F University, 712100, Yangling, Shaanxi Province, China \n4National Engineering Research Center of Water Saving and Irrigation Technology at Yangling, 712100, Shaanxi Province, \n10 \nChina \n5Institute of Green Development Yellow River Drainage Basin, Lanzhou University, 730000, Lanzhou, Gansu Province, \nChina \nCorrespondence to: Xiaodong Gao (gao_xiaodong@nwafu.edu.cn) Abstract. In water-limited areas, planted trees can abstract substantial amounts of soil water from deep layers (>200 cm) to \n15 \nmeet their high water demand, resulting in deep soil desiccation, which influences not only regional water cycling but also \nthe sustainability of trees per se in drylands. However, the limitation in relation to both degree and depth of deep-layer soil \nmoisture (DSM) beyond which root water uptake ceases remains unclear. Whether limitation depends on tree species and \nhow it will affect tree’s xylem hydraulic conductivity are also unclear, restricting our ability to predict the fate of dryland tree Abstract. In water-limited areas, planted trees can abstract substantial amounts of soil water from deep layers (>200 cm) to \n15 \nmeet their high water demand, resulting in deep soil desiccation, which influences not only regional water cycling but also \nthe sustainability of trees per se in drylands. However, the limitation in relation to both degree and depth of deep-layer soil \nmoisture (DSM) beyond which root water uptake ceases remains unclear. Whether limitation depends on tree species and \nhow it will affect tree’s xylem hydraulic conductivity are also unclear, restricting our ability to predict the fate of dryland tree \nplantations. We, therefore, studied the spatiotemporal distribution of deep soil moisture deficit (DSMD, the relative \n20 \ndifference in soil moisture for given trees and referenced cropland or grassland, to minimize the effect of climate differences \nin various sampling years) for two typical planted trees, apple (Malus pumila Mill.) and black locust (Robinia pseudoacacia \nL.), based on the published data and multiple field samplings on China’s Loess Plateau. The results indicated that the lowest \nDSM occurred under the planted trees aged 24-28 years at all sites. The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 The lowest DSMD fluctuated around -0.6, which close to \nthe DSMD at the permanent wilting point (PWP) irrespective of tree species and site, although shallow (<200 cm) soil \n25 \nmoisture was not reduced to the point of limitation. This suggests that PWP is a good indicator of the degree limitation of \nDSM for the tree species examined. The corresponding maximum depth of soil moisture use reached 18.0-22.0 m for these \nold planted trees at different sites while it was more than 25 m for black locust in the drier site of Mizhi. Furthermore, when \nthe degree and depth limitations were reached, the percent loss of xylem hydraulic conductivity of planted tree’s branches \nreached 74.9-96.5%, indicating that tree mortality may occur. The findings will help to predict the sustainability of planted \n30 \ntrees in semiarid regions with thick vadose zone. 1 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Worldwide, tree planting is extensively advocated as a nature-based solution (NbS) to facilitate the realization of Sustainable \nDevelopment Goals (Griscom et al., 2020). China’s Loess Plateau (LOP), the greatest loess deposits in the world with a \nthickness of 50-200 m loess (Shao et al., 2018), has been one of the most active regions for tree planting during the last four \n35 \ndecades, with the aim of controlling severe soil erosion and improving rural lives there (Fu et al., 2016). Many trees of a \nvariety of species (mostly exotic) have been planted during implementation of two large ecological projects: the Three-North \nShelter Forest Program and the Grain-For-Green Program (Gao et al., 2021). So far, the area of tree plantations on the LOP \nhas reached 7.47 million ha; the vegetation cover for this region increased from 31.6% in 1999 to 65.0% in 2017 (official \ndata from the National Forestry and Grassland Administration, http://www.forestry.gov.cn). As a result, a number of \n40 \necosystem services, including soil and water conservation, have been greatly improved on the LOP (Fu et al., 2016; Gao et \nal., 2021). In fact, the success of ecological restoration through tree planting on the LOP greatly depends on the presence of \nan abundant “soil reservoir” deposited in deep loess deposits (200-2000 cm) (Zhu, 2006). The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 They found that the canopy transpiration and the net photosynthetic \n70 \nrate of apple trees was reduced, respectively, by 36% and 20% on average compared with the trees with deep roots in a \nsemiarid site on the LOP. Moreover, embolism resistance increases with a decrease in soil water availability, leading to a \nreduction in xylem hydraulic conductivity and even hydraulic failure of the plantation (Liu et al., 2020; Fuchs et al., 2021). However, it remains unclear on the effect of deep-layer soil desiccation on xylem hydraulic conductivity of planted trees \nwhen degree limitation and depth limitation of DSM are reached. 75 \nTo address the gaps, we built a dataset, including 11980 observations of 380 soil profiles from 34 peer-reviewed publications \nand 4200 observations of 72 soil profiles in-situ sampling across the LOP. The main objectives of this study were (i) to \nidentify the degree and depth limitation of DSM for different tree species and the influencing factors, and (ii) to disentangle \ntheir impact on xylem hydraulic conductivity of planted trees in different climatic zones on the LOP. Our hypotheses were \nthat the degree and depth limitation of deep-layer soil desiccation depends on tree species because of their different \n80 \ndrought-resistance capacity; and that the xylem hydraulic conductivity of planted trees is greatly reduced when degree and \ndepth limitation are reached due to low soil water availability in deep layers. The occurrence of soil desiccation in deep layers can greatly affect the growth and eco-physiological traits of the \naboveground parts of planted trees. It has been widely observed that the aboveground growth of planted trees is greatly \n65 \nconstrained due to deep soil drying, resulting in so-called “dwarf aged trees” on the LOP (Fang et al., 2016). Recently, Li et \nal. (2021) found that when deep soil below 200 cm is dry, the canopy transpiration of apple trees with a rooting depth beyond \n2000 cm is reduced by around 40% relative to that without deep soil desiccation on the LOP. Furthermore, Yang et al. (2022) \nconducted a deep-soil-and-root-partitioning experiment to quantify the effect of deep-layer (>200 cm) root uptake on the \ntranspiration and physiological features of apple tree. They found that the canopy transpiration and the net photosynthetic \n70 \nrate of apple trees was reduced, respectively, by 36% and 20% on average compared with the trees with deep roots in a \nsemiarid site on the LOP. The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 Moreover, embolism resistance increases with a decrease in soil water availability, leading to a \nreduction in xylem hydraulic conductivity and even hydraulic failure of the plantation (Liu et al., 2020; Fuchs et al., 2021). However, it remains unclear on the effect of deep-layer soil desiccation on xylem hydraulic conductivity of planted trees \nwhen degree limitation and depth limitation of DSM are reached. 75 To address the gaps, we built a dataset, including 11980 observations of 380 soil profiles from 34 peer-reviewed publications \nand 4200 observations of 72 soil profiles in-situ sampling across the LOP. The main objectives of this study were (i) to \nidentify the degree and depth limitation of DSM for different tree species and the influencing factors, and (ii) to disentangle \ntheir impact on xylem hydraulic conductivity of planted trees in different climatic zones on the LOP. Our hypotheses were \nthat the degree and depth limitation of deep-layer soil desiccation depends on tree species because of their different \n80 \ndrought-resistance capacity; and that the xylem hydraulic conductivity of planted trees is greatly reduced when degree and \ndepth limitation are reached due to low soil water availability in deep layers. To address the gaps, we built a dataset, including 11980 observations of 380 soil profiles from 34 peer-reviewed publications \nand 4200 observations of 72 soil profiles in-situ sampling across the LOP. The main objectives of this study were (i) to \nidentify the degree and depth limitation of DSM for different tree species and the influencing factors, and (ii) to disentangle \ntheir impact on xylem hydraulic conductivity of planted trees in different climatic zones on the LOP. Our hypotheses were \nthat the degree and depth limitation of deep-layer soil desiccation depends on tree species because of their different \n80 \ndrought-resistance capacity; and that the xylem hydraulic conductivity of planted trees is greatly reduced when degree and \ndepth limitation are reached due to low soil water availability in deep layers. The degree and depth limitation of deep soil desiccation and its \nimpact on xylem hydraulic conductivity in dryland tree plantations \nNana He1, Xiaodong Gao2,3,4, Dagang Guo1, Yabiao Wu1, Dong Ge1, Lianhao Zhao2, Lei Tian5, Xining \nZhao2,3,4 However, inappropriate tree planting can incur other ecological issues, such as soil desiccation due to excessive water \nconsumption in drylands (Wang et al., 2010; Jia et al., 2017; Wu et al., 2021), preventing the sustainable development of \n45 \nrevegetation and may even result in tree mortality (Breshears et al., 2013; Choat et al., 2018; Stovall et al., 2019). Considerable researches have shown that planted trees abstract substantial deep-layer soil moisture (DSM) by developing \ndeep roots to maintain its normal growth on the LOP (e.g., Gao et al., 2018; Su and Shangguan, 2019; Shi et al., 2021). However, continuous use of DSM over a number of years leads to severe soil desiccation (Wang et al., 2011; Gao et al., 2018; Tao et al., 2021), because DSM below 200 cm is hardly recharged by precipitation under tree plantations. In fact, deep-layer \n50 \nsoil desiccation under tree plantations can occur at multiple degrees depending on tree age and local climate (Fang et al., \n2016; Jia et al., 2017). Although soil desiccation of different degrees has been widely reported in the above citations, \nknowledge remains restricted about variation in the lowest amount of soil moisture that limits growth (hereafter “degree \nlimitation”) and/or the maximum depth from which trees are able to extract water (hereafter “depth limitation”) for different 2 2 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The occurrence of soil desiccation in deep layers can greatly affect the growth and eco-physiological traits of the \naboveground parts of planted trees. It has been widely observed that the aboveground growth of planted trees is greatly \n65 \nconstrained due to deep soil drying, resulting in so-called “dwarf aged trees” on the LOP (Fang et al., 2016). Recently, Li et \nal. (2021) found that when deep soil below 200 cm is dry, the canopy transpiration of apple trees with a rooting depth beyond \n2000 cm is reduced by around 40% relative to that without deep soil desiccation on the LOP. Furthermore, Yang et al. (2022) \nconducted a deep-soil-and-root-partitioning experiment to quantify the effect of deep-layer (>200 cm) root uptake on the \ntranspiration and physiological features of apple tree. 2.1.1 Data sources \n85 When analyzing the degree limitation of DSM under apple tree i\n110 \nLuochuan, the extracted data were divided into tree age range for analysis to avoid random errors. Simultan\ncollected basic data from each publication, including data source, geographic coordinates (longitude, latitude\nprecipitation (MAP), tree species, and soil texture. Since there was relatively little published information on\nlocust forests, we also performed supplementary experiments to obtain relevant data, as described in Section 2\nhttps://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. (2) Soil moisture was the gravimetric or volumetric moisture content measured by drying methods or moisture sensors. The volumetric moisture content was uniformly converted to gravimetric moisture content by means of the \naverage soil bulk density in each study area. (3) Experiments with only shrub and grass communities or experiments with irrigation, mulching, pruning and/or \nother measures were excluded. (4) Experiments yielded paired data for DSM in apple orchards or black locust forests and that in farmland or \ngrassland (control). (5) L\ni\nl\ni i\ni\n(\n) f\nd\ni\nl\nl\nd (4) Experiments yielded paired data for DSM in apple orchards or black locust forests and that in farmland or \ngrassland (control). (5) Location, mean annual precipitation (mm), forest age, and vegetation types were clearly presented. 2.1.1 Data sources \n85 Peer-reviewed research papers, published between 1999 and 2021 were extracted from the Web of Science (United States) \nand CNKI (China Knowledge Resource Integrated Database, China; 2000-2021) using the search terms (“Loess plateau” OR \n“Loess hilly region”) AND (“artificial forest” OR “afforestation” OR “plantation”) AND (“soil moisture” OR “soil water \ncontent” OR “soil water deficit”). To avoid publication bias, the following criteria were set to filtrate 718 articles obtained: (1) Soil profiles for moisture extraction under deep-rooted artificial forests must exceed a depth of 500 cm at dense \n90 \nsampling intervals (≤100 cm). The reason for this criteria is that 0-200 cm soil layer is greatly affected by rainfall, \nwhile the phenomenon of 200-500 cm dry soil layer is common (Jia et al., 2020), thus, data from a sampling depth \ngreater than 500 cm were considered in this study in order to explore the state of DSM. 3 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. (2) Soil moisture was the gravimetric or volumetric moisture content measured by drying methods or m\nThe volumetric moisture content was uniformly converted to gravimetric moisture content by\n95 \naverage soil bulk density in each study area. (3) Experiments with only shrub and grass communities or experiments with irrigation, mulching, \nother measures were excluded. (4) Experiments yielded paired data for DSM in apple orchards or black locust forests and that \ngrassland (control). 100 \n(5) Location, mean annual precipitation (mm), forest age, and vegetation types were clearly presented. 2.1.2 Data extraction and classification \nBased on the above criteria, a total of 11980 observations from 380 soil profile, which covered 83 sites in\nmunicipalities (Fig. 1) and extracted from 34 peer-reviewed publications were retrieved, and the specific\nshown in Table 1. The original data were either clearly obtained from tables in the selected papers or intui\n105 \nfrom figures in those papers using GetData Graph Digitizer (version 2.24, http://getdata-graph-digitizer.com\nsoil layers were divided into 200 cm intervals, when more than one soil moisture data sample was obtained\n(e.g., with an interval of 20 cm or 10 cm), we averaged the values of all soil layers to convert them into a 2\nSoil moisture data for the same stand age collected in different sampling years were treated independently \nthe influence of forest age on soil moisture. 2.1.2 Data extraction and classification Based on the above criteria, a total of 11980 observations from 380 soil profile, which covered 83 sites in 4 provinces or \nmunicipalities (Fig. 1) and extracted from 34 peer-reviewed publications were retrieved, and the specific information is \nshown in Table 1. The original data were either clearly obtained from tables in the selected papers or intuitively extracted \n105 \nfrom figures in those papers using GetData Graph Digitizer (version 2.24, http://getdata-graph-digitizer.com). All sampled \nsoil layers were divided into 200 cm intervals, when more than one soil moisture data sample was obtained within 200 cm \n(e.g., with an interval of 20 cm or 10 cm), we averaged the values of all soil layers to convert them into a 200 cm interval. Soil moisture data for the same stand age collected in different sampling years were treated independently when analyzing \nthe influence of forest age on soil moisture. When analyzing the degree limitation of DSM under apple tree in Changwu and \n110 \nLuochuan, the extracted data were divided into tree age range for analysis to avoid random errors. Simultaneously, we also \ncollected basic data from each publication, including data source, geographic coordinates (longitude, latitude), mean annual \nprecipitation (MAP), tree species, and soil texture. Since there was relatively little published information on DSM in black \nlocust forests, we also performed supplementary experiments to obtain relevant data, as described in Section 2.2. 4 \n \nFigure 1: Distribution map of sampling sites on the Loess Plateau. The black dots represent the sampling sites extracted from the article, 115 4 \n \nFigure 1: Distribution map of sampling sites on the Loess Plateau. The black dots represent the sampling sites extracted from the art https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. and the red small crosses represent the sampling sites supplemented in the field. Table 1. Sample information used in this study. 2.1.2 Data extraction and classification Sites \nSpecies \nNEP (pair) \nNSP (pair) \nMAP(mm) \n2021-P (mm) \nClimate type zone \nLuochuan \nApple \n24 \n10 \n608 \n957 \nSemi-humid \nFufeng \nApple \n1 \n- \n606 \n- \nSemi-humid \nBlack locust \n4 \n- \n- \nYongshou \nBlack locust \n4 \n- \n600 \n- \nSemi-humid \nChangwu \nApple \n44 \n8 \n578 \n910 \nSemi-humid \nBlack locust \n6 \n10 \n \nBaishui \nApple \n2 \n- \n577 \n- \nSemi-humid \nJingchuan \nApple \n1 \n- \n571 \n- \nSemi-humid \nQingcheng \nApple \n6 \n- \n561 \n- \nSemi-humid \nYan’an \nApple \n7 \n- \n530 \n- \nSemi-humid \nBlack locust \n15 \n10 \n704 \nDaning \nApple \n2 \n- \n528 \n- \nSemi-humid \nZichang \nApple \n1 \n- \n480 \n- \nSemi-arid \nBlack locust \n1 \n- \n- \nZizhou \nApple \n1 \n- \n424 \n- \nSemi-arid \nMizhi \nApple \n9 \n10 \n421 \n441 \nSemi-arid \nBlack locust \n1 \n10 \n458 \nTotal \n- \n129 \n58 \n- \n- \n- Note: NEP and NSP indicate the number of data pairs from the literature and field sampling, respectively. A data pair refers to the moisture \ncontent of a soil profile from plantation and that of a soil profile from cropland/grassland. MAP is the mean annual precipitation; 250 \nmm<MAP<500 mm and 500 mm<MAP<800 mm are classified as the semi-arid and semi-humid climate zone, respectively (Wang et al., \n2011). The blue text indicates the supplementary sampling in this study with reference to the existing data, and these are representative \nplots for the different climatic regions upon which we focused. 2.2.1 Study area and sampling sites \n125 The LOP covers a total area of 640,000 km2, with an elevation range of 200-3000 m. It belongs to the continental monsoon \nregion where annual precipitation ranges from 150 mm in the northwest to 800 mm in the southeast, 55-78% of which falls \nfrom June to September (Wang et al., 2011). The location of sites from which we collected field data across the entire LOP \n(34°43′-41°16′ N, 100°54′-114°33′ E) are indicated by the red small crosses in Fig. 1 and the blue text in Table 1. 5 \n(34 43 -41 16 N, 100 54 -114 33 E) are indicated by the red small crosses in Fig. 1 and the blue text in Table 1. Geographic coordinates (altitude, longitude, and latitude) for each site were obtained using a GPS (Zhuolin A8, with an \n130 \naccuracy of 1 m). 2.2.2 Soil moisture content \nThe sampling events for soil moisture was done from April 9 to May 6 2022 (the early growing season of trees on the LOP) \nunder the plantations of apple tree and black locust at the ages of 20-30 years old as well as under the crop/grassland as the \ncontrol due to the intact DSM below 200 cm. At first, the sampling depth was set as 0-10 m under different tree plantations; \n135 Geographic coordinates (altitude, longitude, and latitude) for each site were obtained using a GPS (Zhuolin A8, with an \n130 \naccuracy of 1 m). 2.2.2 Soil moisture content The information of the tree plantations reaching the degree limitation was indicated in \nTable 2. The maximum sampling depth was set as 25 m because previous studies reported the maximum rooting depth less \nthan 25 m on the LOP (Wang et al., 2009; Li et al., 2019). In order to minimize the spatial variability in soil moisture caused \nby soil properties, the straight-line distance (SD) between the planted trees and their control was controlled at less than 500 \n140 \nm, and the average SD of the three replicates was shown in Table 2. All soil samples were collected by a hand auger (Φ=6 \ncm) at depth interval of 20 cm and 1.5 m horizontal to the trunk of trees; and three trees with similar growth status were \nselected for each age as repeats. Soil samples without roots was put into an aluminum specimen box and used to measure the \ngravimetric moisture content (g g-1), which was determined by drying method at 105℃ for constant weight. A total of 4200 \nsoil moisture data were collected at 72 soil profiles. Three repeats of soil moisture data under one given tree plantation were \n145 \naveraged when analyzing the degree and depth limitation of DSM under plantations. Moreover, the soil sampling under all \ntree plantations was done once because the temporal variation of DSM below 200 cm under tree plantations was negligible \nin one growing season (Figure S2; Gao et al., 2018; Li et al., 2019). Table 2. Basic information of the sampling trees that causes the lowest deep soil moisture deficit (DSMD). 2.2.2 Soil moisture content Sites \nPlant species \nLongitude \nLatitude \nElevation \n(m) \nSD \n(m) \nStand age \n(yr) \nPH \n(m) \nBD \n(cm) \nDBH \n(cm) \nCD \n(m) \nMizhi \nApple \n110°10′22″ \n37°52′13″ \n1135.1 \n430 \n27 \n3.5±0.25 \n5.8±0.53 \n4.9±0.21 5.73±0.35 \nCropland \n110°11′09″ \n37°52′21″ \n1139.6 \n- \n- \n- \n- \n- \nBlack locust \n110°13′58″ \n37°40′08″ \n1086.2 \n433 \n24 \n9.3±0.40 8.9±0.43 \n7.3±0.17 5.65±0.75 \nGrassland \n110°13′46″ \n37°40′16″ \n1076.4 \n- \n- \n- \n- \n- \nLuochuan \nApple \n109°21′59″ \n35°46′44″ \n1086.9 \n182 \n28 \n2.85±0.35 8.8±0.82 \n8.1±0.52 4.51±16.7 \nCropland \n109°21′58″ \n35°46′38″ \n1083.9 \n- \n- \n- \n- \n- \nYan’an \nBlack locust \n109°23′22″ \n36°40′02″ \n1325.1 \n412 \n25 \n14.35±0.25 9.3±0.19 \n7.9±0.86 5.81±0.77 \nGrassland \n109°22′29″ \n36°40′31″ \n1333.2 \n- \n- \n- \n- \n- \nChangwu \nApple \n107°41′01″ \n35°14′12″ \n1231.7 \n390 \n24 \n4.42±0.25 6.1±0.29 \n5.4±0.73 4.77±0.13 \nCropland \n107°40′43″ \n35°14′20″ \n1232.6 \n- \n- \n- \n- \n- \nBlack locust \n107°41′45″ \n35°12′50″ \n1215.2 \n212 \n28 \n13.35±0.25 9.4±0.66 \n8.1±0.51 5.96±0.01 \nGrassland \n107°42′12″ \n35°12′36″ \n1219.4 \n- \n- \n- \n- \n- \nNote: A±B, A represents the average of the three replicates of each parameter, and B represents the standard deviation. CD-Average crown \n150 \ndiameter; PH-Average plant height; BD-Average basal diameter; DBH-Average diameter at breast height; SD-Average of straight-line \ndistance between three trees and their control (cropland or grassland). Table 2. Basic information of the sampling trees that causes the lowest deep soil moisture deficit (DSMD). 2.2.2 Soil moisture content 5 \nThe sampling events for soil moisture was done from April 9 to May 6 2022 (the early growing season of trees on the LOP) \nunder the plantations of apple tree and black locust at the ages of 20-30 years old as well as under the crop/grassland as the \ncontrol due to the intact DSM below 200 cm. At first, the sampling depth was set as 0-10 m under different tree plantations; \n135 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. however, it was extended down to 25 m when the DSM reached the degree limitation under one given aged plantation as \nwell as under the paired control plots. The information of the tree plantations reaching the degree limitation was indicated in \nTable 2. The maximum sampling depth was set as 25 m because previous studies reported the maximum rooting depth less \nthan 25 m on the LOP (Wang et al., 2009; Li et al., 2019). In order to minimize the spatial variability in soil moisture caused \nby soil properties, the straight-line distance (SD) between the planted trees and their control was controlled at less than 500 \n140 \nm, and the average SD of the three replicates was shown in Table 2. All soil samples were collected by a hand auger (Φ=6 \ncm) at depth interval of 20 cm and 1.5 m horizontal to the trunk of trees; and three trees with similar growth status were \nselected for each age as repeats. Soil samples without roots was put into an aluminum specimen box and used to measure the \ngravimetric moisture content (g g-1), which was determined by drying method at 105℃ for constant weight. A total of 4200 \nsoil moisture data were collected at 72 soil profiles. Three repeats of soil moisture data under one given tree plantation were \n145 \naveraged when analyzing the degree and depth limitation of DSM under plantations. Moreover, the soil sampling under all \ntree plantations was done once because the temporal variation of DSM below 200 cm under tree plantations was negligible \nin one growing season (Figure S2; Gao et al., 2018; Li et al., 2019). however, it was extended down to 25 m when the DSM reached the degree limitation under one given aged plantation as \nwell as under the paired control plots. 2.2.3 Percentage loss of hydraulic conductivity While collecting soil moisture, six current-year branches with 18-20 cm in length and 2-5 mm in diameter were cut off in the \nmorning from apple and black locust trees, age of which reached the degree limitation of DSM; and three trees with similar \n155 \ngrowth status were selected as repeats for each site. Sampled branches were sprayed with water and enclosed in humidified \nblack plastic bags before excising to minimize water loss. After brought these branches to laboratory, we quickly cut 3-5 cm While collecting soil moisture, six current-year branches with 18-20 cm in length and 2-5 mm in diameter were cut off in the \nmorning from apple and black locust trees, age of which reached the degree limitation of DSM; and three trees with similar \n155 \ngrowth status were selected as repeats for each site. Sampled branches were sprayed with water and enclosed in humidified \nblack plastic bags before excising to minimize water loss. After brought these branches to laboratory, we quickly cut 3-5 cm 6 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. from the middle of the branch in water and immediately measured xylem hydraulic conductivity of it by using the xylem \nembolism measurement system (Xylem embolism meter, Bronkhorst, Mon-tigny-les-Cormeilles, France). To prevent water \nspilt from the branch, the initial xylem hydraulic conductivity (Kini, kg s-1 MPa-1) was measured with 1 mmol·L-1 CaCl2 \n160 \nsolution (pH = 6) through a filter with a filter aperture of 0.22 μm at a low pressure of 0.5-1.0 kPa. And at a high pressure of \n0.2 MPa, the branch was flushed with 1 mmol·L-1 CaCl2 solution until there was no air bubble, then the xylem hydraulic \nconductivity was determined again at a low pressure of 0.5-1.0 kPa and the flushing was repeated until attaining a maximum \nconductivity (Kmax, kg s-1 MPa-1). The percentage loss of hydraulic conductivity (PLC, %) was calculated by, (1) \nmax\n1\n100\nini\nK\nPLC\nK\n\n\n\n\n\n\n\n\n\n \n165 max\n1\n100\nini\nK\nPLC\nK\n\n\n\n\n\n\n\n\n\n \n5 (1) 165 2.2.4 Basic parameters of plantation 2.2.4 Basic parameters of plantation In order to accurately target the sample plantation, tree age was determined on the basis of plant growth cones (Haglof, \nCO300-52, Sweden) before sampling, and the crown diameter, basal diameter and diameter at breast height were measured \nwith a tape (Table 2). The collection of plantation root samples was carried out simultaneously with the collection of soil \nmoisture samples, and soil samples with root was stored in a sealed sample bag to minimize the loss of fine roots (≤ 2 mm), and \n170 \nthen gently rinsed in a 100 mesh (0.15 mm) sieve, removing residual roots from the sieve with tweezers. Each fine root sample \nwas dried at 65℃ to constant weight and weighed using an electronic balance with a precision of 0.0001 g to determine root \ndry weight. Finally, the fine root dry weight density (FRDWD) was calculated using the formula given by Zhao et al. (2022). 2.2.5 Soil texture and permanent wilting point The sampling of soil properties was done simultaneously as soil moisture was sampled, and the data was showed in Table 3. 175 \nSoil samples without root were air-dried, ground, and then passed through a 16 mesh sieve for measuring soil particle \ncomposition (i.e., clay, silt and sand contents) and soil pH, and through a 100 mesh sieve for determining soil organic carbon \n(SOC), respectively. Soil particle composition was determined by laser diffraction using a Mastersizer-2000 Laser \nGranularity Analyzer (Malvern Instruments, Malvern, England) (Wang et al., 2008). Soil pH was determined by the DELTA The sampling of soil properties was done simultaneously as soil moisture was sampled, and the data was showed in Table 3. 175 \nSoil samples without root were air-dried, ground, and then passed through a 16 mesh sieve for measuring soil particle \ncomposition (i.e., clay, silt and sand contents) and soil pH, and through a 100 mesh sieve for determining soil organic carbon \n(SOC), respectively. Soil particle composition was determined by laser diffraction using a Mastersizer-2000 Laser \nGranularity Analyzer (Malvern Instruments, Malvern, England) (Wang et al., 2008). Soil pH was determined by the DELTA \n320 pH meter. SOC was measured by using the potassium dichromate volumetric method (Fu et al., 2010), and soil organic \n180 \nmatter (SOM) was converted from SOC (Eq. 4). 2.2.4 Basic parameters of plantation (3) \n(4) \n\n\n10\n10\n1\n1\n10\n2\n46.481\n4.757\n14.028 log (\n) 13.991 log (\n)\n \n42.261 log\n11.763\n19.198\n \n5.448\n0.044\n1.975\ni\ni\ni\ni\ni\ni\ni\ni\ni\ni\ni\nFC\nSOC\nclay\nsand\nSOC\nsand\nSOC\nBD\nSOC\nBD\nSOC\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n \n5 \n1.724\ni\ni\nSOM\nSOC\n\n (3\n\n\n10\n2\ng\n \n5.448\n0.044\n1.975\ni\ni\ni\ni\ni\ni\ni\nBD\nSOC\nBD\nSOC\n\n\n\n\n\n\n (5) \n0.5\n10\n0.5\n2\n1.8284\n0.0429 log (\n)\n0.0205\n0.0125\n \ncos(\n)\n0.0061\n0.0001\n0.0098\n \n0.0071\n0.0505\n0.0002\ni\ni\ni\ni\ni\ni\ni\ni\ni\nBD\nclay\nclay\nclay\nsilt\nsilt\nSG\nSG\nSOC\nSOC\nSOC\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n (5) \n0.5\n10\n0.5\n2\n1.8284\n0.0429 log (\n)\n0.0205\n0.0125\n \ncos(\n)\n0.0061\n0.0001\n0.0098\n \n0.0071\n0.0505\n0.0002\ni\ni\ni\ni\ni\ni\ni\ni\ni\nBD\nclay\nclay\nclay\nsilt\nsilt\nSG\nSG\nSOC\nSOC\nSOC\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n (5) where FC is field capacity, %; SOC and SOM are soil carbon content and soil organic matter, g kg-1; BD is the soil bulk \ndensity, g cm-3; clay, silt and sand are clay, silt and sand content, %, respectively; SG is slope gradient at each sample \nlocation, °; and i represents the ith soil depth. 190 Table 3. Basic soil properties of sampling sites. 2.2.6 Meteorological parameters The precipitation, air temperature, and relative humidity of different regions were from the European Centre for \nMedium-Range Weather Forecasts (ECMWF) ERA5-Land reanalysis (hereafter ERA5-Land) data, and the accuracy of \nwhich has been verified by Araújo et al. (2022). Based on the above meteorological data, vapor pressure deficit (VPD, kPa) \nwas calculated by using Eq.6. The precipitation, air temperature, and relative humidity of different regions were from the European Centre for (6) \nThe precipitation, air temperature, and relative humidity of different regions were from the European Centre for \nMedium-Range Weather Forecasts (ECMWF) ERA5-Land reanalysis (hereafter ERA5-Land) data, and the accuracy of \nwhich has been verified by Araújo et al. (2022). Based on the above meteorological data, vapor pressure deficit (VPD, kPa) \n195 \nwas calculated by using Eq.6. \n\n17.27\n237.3\n0.61078\n1\na\na\nT\nT\nVPD\ne\nRH\n\n\n\n\n\n＋\n \nWhere T is air temperature ℃; and RH is relative humidity % The precipitation, air temperature, and relative humidity of different regions were from the European Centre for \nMedium-Range Weather Forecasts (ECMWF) ERA5-Land reanalysis (hereafter ERA5-Land) data, and the accuracy of \nwhich has been verified by Araújo et al. (2022). Based on the above meteorological data, vapor pressure deficit (VPD, kPa) \n5 \nwas calculated by using Eq.6. 195 (6) \n\n\n17.27\n237.3\n0.61078\n1\na\na\nT\nT\nVPD\ne\nRH\n\n\n\n\n\n＋ (6) \n\n\n17.27\n237.3\n0.61078\n1\na\na\nT\nT\nVPD\ne\nRH\n\n\n\n\n\n＋ (6) Where Ta is air temperature, ℃; and RH is relative humidity, %. Where Ta is air temperature, ℃; and RH is relative humidity, %. 2.2.4 Basic parameters of plantation Sites \nPlant species \nSoil type \nClay \nSilt \nSand \nSoil organic metter (g kg-1) \nBulk density (g cm-3) \npH \nMizhi \nApple \nCultivated loessial soils \n15.5 \n24.3 \n60.2 \n2.94 \n1.28 \n7.64 \nCropland \nCultivated loessial soils \n14.9 \n25.1 \n60.0 \n2.33 \n1.29 \n8.44 \nBlack locust Cultivated loessial soils \n13.6 \n25.0 \n61.4 \n3.09 \n1.28 \n8.03 \nGrassland \nCultivated loessial soils \n13.8 \n25.4 \n60.8 \n2.35 \n1.29 \n8.36 \nLuochuan \nApple \nDark loessial soils \n21.5 \n37.1 \n41.4 \n4.62 \n1.31 \n7.54 \nCropland \nDark loessial soils \n22.2 \n38.5 \n39.3 \n4.38 \n1.33 \n8.17 \nYan’an \nBlack locust Cultivated loessial soils \n18.1 \n33.1 \n48.8 \n4.87 \n1.29 \n8.07 \nGrassland \nCultivated loessial soils \n19.6 \n31.5 \n48.9 \n4.43 \n1.30 \n8.53 \nChangwu \nApple \nDark loessial soils \n24.6 \n38.7 \n36.7 \n4.76 \n1.31 \n7.35 \nCropland \nDark loessial soils \n25.8 \n38.7 \n35.5 \n4.35 \n1.32 \n8.21 \nBlack locust \nDark loessial soils \n22.9 \n38.2 \n38.9 \n5.01 \n1.29 \n7.96 \nGrassland \nDark loessial soils \n22.7 \n38.4 \n38.9 \n4.26 \n1.31 \n8.23 2.2.4 Basic parameters of plantation Due to the challenges of collecting undisturbed soil cores within the 10 m \nprofile, bulk density (BD) and the PWP of each region were estimated using the pedo-transfer functions (PTFs) developed by \nWang et al. (2012) and Balland et al. (2008) based on the above indicators, as follows, 320 pH meter. SOC was measured by using the potassium dichromate volumetric method (Fu et al., 2010), and soil organic \n180 \nmatter (SOM) was converted from SOC (Eq. 4). Due to the challenges of collecting undisturbed soil cores within the 10 m \nprofile, bulk density (BD) and the PWP of each region were estimated using the pedo-transfer functions (PTFs) developed by \nWang et al. (2012) and Balland et al. (2008) based on the above indicators, as follows, (2) https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 2.3 Data analysis 2.3.1 Meta-analysis \n200 2.3.1 Meta-analysis \n200 2.3.1 Meta-analysis \n200 In this study, the differences between soil moisture and RR was considered significant if the 95% CI did not include zero, and \nthe differences were not considered significant if the 95% CI did not include zero. 2.3.2 Quantitative index for degree limitation of soil desiccation \n215 2.3.2 Quantitative index for degree limitation of soil desiccation \n215 \nThe effect of different deep-rooted planted trees RWU on soil moisture was expressed as soil moisture deficit (SMD, the \ncalculation method as shown in Eq. (10)), which could remove the interference of precipitation differences in different \nsampling years. In order to compare the difference between the degree limitation of soil desiccation and the PWP, the SMD \ncorresponding to PWP along the soil profile was calculated by Eq. (11). The effect of different deep-rooted planted trees RWU on soil moisture was expressed as soil moisture deficit (SMD, the \ncalculation method as shown in Eq. (10)), which could remove the interference of precipitation differences in different \nsampling years. In order to compare the difference between the degree limitation of soil desiccation and the PWP, the SMD \ncorresponding to PWP along the soil profile was calculated by Eq. (11). ,\n0,\n,\n0,\n \nj k\nk\nj k\nk\nSMC\nSMC\nSMD\nSMC\n\n\n220 \n0,\n,\n0,\n \nk\nk\npwp k\nk\nPWP\nSMC\nSMD\nSMC\n\n (10) (11) where SMCj,k and SMC0,k represent soil moisture content in the kth layer of the jth plantations and in the kth layer of the \ncontrol (cropland or grassland), %, respectively; and PWPk represents the PWP in the kth layer, %. 2.3.3 Quantitative index for depth limitation of soil desiccation 2.3.1 Meta-analysis \n200 In a traditional meta-analysis, the mean values of the treatment and control with their standard deviations or standard errors 8 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. are extracted from the publications. However, most published papers selected for this study reported their mean values \nwithout standard deviations. To maximize the number of observations, the following unweighted meta-analysis approach \nwas adopted (Li et al., 2021b). (7) \n(\n/\n) \ne\nC\nRR\nLn X\nX\n\n \n205 (7)\n(\n/\n) \ne\nC\nRR\nLn X\nX\n (7) (7) where RR is a unit-free index with positive and negative values indicating either increasing or decreasing soil moisture in \nresponse to changes in planted tree age, and Xe and Xc are values of soil moisture under trees and controls for each study, \nrespectively. To test the significance of differences between soil moisture and RR, a 95% confidence interval (CI) was used. A previously described method (Deng et al., 2016) was used to calculate the 95% CI, as shown in Eqs. (8) and (9), (8) \n(9) \n \ns\ntotal\nV\nSE\nN\n\n \n210 \n95%\n1.96\n \ntotal\nCI\nSE\n\n\n \nWhere SEtotal is the standard error of RR, and Vs and N are the variances of RR and the number of observations, respectively. In this study, the differences between soil moisture and RR was considered significant if the 95% CI did not include zero, and \nthe differences were not considered significant if the 95% CI did not include zero. (8) \n(9) \n \ns\ntotal\nV\nSE\nN\n\n \n210 \n95%\n1.96\n \ntotal\nCI\nSE\n\n\n \nWhere SEtotal is the standard error of RR, and Vs and N are the variances of RR and the number of observations, respectively. In this study, the differences between soil moisture and RR was considered significant if the 95% CI did not include zero, and \nthe differences were not considered significant if the 95% CI did not include zero. s\ntotal\nV\nSE\nN\n\n210 (8) 95%\n1.96\n \ntotal\nCI\nSE\n\n (9) (9) Where SEtotal is the standard error of RR, and Vs and N are the variances of RR and the number of observations, respectively. 3.1 Soil moisture under planted trees of the different ages \n235 The soil moisture under apple orchards (APO) and black locust forest (BLF) varied greatly with tree age across the entire \nsoil layer (Fig. 2). With an increase in tree age, the negative effect of APO (0-21 m soil layer) and BLF (0-10 m soil layer) \ngrowth on deep soil moisture (DSM) increased first and then tended to become stable. Specifically, the negative effect value \nof DSM (4-10 m) under BLF older 30 years decreased by 17.4% compared with 20-30 years. Thus, the tree age at which the \nplantation had an extreme effect on DSM appears to be between 20 and 30 years. 240 Figure 2: Mean effect sizes of soil moisture response to planted trees’ growth with different age classes on the Loess Plateau: (a) tree age \n<10 year, (b) 10 year ≤ tree age<20 year, (c) 20 year ≤ tree age<30 year, and (d) tree age ≥ 30 year. The vertical black dotted lines indicate \nRR=0. Blue and red dots represent black locust forests and apple orchard RRs, respectively, diamonds and circles represent the mean RR of \nblack locust and apple orchard, respectively, and the error bars represent 95% CI. 245 Figure 2: Mean effect sizes of soil moisture response to planted trees’ growth with different age classes on the Loess Plateau: (a) tree age \n<10 year, (b) 10 year ≤ tree age<20 year, (c) 20 year ≤ tree age<30 year, and (d) tree age ≥ 30 year. The vertical black dotted lines indicate \nRR=0. Blue and red dots represent black locust forests and apple orchard RRs, respectively, diamonds and circles represent the mean RR of \nblack locust and apple orchard, respectively, and the error bars represent 95% CI. Figure 2: Mean effect sizes of soil moisture response to planted trees’ growth with different age classes on the Loess Plateau: (a) tree age \n<10 year, (b) 10 year ≤ tree age<20 year, (c) 20 year ≤ tree age<30 year, and (d) tree age ≥ 30 year. The vertical black dotted lines indicate \nRR=0. Blue and red dots represent black locust forests and apple orchard RRs, respectively, diamonds and circles represent the mean RR of \nblack locust and apple orchard, respectively, and the error bars represent 95% CI. 2.3.3 Quantitative index for depth limitation of soil desiccation The paired sample t-test in SPSS was used to analyze the significance of soil moisture between plantations and their controls. 225 \nWhen the significant difference becomes insignificant (p>0.05) as the soil layer deepens, which is considered that the DSM \nconsumption of the plantation has reached the depth limitation. However, the difference is always significant (p<0.05) 9 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. indicates that the maximum water uptake depth has not been reached. indicates that the maximum water uptake depth has not been reached. 2.3.4 Statistical analysis \nRedundancy analysis (RDA) was performed to evaluate the contributions of environmental factors to SMD and soil moisture \n230 \nusing Canoco 5. Least significant difference (LSD) post-hoc tests in SPSS 22.0 software package (SPSS 22.0, SPSS Institute \nLtd., USA) was used to analyze the significant differences in PLC of current-year branches in planted trees, α = 0.05 was \nconsidered statistically significant. Origin 2021 software (OriginLab Corporation, USA) was used to draw the figures. 3.2 Limitation of DSM for planted trees 10 \nThe DSM under a given tree species can vary largely among different years even if at the same age due to varying annual \nprecipitation. Taken the 15-year-old APO in Changwu for example, the DSM varied greatly in different sampling years (Fig. 3a). Soil moisture deficit (SMD), defined as the relative difference of soil moisture under given trees and nearby controls, https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. was used to eliminate the effect of climate differences in various sampling years. Here, the DSM of control adjacent to the \n250 \nplantation served as the background soil moisture, which is not influenced by root water extraction due to the shallow (<100 \ncm) roots of annual crops; hence the DSM in adjacent control can be used to reflect local annual precipitation. We found that \nvariation coefficient (CV) of deep soil moisture deficit (DSMD) (Fig. 3b) was lower than that of DSM, thus, DSMD was \nused for the following in-depth analyses. was used to eliminate the effect of climate differences in various sampling years. Here, the DSM of control adjacent to the \n250 \nplantation served as the background soil moisture, which is not influenced by root water extraction due to the shallow (<100 \ncm) roots of annual crops; hence the DSM in adjacent control can be used to reflect local annual precipitation. We found that \nvariation coefficient (CV) of deep soil moisture deficit (DSMD) (Fig. 3b) was lower than that of DSM, thus, DSMD was \nused for the following in-depth analyses. 250 255 \nFigure 3: Distribution of soil moisture (a) and soil moisture deficit (b) in 15-year-old apple orchards in Changwu in 2002, 2010, 2016 and \n2019. The variation coefficient (CV) of soil moisture and soil moisture deficit below 2 m are shown in the small picture, and the dashed \nlines in the figure represent the mean values of the CV of deep soil moisture and deep soil moisture deficit, respectively. 255 Figure 3: Distribution of soil moisture (a) and soil moisture deficit (b) in 15-year-old apple orchards in Changwu in 2002, 2010, 2016 and \n2019. The variation coefficient (CV) of soil moisture and soil moisture deficit below 2 m are shown in the small picture, and the dashed \nlines in the figure represent the mean values of the CV of deep soil moisture and deep soil moisture deficit, respectively. 3.2.1 Degree limitation of DSM In order to reduce the randomness of the extracted data, the data for apple trees with similar ages in Changwu and \nLuochuan were combined. The gray line indicates SMD at permanent wilting point (SMDPWP) in each sampling site, and the red dashed \n275 \nline indicates the SMD equal to -0.6. Figure 4: Soil moisture deficit (SMD) distribution under apple orchards of different ages in Changwu (a), Luochuan (b) and Mizhi (c); and \nSMD under black locust forests of different ages in Changwu (d), Yan’an (e) and Mizhi (f). The capital letters A and B in the legend \nrepresent apple and black locust, respectively, and the numbers after the letters represent tree age, for example, A-22 represents \n22-year-old apple. In order to reduce the randomness of the extracted data, the data for apple trees with similar ages in Changwu and \nLuochuan were combined. The gray line indicates SMD at permanent wilting point (SMDPWP) in each sampling site, and the red dashed \n275 \nline indicates the SMD equal to -0.6. 3.2.1 Degree limitation of DSM The DSMD fluctuation in APO over 19 years was relatively stable in Changwu, Luochuan and Mizhi (Fig. 4a, b, c). 260 \nInterestingly, with the increase in tree age, there was the lowest DSMD of APO in different climate zones, and tree age \nbetween 24 and 28 years was associated with the lowest DSMD in different regions. After reaching the lowest value, apple \ntrees can no longer extract water from the deep soil. Notably, the lowest value of DSMD in three regions fluctuated around \n-0.6, which was very close to the DSMD value corresponding to the local PWP. In addition, DSMD of the BLF in the \nChangwu, Yan’an and Mizhi regions was similar to that in APO (Fig. 4d, e, f). The consumption of DSM reached the same \n265 \nlowest value in different regions. This suggests that PWP is a good indicator of the degree limitation of DSM, irrespective of \ntree species and site. It is worth noting that, compared with APO, the variation scope of DSMD in Changwu and Yan’an \ndecreased significantly with the age of the BLF, which may be due to the extreme rainfall in 2021, which recharges DSM in \nthe BLF (Table 1). 11 11 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 270 \nFigure 4: Soil moisture deficit (SMD) distribution under apple orchards of different ages in Changwu (a), Luochuan (b) and Mizhi (c); and \nSMD under black locust forests of different ages in Changwu (d), Yan’an (e) and Mizhi (f). The capital letters A and B in the legend \nrepresent apple and black locust, respectively, and the numbers after the letters represent tree age, for example, A-22 represents \n22-year-old apple. In order to reduce the randomness of the extracted data, the data for apple trees with similar ages in Changwu and \nLuochuan were combined. The gray line indicates SMD at permanent wilting point (SMDPWP) in each sampling site, and the red dashed \n275 \nline indicates the SMD equal to -0.6. 270 270 Figure 4: Soil moisture deficit (SMD) distribution under apple orchards of different ages in Changwu (a), Luochuan (b) and Mizhi (c); and \nSMD under black locust forests of different ages in Changwu (d), Yan’an (e) and Mizhi (f). The capital letters A and B in the legend \nrepresent apple and black locust, respectively, and the numbers after the letters represent tree age, for example, A-22 represents \n22-year-old apple. 3.2.2 Depth limitation of DSM , , * and ns in the gray bands indicate the significance level of measured data \nobtained by the paired sample t-test for p< 0.001, p< 0.01, p< 0.05 and no significant differences, respectively. Figure 5: Dynamic distribution of maximum water absorption depth after reaching the degree limitation of deep soil desiccation. (a) 22- \n285 \n(literature data) and 24-year-old (measured data) apple in Changwu; (b) and (c) indicate 28- and 24-year-old apple in Luochuan and Mizhi, \nrespectively; (d), (e) and (f) indicate 28-, 25- and 24-year-old black locust in Changwu, Yan’an and Mizhi, respectively. Gray line \nrepresents soil moisture of grassland or farmland. *, , * and ns in the gray bands indicate the significance level of measured data \nobtained by the paired sample t-test for p< 0.001, p< 0.01, p< 0.05 and no significant differences, respectively. 3.2.2 Depth limitation of DSM Based on the measured and literature data, we analyzed the water consumption depth limit of plantations in different regions \nafter the soil desiccation degree limitation was reached (Fig. 5). With an increase in depth, the soil moisture of plantations \nwas close to or even higher than that of the control, except for the BLF in Mizhi. Thus, we calculated the difference \nsignificance of SM between the plantations and control using the paired sample t-test, treating the points with no significant \n(p>0.05) soil moisture difference as the maximum water uptake depth. And it reached 18.0-22.0 m for APO at different sites; \nand 18.4-18.8 m for BLF in Changwu and Yan’an while in Mizhi it was deeper than 25 m. 12 Figure 5: Dynamic distribution of maximum water absorption depth after reaching the degree limitation of deep soil desiccation. (a) 22- \n285 \nhttps://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 5: Dynamic distribution of maximum water absorption depth after reaching the degree limitation of deep soil desiccation. (a) 22- \n285 \n(literature data) and 24-year-old (measured data) apple in Changwu; (b) and (c) indicate 28- and 24-year-old apple in Luochuan and Mizhi, \nrespectively; (d), (e) and (f) indicate 28-, 25- and 24-year-old black locust in Changwu, Yan’an and Mizhi, respectively. Gray line \nrepresents soil moisture of grassland or farmland. *, , * and ns in the gray bands indicate the significance level of measured data \nobtained by the paired sample t-test for p< 0.001, p< 0.01, p< 0.05 and no significant differences, respectively. Figure 5: Dynamic distribution of maximum water absorption depth after reaching the degree limitation of deep soil desiccation. (a\n285 Figure 5: Dynamic distribution of maximum water absorption depth after reaching the degree limitation of deep soil desiccation. (a) 22- \n285 \n(literature data) and 24-year-old (measured data) apple in Changwu; (b) and (c) indicate 28- and 24-year-old apple in Luochuan and Mizhi, \nrespectively; (d), (e) and (f) indicate 28-, 25- and 24-year-old black locust in Changwu, Yan’an and Mizhi, respectively. Gray line \nrepresents soil moisture of grassland or farmland. 3.3 Effect of factors on the limitation \n290 13\nBased on all extracted and measured data, including SMD, soil moisture (SM), soil texture (clay, silt), and root biomass \n(FRDWD) along with ERA5-Land meteorological data (MAP, mean annual temperature (MAT), relative humidity (RH), \nvapor pressure deficit (VPD)), redundancy analysis (RDA) was used to analyze the key factors that determine the degree and \ndepth limitation of DSM (Fig. 6). The RDA showed that the explanatory variables (soil properties, meteorological variables \nand plant characteristic) account for 67.7% of the total variation of SM and SMD. SMD was significantly (p<0.01) positively \n295 \ncorrelated with VPD, and negatively correlated with clay, silt, RH, MAT, and MAP (Fig. 6a). The clay, VPD, silt, RH, and \nFRDWD provided statistically significant (p<0.01) explanations for the distribution of SMD and SM (Fig. 6b), and clay was 13 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. the most important. Thus, soil texture and climatic conditions were the main factors leading to the degree and depth \nlimitation of deep soil desiccation in different regions. However, the relationship between MAP and SMD was not significant, \nwhich may be due to the MAP of each region weakened the differences of precipitation in each sampling year. 300 the most important. Thus, soil texture and climatic conditions were the main factors leading to the degree and depth \nlimitation of deep soil desiccation in different regions. However, the relationship between MAP and SMD was not significant, \nwhich may be due to the MAP of each region weakened the differences of precipitation in each sampling year. 300 Figure 6: (a) redundancy analysis (RDA) of “response variable (soil moisture deficit (SMD), soil moisture (SM)) and environmental \nfactors”. Descriptors (red arrows) are soil properties (clay and silt), meteorological variables (vapor pressure deficit (VPD), mean annual \nprecipitation (MAP), relative humidity (RH), and mean annual temperature (MAT)), and plant characteristic (fine root dry weight density \n(FRDWD)). Axis 1 (65.67%, p<0.01) and Axis 2 (2.03%, p<0.01) are shown. (b) explanation provided by of each environmental variable \nfor the response variables. *, , and ns following the bar chart indicate that p<0.01, p<0.05, and no significant, respectively. 3.4 Effect of the limitations on hydraulic conductivity In order to further explore how the limitation caused by deep soil desiccation affects the hydraulic conductivity of plants, we \ncompared hydraulic conductance of branches of plantation trees in relation to the degree and depth limitation of deep soil \ndesiccation at different sites (Fig. 7). PLC was higher than 50% for all tree species and regions. Furthermore, the PLC of \n310 \nblack locust in Mizhi was significantly higher than that in other places (p<0.05), which indicates that the risk of mortality of \nblack locust in Mizhi is greatest when deep soil desiccation reaches degree and depth limitation thresholds. Figure 7: Differences in percentage loss of hydraulic conductivity of annual branches of apple (a) and black locust (b) in different regions \n(Changwu, Luochuan, Yan’an, and Mizhi). * or ns indicate that the significance level is p<0.05 or there is no significant difference, \n315 \nrespectively. Figure 7: Differences in percentage loss of hydraulic conductivity of annual branches of apple (a) and black locust (b) in different regions \n(Changwu, Luochuan, Yan’an, and Mizhi). * or ns indicate that the significance level is p<0.05 or there is no significant difference, \n315 \nrespectively. Figure 7: Differences in percentage loss of hydraulic conductivity of annual branches of apple (a) and black locust (b) in different regions \n(Changwu, Luochuan, Yan’an, and Mizhi). * or ns indicate that the significance level is p<0.05 or there is no significant difference, \n315 \nrespectively. 14 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4 Discussion \n4.1 The mechanism underlying for the degree and depth limitation \nAlthough DSM (>200 cm) decreased with an increase of planted trees age, and even reached the lowest limitation, shallow \nsoil moisture (<200 cm) did not change significantly (Fig. 2 and 4). This is ascribed to surface or shallow layer soil moisture \n320 \nis usually greatly influenced by rainfall infiltration and evapotranspiration, while DSM is often in a negative balance, being \nconsumed by deep-rooted trees and unable to be replenished for a long time (Fang et al., 2016; Gao et al., 2020). As the \ncritical water resource for plants to cope with extreme droughts, DSM has reached its lowest limitation, seriously threatening \nthe sustainability of tree growth under future climate conditions. In our study, the lowest DSMD fluctuated around -0.6 irrespective of tree species and site (Fig. 4). 3.4 Effect of the limitations on hydraulic conductivity This contradicts the \n325 \nassumption that degree of deep-layer soil desiccation limitation is dependent on tree species, which is mainly because the \nclimate conditions and soil texture differ between regions (Fig. 6). The precipitation in the LOP showed a decreasing trend \nfrom southeast to northwest, the clay content also follows this trend (Wang et al., 2011). When the soil texture is coarser, it \nhas a larger matric potential or smaller suction under the same soil moisture conditions (Dexter, 2004), resulting in a larger \nmatric potential gradient around fine roots in areas with less rainfall (LRAs), thus effectively enhance soil water availability. 330 \nSoil water potential, when the soil hydraulic conductivity (Ks) drops to lower values than the root hydraulic conductivity (Kr), \ncan be regarded as the critical soil matric potential, at which the soil begins to limit the water flux in the soil-plant continuum \n(Fig. 8; Cai et al., 2022). Thus, the soil with coarser texture has higher Ks when soil starts limiting trees’ RWU, causing the \ncorresponding Kr in LRAs to be higher than that in areas with more rainfall (MRAs) by allocating more biomass to fine root \n(Fig. 8). In addition, VPD in the LRAs is relatively high, and the transpiration traction force increases (Will et al., 2013; \n335 \nHayat et al., 2019), causing a larger water potential gradient between roots and leaves under the same soil moisture, and thus \ndriving roots to absorb more moisture. Thus, although the physiological and growth characteristics of apple and black locust \nin different regions were obviously different (Table 2; Fig. 7), the lowest DSMD of all tree species at each site both \nfluctuated around-0.6. This indicates that the lowest amount of soil moisture absorbed by plants is determined by VPD and \nsoil water availability, which is mainly affected by the precipitation and soil texture. Notably, the lowest DSMD was very \n340 4.1 The mechanism underlying for the degree and depth limitation Our result shows that the maximum RWU depth \n350 \nreached 18.0-22.0 m for APO at different sites, and 18.4-18.8 m for BLF in Changwu and Yan’an while in Mizhi it was more \nthan 25 m (Fig. 5). The above- and under-ground biomass allocation mechanism of plantation determines its’ root: shoot \nratios (Mokany et al., 2005), thus, the FRDWD of the plantation indicates that their water absorption range can only reach \n18-22 m. Furthermore, roots can no longer extract moisture after reaching a certain depth, probably because the hydraulic \npath of water transport in the SPAC is too long and the water potential gradient is insufficient to drive water transport \n355 \nupwards (Ahmed et al., 2014). However, the water consumption depth of BLF in Mizhi was more than 25 m. Wang et al. (2009) also found that the maximum water consumption depth of the artificial Caragana microphylla forest in the semi-arid \nregion was 22.5 m, which indicates that chronic water shortages encourages planted trees to absorb water by growing roots \ndownwards in the semi-arid region. Thus, the RWU depth of plantations in other regions has reached its maximum depth, \neven though BLF in LRAs can obtain water through root extension. 360 Figure 8: Regulatory mechanism of the extreme influence of root water uptake (RWU) on deep soil moisture. The parameters of \nsoil-plant-atmosphere continuum (SPAC) in the white rectangle are quantized, the gray rectangle represents the intermediate variables that \nthe parameters of SPAC cause the result of minimum soil moisture profile (degree limitation), the blue rounded rectangle represents the \nlimitation of degree and maximum depth of RWU (depth limitation). The red arrow, the black solid arrow and the black dotted arrow \nindicate that the parameters of SPAC have a significant (p<0.05) effect on the degree limitation, have an influence on the depth limitation, \nand have an indirect effect on two results, respectively. 4.1 The mechanism underlying for the degree and depth limitation (Fig. 8). In addition, VPD in the LRAs is relatively high, and the transpiration traction force increases (Will et al., 2013; \n335 \nHayat et al., 2019), causing a larger water potential gradient between roots and leaves under the same soil moisture, and thus \ndriving roots to absorb more moisture. Thus, although the physiological and growth characteristics of apple and black locust \nin different regions were obviously different (Table 2; Fig. 7), the lowest DSMD of all tree species at each site both \nfluctuated around-0.6. This indicates that the lowest amount of soil moisture absorbed by plants is determined by VPD and 15 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. the LOP. After soil moisture use in the profile reaches the degree limitation, plants extend their roots into the deep soil to expand their \nwater hunting range during the dry season (Fig. 8; Ivanov et al., 2012). Our result shows that the maximum RWU depth \n350 \nreached 18.0-22.0 m for APO at different sites, and 18.4-18.8 m for BLF in Changwu and Yan’an while in Mizhi it was more \nthan 25 m (Fig. 5). The above- and under-ground biomass allocation mechanism of plantation determines its’ root: shoot \nratios (Mokany et al., 2005), thus, the FRDWD of the plantation indicates that their water absorption range can only reach \n18-22 m. Furthermore, roots can no longer extract moisture after reaching a certain depth, probably because the hydraulic \npath of water transport in the SPAC is too long and the water potential gradient is insufficient to drive water transport \n355 \nupwards (Ahmed et al., 2014). However, the water consumption depth of BLF in Mizhi was more than 25 m. Wang et al. (2009) also found that the maximum water consumption depth of the artificial Caragana microphylla forest in the semi-arid \nregion was 22.5 m, which indicates that chronic water shortages encourages planted trees to absorb water by growing roots \ndownwards in the semi-arid region. Thus, the RWU depth of plantations in other regions has reached its maximum depth, \neven though BLF in LRAs can obtain water through root extension. 360 the LOP. After soil moisture use in the profile reaches the degree limitation, plants extend their roots into the deep soil to expand their \nwater hunting range during the dry season (Fig. 8; Ivanov et al., 2012). 4.3 Limitations and practical implications \n380 4.3 Limitations and practical implications \n380 \nAffected by objective factors such as differences in the time of planting and vegetation succession in different regions, \nsampling of the same tree species in different regions cannot guarantee the same tree age. In addition, the collection of large \namounts of DSM data requires a great deal of resources, the number of samples available in this study is limited. Thus, the \nresults of this study need to be in-depth verified in different tree ages and in different regions to guide vegetation \nmanagement under local conditions. 385 \nLarge numbers of tree deaths caused by prolonged drought are of great concern because trees fulfil a critical ecological role \nand have the largest biomass and storage of carbon (Feng et al., 2016; Bai et al., 2021). Thus, it is essential to understand the \ndegree and depth limitation of deep-layer soil desiccation for maintaining the sustainability of vegetation restoration under \nfuture climatic drying. Our study shows that 24-28-year-old plantations, both APO and BLF, have reached the degree \nlimitation of deep soil desiccation; and the maximum DSMD is about -0.6, which corresponds to the DSMD at local PWP, \n390 \nirrespective of tree species and site. On the one hand, this confirms that PWP is a good indicator of the degree limitation of \ndeep-layer soil desiccation for the tree species examined; on the other hand, when the precise wilting point of deep soil in \ndifferent regions is difficult to determine, the lowest DSMD (0.6) can be used as an index for judging whether DSM can \nsustain the survival of the typical plantations on the LOP during prolonged drought. Furthermore, except for the BLF in \nMizhi, all plantations have reached the depth limitation of deep-layer soil desiccation, and after reaching the degree and \n395 \ndepth limitation thresholds, plantations of different sites suffer different degrees of embolism. Therefore, plantations of 24 \nyears and above face the greatest risk of dieback and death in drought years or prolonged dry periods, especially in semi-arid \nregions like Mizhi. This study provides practical insights into the sustainable development of vegetation restoration on the \nLOP in future extreme climate. 4.2 Effects of the limitations on the hydraulic conductivity of plantations 16 \nDuring prolonged dry periods, deep-rooted trees often rely on DSM to avoid drought stress (Ding et al., 2021; Wang et al., \n2021). However, deep soil desiccation reduces the moisture supply capacity of the soil, which can lead to xylem embolism, \nand even tissue dehydration and, consequently, hydraulic failure (Yang et al., 2022). At the same time, water stress leads to \nstomatal closure, and thus photosynthesis and other physiological processes are inhibited (McDowell et al., 2018). When the \nxylem hydraulic conductance drops below a certain threshold, plant may occurs dieback or even die completely due to the 16 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. combined occurrence of hydraulic failure and carbon starvation (Arend et al., 2021). Our results show that branches of \nsimilar aged trees that causes the DSM to reach the degree and depth limitation suffered embolism to different degrees (Fig. 375 \n7), which is consistent with the hypothesis about the hydraulic conductivity of planted trees. Furthermore, the embolism of \ntrees is more severe LRAs because higher VPD and lower water availability increase the risk of embolism (Breshears et al., \n2013). This indicates that mature (>24 year) plantations on the LOP are already facing a great risk of dieback and even death \ndue to embolism, especially in semi-arid areas. combined occurrence of hydraulic failure and carbon starvation (Arend et al., 2021). Our results show that branches of \nsimilar aged trees that causes the DSM to reach the degree and depth limitation suffered embolism to different degrees (Fig. 375 \n7), which is consistent with the hypothesis about the hydraulic conductivity of planted trees. Furthermore, the embolism of \ntrees is more severe LRAs because higher VPD and lower water availability increase the risk of embolism (Breshears et al., \n2013). This indicates that mature (>24 year) plantations on the LOP are already facing a great risk of dieback and even death \ndue to embolism, especially in semi-arid areas. 5 Conclusions \n400 Deep soil moisture plays an important role in maintaining the survival of deep-rooted plantations during prolonged dry \nperiods on the water-limited Loess Plateau. Our results show that the lowest deep soil moisture deficit (DSMD) was close to Deep soil moisture plays an important role in maintaining the survival of deep-rooted plantations during prolonged dry \nperiods on the water-limited Loess Plateau. Our results show that the lowest deep soil moisture deficit (DSMD) was close to 17 https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/hess-2023-12\nPreprint. Discussion started: 31 January 2023\nc⃝Author(s) 2023. CC BY 4.0 License. the DSMD at the permanent wilting point (PWP), irrespective of tree species and site. This confirms that PWP is a good \nindicator of the degree limitation of DSM for apple and black locust trees. The corresponding maximum water consumption \ndepth reached 18.0-22.0 m for apple trees at different sites, and 18.4-18.8 m for black locust in Changwu and Yan’an while it \n405 \nwas more than 25 m in the drier site of Mizhi, thus relying on the availability of DSM and the ability of deep roots to \ntransport water. Furthermore, when the degree and depth limitation thresholds were reached, the percentage loss of hydraulic \nconductivity of planted trees’ branches was between 74.9% and 96.5%, indicating that dieback and tree death may occur. Our \nstudy provides insights into the management and sustainable development of revegetated sites on the water-limited regions \nincluding the Loess Plateau. 410 the DSMD at the permanent wilting point (PWP), irrespective of tree species and site. This confirms that PWP is a good \nindicator of the degree limitation of DSM for apple and black locust trees. The corresponding maximum water consumption \ndepth reached 18.0-22.0 m for apple trees at different sites, and 18.4-18.8 m for black locust in Changwu and Yan’an while it \n405 \nwas more than 25 m in the drier site of Mizhi, thus relying on the availability of DSM and the ability of deep roots to \ntransport water. Furthermore, when the degree and depth limitation thresholds were reached, the percentage loss of hydraulic \nconductivity of planted trees’ branches was between 74.9% and 96.5%, indicating that dieback and tree death may occur. Our \nstudy provides insights into the management and sustainable development of revegetated sites on the water-limited regions \nincluding the Loess Plateau. 410 Data availability The basic data, including extracted and measured set of soil moisture, environmental factors, and percentage loss of \nhydraulic conductivity of trees’ branches, are available for download at https://doi.org/10.5281/zenodo.7412472. References \n425 \nAhmed, M. A., Kroener, E., Holz, M., Zarebanadkouki, M., and Carminati, A.: Mucilage exudation facilitates root water \nuptake in dry soils, Func. plant boil., 41(11), 1129-1137, 10.1071/FP13330, 2014. Acknowledgments \n420 Acknowledgments \n420 \nThe authors thank Min Yang and Shaofei Wang for their help. This work was jointly supported by the National Natural \nScience Foundation of China (42125705), Natural Science Basic Research Program of Shaanxi Province (2021JC-19), the \nCyrus Tang Foundation, the Shaanxi Key Research and Development Program (2020ZDLNY07-04), Shaanxi Province Key \nR&D Plan (2022NY-064) and Chinese Universities Scientific Fund (2452020242). The authors thank Min Yang and Shaofei Wang for their help. This work was jointly supported by the National Natural \nScience Foundation of China (42125705), Natural Science Basic Research Program of Shaanxi Province (2021JC-19), the \nCyrus Tang Foundation, the Shaanxi Key Research and Development Program (2020ZDLNY07-04), Shaanxi Province Key \nR&D Plan (2022NY-064) and Chinese Universities Scientific Fund (2452020242). References \n425 \nAhmed, M. A., Kroener, E., Holz, M., Zarebanadkouki, M., and Carminati, A.: Mucilage exudation facilitates root water \nuptake in dry soils, Func. plant boil., 41(11), 1129-1137, 10.1071/FP13330, 2014. Author contributions XG and XZ conceived the study; NH, DG, YW and DG performed field experiments and collected the data; NH performed \n415 \nthe analysis and prepared the first draft of the manuscript with the help of LT and LZ; XG edited and commented on the \nmanuscript. The authors declare that they have no conflict of interest. References \n425 18 https://doi.org/10.5194/hess-2023-12\nPreprint. 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https://openalex.org/W4385143920	https://digital-pedagogy.eu/article/JDP_2022_Cismaru_Croce_role_of_video_games.pdf	English	null	Essay on the Role of Video Games in Teaching Literature	Journal of Digital Pedagogy	2,022	cc-by	2,803	Essay on the Role of Video Games in Teaching Literature Giulia-Miruna Cismaru-Croce Giulia-Miruna Cismaru-Croce University of Bucharest, Faculty of Psychology and Education Sciences University of Bucharest, Faculty of Psychology and Education Sciences Abstract: In an era of continuous digitalization, teachers turn to new technology in order to adapt to the universe of their students, to remain relevant in this constant change of interests. Students are motivated by elements of popular culture, being constantly exposed to an environment that is rich in information and textual patterns. And yet, what is the connection between literature and video games? Is there such a connection in the first place? Students need guidance when it comes to developing their literary skills of analysis and communication, as well as literary thinking. Hours dedicated to integrating video games in our educational environment corresponds to students’ passions, highlighting one of their major interest, represented by entertainment in a virtual environment. We can all see this happening when it comes to our students. They can be easily absorbed by any kind of virtual reality that may encounter, but it is so hard to get the same reaction from them when it comes to a classical way of teaching. Until now, reading competences and literary skills were applied to books. But the same skills can be applied to many other mediums in which storytelling plays out the main role, such as comics, movies, and video games. Video games have evolved from a simple means of entertainment to a tool of deep quality learning. Often, students feel that they are a more significant part of a story within a video game that in the environment created by books. Let’s face it! Students love to play, but sometimes they hate to read, especially when they feel forced to do so. The need for teachers to examine video games as literature starts from students’ lack of interest in reading, which comes from the way they lived their lives until now, growing up spending more time playing video games than reading books. This is a reality that we do not only need to accept, but also to adapt to it and to make the best out of it. But how can we use video games to teach literature and develop the analytical skills of our students? i Cismaru-Croce, G.-M. (2022). Essay on the Role of Video Games in Teaching Literature. Journal of Digital Pedagogy, 1(1) 11-14. Bucharest: Institute for Education. https://doi.org/10.61071/JDP.0378 Digital-Pedagogy.eu Institute for Education Journal of Digital Pedagogy ISSN 3008-2021 2022, Vol. 1, No. 1, pp. 11-14 https://doi.org/10.61071/JDP.0378 Digital-Pedagogy.eu Institute for Education Journal of Digital Pedagogy ISSN 3008-2021 2022, Vol. 1, No. 1, pp. 11-14 https://doi.org/10.61071/JDP.0378 Institute for Education Digital-Pedagogy.eu Journal of Digital Pedagogy ISSN 3008-2021 Journal of Digital Pedagogy ISSN 3008-2021 2022, Vol. 1, No. 1, pp. 11-14 https://doi.org/10.61071/JDP.0378 Received: 17.12.2022. Accepted: 20.12.2022 © Giulia-Miruna Cismaru-Croce, 2022. Published by the Institute for Education (Bucharest). This open access article is distributed under the terms of the Creative Commons Attribution Licence CC BY, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: Essay on the Role of Video Games in Teaching Literature Keywords: serious games, teaching literature, video games, digital pedagogy Video games can be seen as interactive stories, most of the time involving the player directly and giving them control over how the game evolves. The game, as a way of interpreting the text, contributes to the development of creativity and to the stimulation of the ability to make decisions based on logical reasoning. The video games are influenced by a primary form of storytelling, but they influence storytelling as well. Many games have their source of inspiration in Received: 17.12.2022. Accepted: 20.12.2022 © Giulia-Miruna Cismaru-Croce, 2022. Published by the Institute for Education (Bucharest). This open access article is distributed under the terms of the Creative Commons Attribution Licence CC BY, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited: ______________________________________ Received: 17.12.2022. Accepted: 20.12.2022 Digital Identity In order to motivate students to be actively involved in the learning process, it is necessary to explore the concept of identity within video games. Popular culture, through its branches represented by media, television, film and the music industry, influences the formation of identity. But video games have the biggest impact on user identity. They encourage identity work and identity-based reflections in clear and meaningful ways. To introduce the concept of gamification, it is necessary to lay the foundations of this approach by creating and analysing a character, an element known as an avatar in the environment of video games. Most activities based on the use of an electronic environment require creating a virtual representation of the user – an avatar, defined by Klevjer (2007) as a replaceable body that allows the player to be present in the fictional world. In education, avatar is a fun and handy way to engage students in the activity, giving them the chance to express themselves and to create a character according to their personal desires and ideals. Going beyond stereotypes and standardized representations, theta are commonly used, limiting the creativity of creating one-self, the student discovers its defining elements, contributing to the creation of a character that will represent him, but will not replace him in the process of learning. Citation: They feel part of the story, of the adventure, and this sense of purpose has a profound effect of motivating them. There is a sense of control that they notice when it comes to playing and this gives them a powerful feeling. But there is also a balance in the ratio between creativity and control. Games promote mistakes as a means of finding creative solutions. And this is what we want our students to learn, creative problem-solving, having a sense of purpose as a guidance, being motivated by wins and not getting discouraged by failures, always trying to do better, to improve their abilities. Citation: Citation: Cismaru-Croce, G.-M. (2022). Essay on the Role of Video Games in Teaching Literature. Journal of Digital Pedagogy, 1(1) 11-14. Bucharest: Institute for Education. https://doi.org/10.61071/JDP.0378 11 ISSN 3008 – 2021, Journal of Digital Pedagogy 1(1) 2022 books, and joining these mediums of presenting common themes can lead students to divergent, complex thinking that enables them to identify defining elements in both environments. books, and joining these mediums of presenting common themes can lead students to divergent, complex thinking that enables them to identify defining elements in both environments. The first method by which we can use video games as a didactic method is represented by the use of materials that teach content. These games use skill collections, practices, values and identities. Teaching through content-based games and, to be more specific, epistemic games, promotes understanding, effective social practice, strong identities. i The first method by which we can use video games as a didactic method is represented by the use of materials that teach content. These games use skill collections, practices, values and identities. Teaching through content-based games and, to be more specific, epistemic games, promotes understanding, effective social practice, strong identities. Another way in which video games are useful to us in our literature classes is represented by their interpretation as texts. We got used to only using texts from books when it came to decoding the meaning behind words and analysis. Written fiction is the most widely used form of text that is meant for interpretation. But limiting ourselves to this type of text limits the very capacity for interpretation. When a student plays a complex narrative game, this involves exploration, rules, taking risks, developing and testing hypotheses, reading words, interpreting sounds and images. What makes gaming such an interesting alternative? In addition to their educational character, the motivational component it develops among students has a significant contribution to the learning process. Students are motivated to achieve their goals and to persevere in their attempts, despite the failures that might occur. Games also develop cooperative problem-solving skills and recognition of the efforts of others. Games are, most of the time, a challenge for children, and they don’t play them because they are simple, but, on the contrary, because they are difficult, causing them to make a sustained effort in achieving their objectives. Exploring Narrative in Video Games The use of video games as literary texts, subject to interpretation and analysis, encourages student participation and connect literary concepts through multimodal learning. To play a game, the player must interact with it, both physically, via controller, mouse or keyboard, as well as intellectually, by the way the narrative is directed. Games provide the participant the chance to identify at an advanced level with the player, or virtual identity. Movies give you the opportunity to see the characters in action and follow their journey, however games allow you to direct this action, to control the character, playing a role that makes you get attached and experience the emotion at a high level. This kind of involvement determines the student to connect deeply to the narrative of the video game. The evolution of narrative starts from oral storytelling, going to written structure, then to the development of various techniques and ideas that facilitate understanding. New media allowed storytelling to evolve and express itself in many ways in a relatively short amount of time. In this new reality of technology, one of the underrated modes of expression is represented by the video games. Video games are a combination between film elements and the innovative structure of graphic novels. They manage to do things related to narrative that no other medium has done before. 12 Giulia-Miruna Cismaru-Croce – Essay on the Role of Video Games in Teaching Literature First, the sense of control you can get from playing is really attractive for players, engaging them on a deeply emotional level. Reading books allows you to take part in the action, but video games can let you create it. This gives the players a new perspective on the relationship between characters and action, which cannot be experienced through written means. Take, for example, the game World of Warcraft, a popular game among teenagers, lacking, at first glance, the necessary depth for decryption. In this game, participants make their choice characters belonging to a fantasy world (elves, dwarves, orcs, mythical creatures) and assume the role of heroes, completing missions, forming organized groups called guilds, in which they interact with other players, earn points and forge a well-established path through the endless Azeroth, an imaginary, fantastic world. The game revolves around missions that challenge players to explore new locations, find certain objects and interact with the presented narrative. Interaction as a Form of Art Video games, often viewed as a childish, time-consuming pastime, manage to offer more than that. This medium carries with it a huge potential for expression. We can perceive video games as a form of literature, thus giving rise to different approaches and some fascinating new stories among students. What does the interaction that a video game proposes gives us? First of all, individual stories, the chance to create a personal story around a character that represents the player, being able to experience the story in an individual way. Also, fascinating frames that can serve as a background or a point where we can focus attention in order to act according to our own goals. A writer’s ability to create a complex spatial setting that can make the player fully engage, where he can explore and create new possibilities regarding his own narrative. The balance between entertainment and the technical side of the game translates into authentically connecting the player to experiencing the narrative. Exploring Narrative in Video Games In the studies conducted on the skills acquired while playing this game by researcher Jonathan Alexander, several fundamental skills were noted, these being: critical analysis, multicultural communication, collaborative writing, reflection on the relationship between the skills required in the game and those in real life. The game’s narrative can be deciphered in parallel with certain books that allow students to make certain connections at the content level. An example of a book that can be used for this purpose is The Hobbit by J.R.R. Tolkien. Students can draw parallels between the characters in the book and the characters in the game, as well as write short stories based on these characters to explore various themes. In the this way, they engage on a deep level, enrich their vocabulary and develop creativity. Some of the literary references that can be found in this game are Alice in Wonderland ( Noggenfogger Elixir), The Bible, British Poetry, Crime and Punishment, Don Quixote, 20,000 Leagues Under the Sea and many others. Analysis and Critical Thinking To truly understand the fundamentals of any video game, we need some careful and detailed analysis of the constituent elements. What can we really analyse within a video game and what literary skills can we use in this endeavour? As in any literary text, we start with the theme of the game. Theme plays a fundamental role within any environment, influencing every character, every part of the narrative, the spatial and temporal elements. We can also focus our attention to symbolism, to identify meaningful symbolic elements, chosen in order to successfully represent abstract ideas or fundamental concepts. Characters can be characterized in various ways, we can use archetypes to highlight their defining features. Action is often marked by conflict, which we can capitalize on by unfolding narrative threads in new directions, meant to expand the viewer’s universe of expectation. In order to evaluate and analyse a text belonging to a video game, we need tools that can help us identify the genre and through which we can contribute by adding critical value. We must keep in mind the figurative representations of the games, the intertextual references, the procedural and fictional adaptations of them, for being able to penetrate beyond the universe of meanings. Among the fundamental qualities that video games develop in their players are critical thinking, confidence in one’s own decisions and inner voice, and insight in terms of emotions and motivation, but also when it comes to the actions that are taken. So what can we expect our students to gain through the use of video games in the study of literature and composition? The answer is that video games are powerful environments where players are given the opportunity to 13 ISSN 3008 – 2021, Journal of Digital Pedagogy 1(1) 2022 see the world through the eyes of others, being simultaneously the heroes of their own story, by making decisions on behalf of the character that they represent. In this way, they are given the freedom to explore on their own, to act and influence the course of the narrative. They are also required to find solutions to the problems that arise, to make full use of critical thinking in order to make their way through the story. The narrative becomes a frame of the development of individuality, of creativity. Analysis and Critical Thinking The story makes them part of a fascinating universe, engaging them in activities with clear goals that allow them to develop their own way of acting, having control over the world around them. The game becomes a multimodal space, in which the player constantly interacts with others, with the story and with his own way of thinking, trying different approaches, exploring new paths and learning more about himself. see the world through the eyes of others, being simultaneously the heroes of their own story, by making decisions on behalf of the character that they represent. In this way, they are given the freedom to explore on their own, to act and influence the course of the narrative. They are also required to find solutions to the problems that arise, to make full use of critical thinking in order to make their way through the story. The narrative becomes a frame of the development of individuality, of creativity. The story makes them part of a fascinating universe, engaging them in activities with clear goals that allow them to develop their own way of acting, having control over the world around them. The game becomes a multimodal space, in which the player constantly interacts with others, with the story and with his own way of thinking, trying different approaches, exploring new paths and learning more about himself. Further readings Lucas, B. (2017). Teaching Creative Thinking: Developing learners who generate ideas and can think critically (Pedagogy for a Changing World) youtube.com/watch?v=4qlYGX0H6Ec futurelearn.com/courses/teaching-digital-literacies-video-games-in-education theconversation.com/game-designers-are-beating-teachers-at-their-own-game-31398 frontiersin.org/articles/10.3389/fcomp.2022.968137/full Lucas, B. (2017). Teaching Creative Thinking: Developing learners who generate ideas and can think critically (Pedagogy for a Changing World) youtube.com/watch?v=4qlYGX0H6Ec Lucas, B. (2017). Teaching Creative Thinking: Developing learners who generate ideas and can think critically (Pedagogy for a Changing World) youtube.com/watch?v=4qlYGX0H6Ec futurelearn.com/courses/teaching-digital-literacies-video-games-in-education ii youtube.com/watch?v=4qlYGX0H6Ec 14
https://openalex.org/W3005946883	https://europepmc.org/articles/pmc7053313?pdf=render	English	null	Tooth loss early in life induces hippocampal morphology remodeling in senescence-accelerated mouse prone 8 (SAMP8) mice	International journal of medical sciences	2,020	cc-by	5,370	Received: 2019.09.12; Accepted: 2020.01.15; Published: 2020.02.10 Received: 2019.09.12; Accepted: 2020.01.15; Published: 2020.02.10 Abstract Long-term tooth loss is associated with the suppression of hippocampal neurogenesis and impairment of hippocampus-dependent cognition with aging. The morphologic basis of the hippocampal alterations, however, remains unclear. In the present study, we investigated whether tooth loss early in life affects the hippocampal ultrastructure in senescence-accelerated mouse prone 8 (SAMP8) mice, using transmission electron microscopy. Male SAMP8 mice were randomized into control or tooth-loss groups. All maxillary molar teeth were removed at 1 month of age. Hippocampal morphologic alterations were evaluated at 9 months of age. Tooth loss early in life induced mitochondrial damage and lipofuscin accumulation in the hippocampal neurons. A thinner myelin sheath and decreased postsynaptic density length were also observed. Our results revealed that tooth loss early in life may lead to hippocampal ultrastructure remodeling and subsequent hippocampus-dependent cognitive impairment in SAMP8 mice with aging. Key words: tooth loss, hippocampus, transmission electron microscopy, mitochondria, myelin sheath, synapse Ivyspring International Publisher Ivyspring International Publisher International Journal of Medical Sciences 2020; 17(4): 517-524. doi: 10.7150/ijms.40241 Tooth loss early in life induces hippocampal morphology remodeling in senescence-accelerated mouse prone 8 (SAMP8) mice Masahisa Katano1, Kyoko Kajimoto1, Mitsuo Iinuma1, Kagaku Azuma2, Kin-ya Ku 1. Department of Pediatric Dentistry, Asahi University School of Dentistry, 1851 Hozumi, Mizuho, Gifu, 501-0296, Japan 2 Department of Anatomy School of Medicine University of Occupational and Environmental Health 1-1 Iseigaoka Yahatanishi-ku Kitakyushu Fukuoka . Department of Pediatric Dentistry, Asahi University School of Dentistry, 1851 Hozumi, Mizuho, Gifu, 501-0296, Japan 2 D t t f A t S h l f M di i U i it f O ti l d E i t l H lth 1 1 I i k Y h t p y y y p . Department of Anatomy, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatan 807-8555, Japan n Life Science, Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya, Aichi, 467-8610, Japan . Graduate School of Human Life Science, Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya, Aichi, 467-8610,  Corresponding author: Kagaku Azuma, Department of Anatomy, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka, 807-8555, Japan. Tel. +81-93-691-7418; Fax. +81-93-691-8544; E-mail address: kazuma@med.uoeh-u.ac.jp © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. 517 517 Int. J. Med. Sci. 2020, Vol. 17 Ivyspring International Publisher Introduction dementia and Alzheimer’s disease [2, 3, 6]. Previous studies revealed that long-term tooth loss, representing a chronic psychologic stressor, induces stress reactions, activates the hypothalamic-pituitary- adrenal axis, and stimulates the adrenal cortex to secrete excess glucocorticoids, corticosterone in rodents and cortisol in humans [7-10]. Animal experi- ments demonstrate that the blood corticosterone concentration in animals with long-term tooth loss is significantly higher than that in controls [11, 12]. The lipophilic nature of glucocorticoids facilitates their rapid entry into the brain. The hippocampus contains the highest level of glucocorticoid receptors, making it a target of stress hormone effects [5, 13]. Mice and rats with tooth loss exhibit impaired hippocampal neurogenesis as well as impaired spatial learning and memory in the Morris water maze test [12, 14-16]. The The rapid growth of elderly populations is associated with an increase in the number of people with senile dementia throughout the world. Currently, 47 million patients have dementia and the number will increase to 75 million globally by 2030. Dementia is a major health and socioeconomic problem [1]. Understanding the pathogenesis of dementia will facilitate the development of novel therapeutic strategies. Numerous studies indicate an association between masticatory dysfunction and cognitive impairment [2-4]. Teeth are important for mastication. Masticatory activity is crucial for overall nutrition, general health, and hippocampus-dependent cogni- tion [4, 5]. The prevalence of tooth loss increases with age. Impaired masticatory function induced by tooth loss is a risk factor for cognitive impairment, such as http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 518 immunosorbent assay kit (AssayPro, Saint Charles, MO, USA), as previously reported [21, 22]. immunosorbent assay kit (AssayPro, Saint Charles, MO, USA), as previously reported [21, 22]. effect of early tooth loss on the hippocampal morpho- logy during aging, however, is largely unknown. Identifying the effects of long-term tooth loss on hippocampal structural remodeling will improve our understanding of the relationship between mastica- tory activity and hippocampus-dependent cognition in the elderly. Transmission electron microscopy Mice were perfused through the aorta with 0.9% sodium chloride, followed by 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were dissected and removed from the skulls quickly and carefully, and immersed further in the same fixative overnight at 4℃. After rinsing with phosphate buffer, the brain tissues were then post-fixed with 1% aqueous osmium tetroxide solution for 1 h. The tissues were then dehydrated by passing through a graded series of acetone. Specimens were embedded using epoxy resin. The ultra-thin sections were prepared with a diamond knife on a Porter-Blum MT-1 ultramicrotome (Ivan Sorvall, Inc., Norwalk, CT, USA), stained with uranyl acetate and lead salts, and observed under a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan). Animals and experimental design SAMP8 mice obtained from Japan SLC (Hamamatsu, Shizuoka, Japan) were used. Mice were maintained on standard rodent pellet chow (CE-2, CLEA Japan, Inc., Tokyo, Japan) available ad libitum. Mice were housed in plastic cages under controlled temperature and humidity (23 ± 1°C, 55 ± 5%) with a 12-h light-dark cycle. The experimental protocol complied with the guidelines for laboratory animal care and use of Asahi University. Mice were randomly divided into control and tooth-loss groups (n=7). Extraction of the maxillary molar teeth was performed as previously described [7, 11, 12, 19]. Briefly, 1-month-old male mice were anesthetized with sodium pentobarbital and all maxillary molar teeth were removed using dental tweezers. The development of mouse teeth continues up to postnatal day 25 [20]. To ensure complete tooth removal, we extracted the molar teeth at 1 month of age. Control mice underwent the same anesthesia procedures, but their maxillary molar teeth were not removed. After surgery, the animals were raised under standard conditions for 8 months. The average daily food intake and body weights were measured once per month. p (J J y J p ) The areas of the cytoplasm, mitochondria, and lipofuscin in the pyramidal neurons of the hippocampal CA3 region were determined using imaging software (CellSens, Olympus Corporation, Tokyo, Japan). Ten images per animal were selected and the volume density of the cytoplasm occupied by mitochondria and lipofuscin was estimated. The volume density of mitochondria and lipofuscin was determined according to standard stereological principles described previously [23]. The number of lipofuscin granules per neuron and the number of microtubules per axon were also calculated. Myelin structures in the CA3 region were quantitatively analyzed in 10 images per animal, which in total contained 210 axons. The G-ratio of the inner axonal diameter to the outer diameter was determined on the same myelin axonal structures as described previously [24, 25]. We identified synapses through the existence of synaptic vesicles within the presynaptic terminals and the postsynaptic density (PSD) in the postsynaptic elements. Measurement of PSD length was carried out using a previously described method [25, 26]. Fifty synapses per animal were counted. Light microscopy Mice were given an overdose of sodium pentobarbital and perfused with 0.9% sodium chloride, followed by 4.0% paraformaldehyde through the aorta. The brains were carefully removed and immersed further in the same fixative overnight at 4℃. The brains were coronally sectioned at 10 µm using a cryostat. Sections were stained cresyl violet for histological observation. The present study examined the effects of tooth loss early in life on the hippocampal ultrastructure in senescence-accelerated mouse strain P8 (SAMP8) mice. SAMP8 mice exhibit age-related behavioral and morphologic alterations, as well as spatial learning deficits [12, 17] . The SAMP8 mouse is a murine model of senile dementia [18]. In order to explore the underlying mechanisms of the senile dementia, we examined the influence of long-term tooth loss on hippocampal morphologic remodeling in aged SAMP8 mice. Blood corticosterone analysis At 9 months of age, all mice were injected intraperitoneally with an overdose of sodium pentobarbital. The blood was extracted at the beginning of the dark cycle around 20:00, when the mouse blood corticosterone level is highest [16]. The serum corticosterone concentration was measured using a mouse corticosterone enzyme-linked http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 519 Figure 1. The daily food intake (A) and body weight (B) in the control and tooth-loss mice. There were no significant differences between the control and tooth-loss mice throughout the experimental period. Figure 1. The daily food intake (A) and body weight (B) in the control and tooth-loss mice. There were no significant differences between the control and tooth-loss mice throughout the experimental period. ) and body weight (B) in the control and tooth-loss mice. There were no significant differences between the control and tooth-loss riod Figure 2. The serum corticosterone level in the control and tooth-loss mice.The serum corticosterone level was significantly higher in the tooth-loss mice than in the control mice. p < 0.05. differences between the control and tooth-loss groups throughout the experimental period (Fig. 1). The serum corticosterone levels in both the control and tooth-loss groups are shown in Fig. 2. The serum corticosterone level was significantly higher in the tooth-loss group than in the control group (Fig. 2). Statistical Analysis All values are reported as means ± standard deviation (SD). Statistical analysis was performed using SPSS version 22. Differences between the control and tooth-loss groups were evaluated using an unpaired t-test. Differences were considered statistically significant at p < 0.05. Lipofuscin is an irregularly shaped structure with varying degrees of electron density. Lipofuscin granules are scattered in the cytoplasm. We observed few lipofuscin granules in the hippocampal neurons of the control mice. Several lipofuscin deposits, however, were observed in the hippocampal neurons of the tooth-loss group (Fig. 4). We calculated the volume density and number of lipofuscin granules per neuron. Compared with the control group, the volume density and number of the lipofuscin granules were significantly higher in the tooth-loss group (Fig. 5B, C). Morphologic features of the hippocampal neurons The general morphology of the hippocampal cornu ammonis (CA1, CA3) regions and the dentate gyrus (DG) was similar between the control and tooth-loss groups (Fig. 3A). Cresyl violet staining revealed that there were no observable morphological differences in the hippocampal CA3 pyramidal cells between the control and tooth-loss groups (Fig. 3B). Neuronal polarity was preserved in the tooth-loss group. The rough endoplasmic reticulum, Golgi complex and mitochondria were well-developed in both groups. We observed numerous swollen mitochondria, missing cristae, and ring-shaped mitochondria in the tooth-loss group (Fig. 4). There was no significant difference in the volume density of mitochondria between the control and tooth-loss groups (Fig. 5A). The rough endoplasmic reticulum and Golgi complex exhibited no marked difference between the control and tooth-loss groups. Figure 2. The serum corticosterone level in the control and tooth-loss mice.The serum corticosterone level was significantly higher in the tooth-loss mice than in the control mice. p < 0.05. Daily food intake, body weight and serum corticosterone levels Figure 4. The ultrastructural features of the mouse hippocampal neurons. There were many mitochondria with intact cristae neurons of the control mice. Numerous lipofuscins, swollen and ring-shaped mitochondria were observed in the tooth-loss mice. Arrow features of the mouse hippocampal neurons. There were many mitochondria with intact cristae and few lipofuscins in the hippocampa merous lipofuscins, swollen and ring-shaped mitochondria were observed in the tooth-loss mice. Arrows: lipofuscins. Scale bars: 1 µm. Daily food intake, body weight and serum corticosterone levels The time course of the average daily food intake and body weight in control and tooth-loss group is shown in Fig. 1. The food intake and body weight of the tooth-loss group tended to decrease for a few days after the extraction and then returned to the preoperative levels. There were no significant http://www.medsci.org 520 Int. J. Med. Sci. 2020, Vol. 17 Figure 3. Light micrographs of the mouse hippocampus (A). The general morphology of the dentate gyrus (DG) and cornu ammonis (CA1, CA3) regions is similar between the control and tooth-loss mice. Scale bars: 1 mm. Light micrographs of the CA3 pyramidal cell layer (B). There are no observable morphological differences in the hippocampal CA3 pyramidal cells between the control and tooth-loss mice. Scale bars: 20 µm. Figure 3. Light micrographs of the mouse hippocampus (A). The general morphology of the dentate gyrus (DG) and cornu ammo etween the control and tooth-loss mice. Scale bars: 1 mm. Light micrographs of the CA3 pyramidal cell layer (B). There are no obse n the hippocampal CA3 pyramidal cells between the control and tooth-loss mice. Scale bars: 20 µm. of the mouse hippocampus (A). The general morphology of the dentate gyrus (DG) and cornu ammonis (CA1, CA3) regions is simila ss mice. Scale bars: 1 mm. Light micrographs of the CA3 pyramidal cell layer (B). There are no observable morphological difference al cells between the control and tooth-loss mice. Scale bars: 20 µm. Figure 3. Light micrographs of the mouse hippocampus (A). The general morphology of the dentate gyrus (DG) and cornu ammonis (CA1, CA3) regions is similar between the control and tooth-loss mice. Scale bars: 1 mm. Light micrographs of the CA3 pyramidal cell layer (B). There are no observable morphological differences in the hippocampal CA3 pyramidal cells between the control and tooth-loss mice. Scale bars: 20 µm. Figure 4. The ultrastructural features of the mouse hippocampal neurons. There were many mitochondria with intact cristae and few lipofuscins in the hippocampal neurons of the control mice. Numerous lipofuscins, swollen and ring-shaped mitochondria were observed in the tooth-loss mice. Arrows: lipofuscins. Scale bars: 1 µm. Figure 4. The ultrastructural features of the mouse hippocampal neurons. There were many mitochondria with intact cristae and few lipofuscins in the hippocampal neurons of the control mice. Numerous lipofuscins, swollen and ring-shaped mitochondria were observed in the tooth-loss mice. Arrows: lipofuscins. Scale bars: 1 µm. Myelin sheath and PSD length Microtubules play a critical role in transporting neurotransmitters within the axons of target neurons. Axons were filled with numerous microtubules arranged in parallel (Fig. 6). The morphologic features of the microtubules, such as microtubule number, length, orientation, and distribution, were similar between groups (Fig. 6A). Quantitative analysis revealed that there was no significant difference in the number of microtubules between the control and tooth-loss groups (Fig. 6B). Myelinated fibers in the central nervous system are essential for neuronal communication and are involved in neurotransmission [27]. The thickness of the myelin sheath is a determinant of neuronal conduction velocity [28]. Myelinated nerve fibers in the control mice appeared normal, with a concentric dense multi-lamellar structure (Fig. 7). There were numerous irregular myelin sheaths in the tooth-loss mice (Fig. 7A). Myelin sheaths were structured http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 521 average PSD length was significantly shorter in tooth-loss mice (Fig. 8A, B). loosely and disordered in texture. Some myelin sheaths in the tooth-loss mice had a collapsed, disrupted, and disordered configuration. Quantitative analysis showed that the G-ratio was significantly higher in the tooth-loss mice than in the control mice, revealing a thinner myelin sheath in the tooth-loss mice (Fig. 7B). Discussion 2020, Vol. 17 Figure 7. Electron micrographs of the mouse hippocampal myelin sheath (A) and the G-ratio (B). The myelin sheaths were structured loosely and disordered in texture in the tooth-loss mice. The G-ratio was significantly higher in the tooth-loss mice, revealing a thinner myelin sheath. Scale bars: 1 µm. p < 0.05. Figure 7. Electron micrographs of the mouse hippocampal myelin sheath (A) and the G-ratio (B). The myelin sheaths were structured loosely and disordered in texture in the tooth-loss mice. The G-ratio was significantly higher in the tooth-loss mice, revealing a thinner myelin sheath. Scale bars: 1 µm. p < 0.05. Figure 8. Electron micrographs of the mouse hippocampal synapses (A) and the PSD length (B). Compared with the control mice, the average PSD length was significantly shorter in the tooth-loss mice. Scale bars: 100 nm. p < 0.01. Figure 8. Electron micrographs of the mouse hippocampal synapses (A) and the PSD length (B). Compared with the con significantly shorter in the tooth-loss mice. Scale bars: 100 nm. p < 0.01. hippocampal synapses (A) and the PSD length (B). Compared with the control mice, the average PSD length was s: 100 nm. p < 0.01. Previous studies showed that tooth loss early in life induced an increase in the corticosterone level, consistent with the present finding. Tooth loss early in life reduces spine density and synaptophysin expression in the hippocampus, and leads to impaired hippocampus-dependent cognition with aging [7, 10, 12, 14]. are involved in the regulation of the hippocampal neurogenesis and synaptic plasticity [11, 33]. Animal studies indicated that tooth loss decreased hippocampal BDNF expression levels both in C57BL/6J and SAMP8 mice [11, 33]. We consider that the effect of tooth loss on the hippocampus was similar for genetically different inbred murine strains. There is no consensus on the effects of tooth loss on body weight. Kawahata et al. extracted molar teeth at 8 weeks of SAMP8 mice and found the body weights decreased significantly from 10 to 24 weeks after surgery [10]. Several other studies, however, demonstrated that tooth loss in SAMP8 mice may lead to temporary weight reduction immediately after tooth loss, with no significant change in body weight The adult hippocampal neurogenesis is important for learning and memory [11, 12, 32]. Immuno fluorescence study demonstrated that tooth loss inhibited the hippocampal neurogenesis both in the normal CD-1 mice and SAMP8 mice [11, 12, 32]. Discussion This is the first study to investigate hippocampal ultrastructural changes induced by long-term tooth loss in aged SAMP8 mice. In the present study, we found that the main characteristics of the ultra- structural remodeling in hippocampus of tooth-loss mice were mitochondrial damage, lipofuscin accumulation, thinning of the myelin sheath and shortening of postsynaptic density. These findings indicate that hippocampal structural remodeling can be induced by tooth loss early in life. Alterations of synapse size are linked to neurodevelopmental disorders [29]. We determined the synapse size by measuring the PSD length, which may be associated with alterations in synaptic efficacy, synaptic plasticity, and intellectual disability [26, 30, 31]. A representative ultrastructure of the hippocampal synapses in SAMP8 mice is shown in Figures 6. Compared with the control mice, the Figure 5. Volume density of mitochondria (A) and lipofuscin (B) in the cytoplasm, and the average lipofuscin number (C) per neuron of the mouse hippocampus. There was no significant difference in the volume density of mitochondria between the control and tooth-loss mice. Compared with the control group, the volume density and number of lipofuscin granules were significantly higher in the tooth-loss mice. p < 0.01. Figure 5. Volume density of mitochondria (A) and lipofuscin (B) in the cytoplasm, and the average lipofuscin number (C) per neuron of the mouse hippocampus. There was no significant difference in the volume density of mitochondria between the control and tooth-loss mice. Compared with the control group, the volume density and number of lipofuscin granules were significantly higher in the tooth-loss mice. **p < 0.01. Figure 6. Electron micrographs of the mouse hippocampal microtubules (A) and the average number of microtubules per axon (B). Axons were filled with numerous microtubules arranged in parallel. The morphologic features of the microtubules are similar in the control and tooth-loss mice. Quantitative analysis revealed that there was no significant difference in the number of microtubules between the control and tooth-loss mice. Arrows: microtubules. Scale bars: 1 µm. Figure 6. Electron micrographs of the mouse hippocampal microtubules (A) and the average number of microtubules per axon (B). Axons were filled with numerous microtubules arranged in parallel. The morphologic features of the microtubules are similar in the control and tooth-loss mice. Quantitative analysis revealed that there was no significant difference in the number of microtubules between the control and tooth-loss mice. Arrows: microtubules. Scale bars: 1 µm. http://www.medsci.org 522 Int. J. Med. Sci. Discussion Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which is widely expressed in the brain. The hippocampal BDNF levels http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 523 thereafter [12, 16, 34]. In the present study, we observed that after surgery, the daily food intake and body weight of the tooth-loss group tended to decrease compared with that of the control group. However, there were no significant differences between the two groups throughout the study. Abnormal myelin sheaths, including thinning, collapse, disruption, as well as disordered arrangement of myelin sheaths could result in decreased neuronal conduction [24]. Structural alterations of hippocampal myelin sheaths are associated with slowed impulse conduction between the hippocampus and other brain regions, triggering hippocampus-dependent cognitive impairment [25, 40]. Like mitochondria, myelin sheath is selectively vulnerable to oxidative stress [41]. The central myelin sheaths are produced by oligodendrocytes. We previously demonstrated that stress exposure affects the hippocampal oligodendrocytes and myelin sheaths [25]. We speculate that the abnormal myelin sheaths are linked to oligodendrocyte degeneration in long-term tooth-loss SAMP8 mice, although we did not evaluate oligodendrocytes in this study. Mitochondrial dysfunction is a common characteristic of aging and neurodegenerative disorders [35, 36]. Improvement of mitochondrial function could be a strategy to slow aging and age-related neurodegenerative diseases. In the present study, we identified the presence of abnormal mitochondria in the hippocampus of tooth-loss mice. The morphology of other organelles, including the rough endoplasmic reticulum and Golgi complex, as well as the plasma membrane remained unaltered in the hippocampal neurons of tooth-loss mice, suggesting that mitochondria is more vulnerable to damage caused by tooth loss. Synapses are crucial for transmitting nerve signals. Synaptic plasticity plays an essential role in learning and memory [42]. The PSD is a complex molecular assembly below the postsynaptic membrane. Induction of long-term potentiation is related to increases in PSD length [43]. PSD length is a crucial parameter for synaptic plasticity and neurobehavioral assessment [30]. We found that long-term tooth loss shortened the hippocampal PSD length, and impaired synaptic plasticity, leading to hippocampus-dependent learning deficits. Lipofuscin deposits, as a marker of aging, are often observed in hippocampal neurons of the aged animals [37, 38]. Lipofuscin deposits are observed in the hippocampal neurons of aged SAMP8 mice, particularly after kainite administration. Senile SAMP8 mice are highly vulnerable to oxidative stress. References Kausar S, Wang F, Cui H. The role of mitochondria in reactive oxygen species generation and its implications for neurodegenerative diseases. Cells. 2018;7:274. 8. Levine S. Developmental determinants of sensitivity and resistance to stress. Psychoneuroendocrinology. 2005;30:939-946. 40. Fan LW, Chen RF, Mitchell HJ, et al. alpha-Phenyl-n-tert-butyl-nitrone attenuates lipopolysaccharide-induced brain injury and improves neurological reflexes and early sensorimotor behavioral performance in juvenile rats. J Neurosci Res. 2008;86:3536-3547. 9. Aoki H, Kimoto K, Hori N, et al. Molarless condition suppresses proliferation but not differentiation rates into neurons in the rat dentate gyrus. Neurosci Lett. 2010;469:44-48. 10. Kawahata M, Ono Y, Ohno A, et al. Loss of molars early in life develops behavioral lateralization and impairs hippocampus-dependent recognition memory. BMC Neurosci. 2014;15:4. j 41. Bradl M, Lassmann H. Oligodendrocytes: biology and pathology. Acta Neuropathol. 2010;119:37-53. 42. Muller D, Nikonenko I, Jourdain P, Alberi S. LTP, memory and structural plasticity. Curr Mol Med. 2002;2:605-611. 11. Kondo H, Kurahashi M, Mori D, et al. Hippocampus-dependent spatial memory impairment due to molar tooth loss is ameliorated by an enriched environment. Arch Oral Biol. 2016;61:1-7. 43. Sheng M, Kim E. The postsynaptic organization of synapses. Cold Spring Harb Perspect Biol. 2011; 3:a005678. 12. Kubo KY, Murabayashi C, Kotachi M, et al. Tooth loss early in life suppresses neurogenesis and synaptophysin expression in the hippocampus and impairs learning in mice. Arch Oral Biol. 2017;74:21-27. 44. Kapitein LC, Hoogenraad CC. Building the Neuronal Microtubule Cytoskeleton. Neuron. 2015; 87:492-506. 45. Eskandari Sedighi G, Riazi GH, Vaez Mahdavi MR, et al. Chronic, long-term social stress can cause decreased microtubule protein network activity and dynamics in cerebral cortex of male Wistar rats. J Mol Neurosci. 2015;55:579-586. g 13. De Kloet ER, Vreugdenhil E, Oitzl MS, Joëls M. Brain corticosteroid receptor balance in health and disease. Endocr Rev. 1988;19:269-301. 14. Onozuka M, Watanabe K, Mirbod SM, et al. Reduced mastication stimulates impairment of spatial memory and degeneration of hippocampal neurons in aged SAMP8 mice. Brain Res. 1999;826:148-153. g 15. Watanabe K, Tonosaki K, Kawase T, et al. Evidence for involvement of dysfunctional teeth in the senile process in the hippocampus of SAMP8 mice. Exp Gerontol. 2001;36:283-295. p 16. Onozuka M, Watanabe K, Fujita M, et al. Evidence for involvement of glucocorticoid response in the hippocampal changes in aged molarless SAMP8 mice. Behav. Brain Res. 2002;131:125-129. 17. Takeda T. Senescence-accelerated mouse (SAM) with special references to neurodegeneration models, SAMP8 and SAMP10 mice. Neurochem Res. References g adult mice. Neural Regen Res. 2014;9:1606-1609. 33. Takeda Y, Oue H, Okada S, et al. Molar loss and powder diet leads to memory deficit and modifies the mRNA expression of brain-derived neurotrophic factor in the hippocampus of adult mice. BMC Neurosci. 2016;17:81. 1. Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011;7:137 152 1. Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nat Rev Neurol. 2011;7:137-152. Neurol. 2011;7:137-152. 2. Lexomboon D, Trulsson M, Wårdh I, Parker MG. Chewing ability and tooth loss: association with cognitive impairment in an elderly population study. J Am Geriatr Soc. 2012;60:1951-1956. 2. Lexomboon D, Trulsson M, Wårdh I, Parker MG. Chewing ability and tooth loss: association with cognitive impairment in an elderly population study. J Am Geriatr Soc. 2012;60:1951-1956. pp p 34. Jiang QS, Liang ZL, Wu MJ, et al. Reduced brain-derived neurotrophic factor expression in cortex and hippocampus involved in the learning and memory deficit in molarless SAMP8 mice. Chin Med J (Engl). 2011;124:1540-1544. 3. Teixeira FB, Pereira Fernandes Lde M, Noronha PA, et al. Masticatory deficiency as a risk factor for cognitive dysfunction. Int J Med Sci. 2014;11:209-214. 35. Wang Y, Xu E, Musich PR, Lin F. Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure. CNS Neurosci Ther. 2019;25:816-824. 4. Chen H, Iinuma M, Onozuka M, Kubo KY. Chewing maintains hippocampus-dependent cognitive function. Int J Med Sci. 2015;12:502-509. 36. Perez Ortiz JM, Swerdlow RH. Mitochondrial dysfunction in Alzheimer's disease: Role in pathogenesis and novel therapeutic opportunities. Br J Pharmacol. 2019;176:3489-3507. 5. Azuma K, Zhou Q, Niwa M, Kubo KY. Association between mastication, the hippocampus, and the HPA axis: A comprehensive review. Int J Mol Sci. 2017;18:1687. 37. Kim HC, Bing G, Jhoo WK, et al. Oxidative damage causes formation of lipofuscin-like substances in the hippocampus of the senescence-accelerated mouse after kainate treatment. Behav Brain Res. 2002;131:211-220. 6. Shimazaki Y, Soh I, Saito T, et al. Influence of dentition status on physical disability, mental impairment, and mortality in institutionalized elderly people. J Dental Res. 2001;80:340-345. mouse after kainate treatment. Behav Brain Res. 2002;131:211-220. 38. Richardson JC, Kendal CE, Anderson R, et al. Ultrastructural and behavioural changes precede amyloid deposition in a transgenic model of Alzheimer's disease. Neuroscience. 2003;122:213-228. p p 7. Kubo KY, Iwaku F, Watanabe K, Fujita M, Onozuka M. Molarless-induced changes of spines in hippocampal region of SAMP8 mice. Brain Res. 2005;1057:191-195. 39. Acknowledgements 26. Hara Y, Rapp PR, Morrison JH. Neuronal and morphological bases of cognitive decline in aged rhesus monkeys. Age. 2012;34:1051-1073. g g y g 27. Ye Y, Xiong J, Hu J, et al. Altered hippocampal myelinated fiber integrity in a lithium-pilocarpine model of temporal lobe epilepsy: a histopathological and stereological investigation. Brain Res. 2013;1522:76-87. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI 16H05560) form the Japan Society of the Promotion of Science (JSPS). g g 28. Michailov GV, Sereda MW, Brinkmann BG, et al. Axonal neuregulin-1 regulates myelin sheath thickness. Science. 2004;304:700-703. 29. Fiala JC, Spacek J, Harris KM. Dendritic spine pathology: cause or consequence of neurological disorders? Brain Res Rev. 2002;39:29-54. Discussion Kainite is considered to induce oxidative damage, which might contribute to neuronal lipofuscin accumulation. The accumulation of lipofuscin granules is considered an oxidative stress response. The present study showed that the volume density and number of lipofuscin granules were significantly higher in tooth-loss mice than in control mice. We consider that long-term tooth loss induces chronic stress, and accelerates the hippocampal neuronal aging process, due to the accumulation of lipofuscin granules. Microtubules comprise a major component of the neuronal cytoskeleton, which plays a critical role in facilitating long-distance transport of neurotrans- mitters to synapses [44]. Neurons are highly susceptible to microtubule defects, which occur in various neurodegenerative diseases. Microtubule defects include abnormalities of microtubule number, length, orientation, distribution and bundling. In our work, we observed no morphologic alterations of the microtubules in aged tooth-loss mice. A previous study indicated that chronic social stress decreased the brain microtubule protein network dynamics and polymerization ability in rats [45]. Whether long-term tooth loss affects microtubule protein network activity and dynamics needs to be confirmed. The mitochondrion is an important organelle for maintaining normal cell function, and plays a main role in the response to oxidative stress [39]. Mitochon- drial dysfunction could cause the overproduction of reactive oxygen species, resulting in oxidative stress. As the formation of lipofuscin is involved in oxidative stress, lipofuscin accumulation might be associated with mitochondrial damage. In this study, we observed many abnormal mitochondria, including those without cristae, swollen mitochondria, and ring-shaped mitochondria in the hippocampal neurons of long-term tooth-loss mice. While the morphology of the rough endoplasmic reticulum, Golgi complex, and cytoplasmic membrane remained intact. These findings suggest that the mitochondria are particularly vulnerable to oxidative damage due to long-term tooth loss in aged SAMP8 mice. In conclusion, the present findings indicate that tooth loss early in life causes mitochondrial damage and lipofuscin accumulation in the hippocampal neurons, accompanied by a thinner myelin sheath and decreased postsynaptic density length in the hippocampus of the aged SAMP8 mice. These findings demonstrate that tooth loss early in life can lead to hippocampal ultrastructure remodeling, resulting in hippocampus-dependent cognitive impairment in SAMP8 mice with aging. The central myelin sheaths contribute to the conduction of neuronal impulses in myelinated fibers. http://www.medsci.org Int. J. Med. Sci. 2020, Vol. 17 524 Competing Interests q g 30. Moretti P, Levenson JM, Battaglia F, et al. Learning and memory and synaptic plasticity are impaired in a mouse model of Rett syndrome. J Neurosci. 2006;26:319-327. The authors have declared that no competing interest exists. 31. Watson DJ, Ostroff L, Cao G, et al. LTP enhances synaptogenesis in the developing hippocampus. Hippocampus. 2016;26:560-576. p g pp p pp p 32. Su S, Qi T, Su B, et al. Tooth loss inhibits neurogenesis in the dentate gyrus of adult mice. Neural Regen Res. 2014;9:1606-1609. References 2009;34:639-659. 18. Akiguchi I, Pallàs M, Budka H, et al. SAMP8 mice as a neuropathological model of accelerated brain aging and dementia: Toshio Takeda's legacy and future directions. Neuropathology 2017; 37:293-305. p gy 19. Kurahashi M, Kondo H, Iinuma M, et al. Tooth loss early in life accelerates age-related bone deterioration in mice. Tohoku J Exp Med. 2015;235:29-37. 20. Gaete M, Lobos N, Torres-Quintana MA. Mouse tooth development time sequence determination for the ICR/Jcl strain. J Oral Sci. 2004;46:135-141. 21. Furuzawa M, Chen H, Fujiwara S, et al. Chewing ameliorates chronic stress-induced bone loss in senescence-accelerated mouse (SAMP8), a murine model of senile osteoporosis. Exp Gerontol. 2014;55:12-18. model of senile osteoporosis. Exp Gerontol. 2014;55:12-18 22. Onishi M, Iinuma M, Tamura Y, Kubo KY. Learning deficits and suppression of the cell proliferation in the hippocampal dentate gyrus of offspring are attenuated by maternal chewing during prenatal stress. Neurosci Lett. 2014;560:77-80. 23. Weible ER. Stereological methods. Practical methods for biological morphometry. London-New York-Toronto: Academic Press; 1979. p y 24. Chomiak T, Hu B. What is the optimal value of the g-ratio for myelinated fibers in the rat CNS? A theoretical approach. PLoS One. 2009;4:e7754. p y homiak T, Hu B. What is the optimal value of the g-ratio for mye 25. Suzuki A, Iinuma M, Hayashi S, et al. Maternal chewing during prenatal stress ameliorates stress-induced hypomyelination, synaptic alterations, and learning impairment in mouse offspring. Brain Res. 2016;1651:36-43. http://www.medsci.org http://www.medsci.org
https://openalex.org/W3159660080	http://dogadergi.ksu.edu.tr/tr/download/article-file/1526939	Latin	null	Evalution of Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Species Distributed in Sinop and Kastamonu (Turkey) in Taxonomic Terms	Kahramanmaraş Sütçü İmam Üniversitesi tarım ve doğa dergisi	2,022	cc-by	4,379	ABSTRACT In this study, specimens belonging to Cercopidae collected from Sinop and Kastamonu in 2016-2018 were evaluated. Three species belonging to to genus Cercopis and Triecphorella were identified. General morphological characteristics, drawings of male and female of genital structures, measurements of some body parts, distributional data (Turkey and in the world) and collection localities of the species were given. Brief ecological notes on examples are mentioned. The data about the elytral color/pattern variation of identified species were also examined. To Cite: Tanyeri R, Zeybekoğlu Ü 2022. Evalution of Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Species in Sinop and Kastamonu (Turkey) in Taxonomic Terms. KSU J. Agric Nat 25 (1): 133-139. DOI: 10.18016/ksutarimdoga.vi.866486. To Cite: Tanyeri R, Zeybekoğlu Ü 2022. Evalution of Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Species in Sinop and Kastamonu (Turkey) in Taxonomic Terms. KSU J. Agric Nat 25 (1): 133-139. DOI: 10.18016/ksutarimdoga.vi.866486. KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 DOI:10.18016/ksutarimdoga.vi.866486 KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 DOI:10.18016/ksutarimdoga.vi.866486 Sinop ve Kastamonu (Türkiye)’da Dağılım Gösteren Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Türlerinin Taksonomik Yönden Değerlendirilmesi Sinop ve Kastamonu (Türkiye)’da Dağılım Gösteren Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Türlerinin Taksonomik Yönden Değerlendirilmesi Rukiye TANYERİ1 , Ünal ZEYBEKOĞLU2 1Department of Biology, Faculty of Arts and Science, Sinop University, Sinop, Turkey, 2Department of Biology, Faculty of Arts and Science, Ondokuz Mayıs University, Samsun, Turkey 1https://orcid.org/0000-0001-9994-8783, 2https://orcid.org/0000-0003-1646-5999 : rtanyeri@sinop edu tr Rukiye TANYERİ1 , Ünal ZEYBEKOĞLU2 1Department of Biology, Faculty of Arts and Science, Sinop University, Sinop, Turkey, 2Department of Biology, Faculty of Arts and Science, Ondokuz Mayıs University, Samsun, Turkey 1https://orcid.org/0000-0001-9994-8783, 2https://orcid.org/0000-0003-1646-5999 : rtanyeri@sinop.edu.tr ÖZET Bu çalışmada 2016-2018 yıllarında Sinop ve Kastamonu’dan toplanan Cercopidae familyasına ait örnekler incelenmiştir. Cercopis ve Triechphorella cinslerine ait 3 tür tespit edilmiştir. Tespit edilen türlerin genel morfolojik özellikleri, dişi ve erkek genital yapılarının çizimleri, bazı vücut kısımlarının ölçümleri, Türkiye ve dünyadaki dağılımları verilmiştir. Örneklerle ilgili kısa ekolojik notlardan bahsedilmektedir. Ayrıca tespit edilen türlerin elitral renk/desen varyasyonuna dair veriler de incelenmiştir. Evalution of Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Species Distributed in Sinop and Kastamonu (Turkey) in Taxonomic Terms Research Article Article History Received : 22.01.2021 Accepted : 06.05.2021 Keywords Cercopidae Turkey Taxonomy To Cite: Tanyeri R, Zeybekoğlu Ü 2022. Evalution of Cercopidae (Hemiptera: Auchenorrhyncha: Cicadomorpha) Species in Sinop and Kastamonu (Turkey) in Taxonomic Terms. KSU J. Agric Nat 25 (1): 133-139. DOI: 10.18016/ksutarimdoga.vi.866486. INTRODUCTION vulnerata Rossi, 1807, Haematoloma dorsata (Ahrens, 1812) and Triecphorella geniculata (Horvath, 1881) (Lodos and Kalkandelen, 1988; Önder et al., 2011). intervals between May and July. The spittlebugs were frequently collected from the herbaceous plants in humid habitats near water sources, such as mini- meadows, streamsides and roadsides. In the sampling of the adults, a standard sweeping net and a handheld aspirator were used. The specimens were prepared by standard insect preparation and identified according to (Dusolier, 2004; Holzinger, 2003; 2008) by the first author. An overview photograph of dry samples was taken with Canon EOS 70D model camera connected to Zeiss Stem 2000-C stereomicroscope. The shapes of genital structures were drawn using Zeiss discovery V- 20 stereomicroscope attached drawing attachment. The purpose of this study is to determine the species of Cercopidae distributed in Sinop and Kastamonu provinces in the West Black Sea Region of Turkey. And it has also been intended to evaluate the species in taxonomic terms and take data about the elytral color/pattern variation of the species. MATERIAL and METHOD The specimens were collected from Sinop and Kastamonu provinces in the West Black Sea Region of Turkey between 2016 and 2018 (Figure 1). The sampling of the adults was implemented biweekly The specimens are deposited in Sinop University, Faculty of Arts and Sciences, Department of Biology, Invertebrata Laboratory. Figure 1. Study area Şekil 1. Çalışma alanı Figure 1. Study area Şekil 1. Çalışma alanı 42 ♀♀); 14.05.2017, 41° 53ˈ 05.9̎ N 34° 33ˈ 53.6̎ E (24 ♂♂, 37 ♀♀); 27.05.2017 , 41° 44ˈ 52.0̎ N 34° 57ˈ 40.9̎ E (23 ♂♂, 38 ♀♀), 41° 32ˈ 45.1̎ N34° 47ˈ 0.01̎ E (17 ♂♂, 13 ♀♀), 41° 50ˈ 22.0̎ N 35° 03ˈ 02.2̎ E (75 ♂♂, 54 ♀♀); 01.06.2017 41° 44ˈ 21.9̎ N 35° 13ˈ 53.1̎ E (8 ♂♂, 3 ♀♀); 09.06.2017 42° 00ˈ 52.1̎ N 34° 58ˈ 24.4̎ E (8 ♂♂, 13 ♀♀), 41° 59ˈ 41.7̎ N 34° 54ˈ 00.2̎ E (83 ♂♂, 13 ♀♀) INTRODUCTION The posterior tibia is round, has 1-2 spines. The head is narrower than the pronotum (Holzinger et al., 2003). The posterior tibia is round, has 1-2 spines. The head is narrower than the pronotum (Holzinger et al., 2003). Cercopidae is the most diverse group in the Cercopoidea (Hemiptera: Auchenorrhyncha) superfamily. This family includes xylem-sucking insects containing 1500 species belonging to 150 genera. Since Cercopidae nymphs produce a frothy secretion, the family name is foamy cicadas or spittlebugs and also known as froghoppers because adults have well jumping ability (Cryan and Svenson, 2010). thşs group has a dominant distribution in tropical and subtropical areas of the world. Almost 70% of the identified species have tropical distribution. In most species, adults are characterized by having colorful elytral patterns. Adults feed on leaves and stems of various plants, and nymphs on the roots (Liang and Fletcher, 2002; Liang and Webb, 2002). The family of members is 6-12 mm in size and characterized by their colored elytra. There are two simple eyes on the vertex. The femur is not thick in the front legs. Pronotum is usually flat, slightly curved. Some Cercopidae species have potential to cause significant damage to economically important plants. Some Cercopidae species have potential to cause significant damage to economically important plants. In particular, they cause harm in rice either through by feeding or as a vector of phytoplasmas (Paladini et al., 2018). Several species are known to cause significant damage to forage grasses and sugarcane by feeding (Peck et al., 2001). The first records from our country belonging to the Cercopidae family, which has 93 species in the Palearctic Region, were given by Fieber (1872) and Fahringer (1922). Metcalf (1961), listed 4 species from Turkey. Lodos and Kalkandelen (1981), prepared a checklist of Cercopidae family of Turkey. Kartal et al., (1994) conducted a taxonomic study on Cercopidae species in Samsun and its surroundings. Turkey Cercopidae fauna comprises of 7 species: Cercopis distincta (Melichar, 1896), C. intermedia Kirschbaum, 1868, C. sanguinolenta (Scopoli, 1763), C. KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 Araştırma Makalesi Research Article septemmaculata (Melichar, 1903), C. vulnerata Rossi, 1807, Haematoloma dorsata (Ahrens, 1812) and Triecphorella geniculata (Horvath, 1881) (Lodos and Kalkandelen, 1988; Önder et al., 2011). septemmaculata (Melichar, 1903), C. RESULTS The specimens collected during fieldwork in Sinop and Kastamonu provinces were identified, and it has been determined that 3 species were found in the region belonging to Cercopidae family. One of these Cercopis intermedia Kirschbaum, 1868 is that has a large distribution area in Turkey; the other is Cercopis vulnerata Rossi, 1807 and the third one is Triecphorella geniculata (Horvath, 1881). It was determined that the samples belonging to the Cercopis genus have a high rate of color and pattern variation (Figures 2, 4). Morphometric measurements some body parts of species are shown in Table 1, 2 and 3. With this study, it can be stated that C. intermedia is recorded for the first time from Sinop and Kastamonu, T. geniculata is the first record for the local fauna of Kastamonu. Kastamonu: 18.05.2017 41° 27ˈ 35.2̎ N 33° 39ˈ 38.9̎ E (63 ♂♂, 47 ♀♀); 19.05.2017 41° 42ˈ 08.7̎ N 33° 28ˈ 56.3̎ E (78 ♂♂, 67 ♀♀), 41° 42ˈ 08.4̎ N 33° 26ˈ 37.0̎ E (53 ♂♂, 33 ♀♀), (48 41° 38ˈ 35.8̎ N 33° 07ˈ 13.9̎ E (♂♂, 34 ♀♀), 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E(73 ♂♂, 54 ♀♀), 41° 36ˈ 45.4̎ N 33° 07ˈ 02.9̎ E 41° 58ˈ 25.00̎ N 33° 48ˈ 27.9̎ E (23 ♂♂, 13 ♀♀); 20.05.2017 41° 58ˈ 10.7̎ N 34° 05ˈ 22.7̎ E41° 55ˈ 31.9̎ N 34° 10ˈ 56.2̎ E (54 ♂♂, 33 ♀♀), 41° 41ˈ 34.8̎ N 33° 57ˈ 43.3̎ E (90 ♂♂, 65 ♀♀) Distribution in Turkey: Adıyaman, Amasya, Artvin, Ankara, Antalya, Bitlis, Çanakkale, Çorum, Diyarbakır, Elazığ, Eskişehir, Gaziantep, Giresun, Hakkari, Hatay, Isparta, İstanbul, İzmir, Kahramanmaraş, Kayseri, Konya, Kütahya, Kırklareli, Mardin, Niğde, Rize, Samsun, Siirt, Şanlıurfa, Trabzon, Uşak (Önder et al., 2011; Demir, 2019) Genus: Cercopis Fabricius, 1775 Cercopis intermedia Kirschbaum, 1868 (Figures 2-3) Material Sinop: 18.06.2016, 41° 56' 995" N 34° 48' 238" E (32 ♀♀); 13.05.2017, 41° 52ˈ 31.3̎ N 34° 51ˈ 00.6̎ (15 ♂♂, Genus: Cercopis Fabricius, 1775 Cercopis intermedia Kirschbaum, 1868 (Figures 2-3) Material Sinop: 18.06.2016, 41° 56' 995" N 34° 48' 238" E (32 ♀♀); 13.05.2017, 41° 52ˈ 31.3̎ N 34° 51ˈ 00.6̎ (15 ♂♂, Genus: Cercopis Fabricius, 1775 Cercopis intermedia Kirschbaum, 1868 (Figures 2-3) Material Sinop: 18.06.2016, 41° 56' 995" N 34° 48' 238" E (32 ♀♀); 13.05.2017, 41° 52ˈ 31.3̎ N 34° 51ˈ 00.6̎ (15 ♂♂, Africa, Poland, Portugal, Romania, Russia, Slovakia, Syria, Spain, Switzerland, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021) Material Body length 9.5 10.7 9.9 9 11 9.74 Head width 2 2.3 2.2 2.1 2.5 2.3 Vertex length 0.5 0.7 0.6 0.5 0.7 0.6 Pronotum length 1.4 1.9 1.7 1.6 2 1.7 Mesonotum length 1.2 1.7 1.5 1.4 1.7 1.5 Table 3. Measurements of body parts of Triechphorella geniculata (mm) (N=number of individuals). Çizelge 3. Triechphorella geniculata’nın vücut kısımlarının ölçüleri (mm) (N=birey sayısı). Male (N=31) Female (N=14) Body parts (mm) Min. Max. Ave. Min. Max. Ave. Body length 6 7 6.57 6.5 7.1 6.8 Head width 1.6 1.8 1.7 1.7 1.9 1.8 Vertex length 0.3 0.5 0.46 0.4 0.5 0.48 Pronotum length 1 1.3 1.2 1.2 1.4 1.3 Mesonotum length 1 1.2 1.06 0.9 1.3 1.1 Cercopis vulnerata Rossi, 1807 (Figures 4-5) Material Sinop: 13.05.2017 41° 53ˈ 05.9̎ N 34° 33ˈ 53.6̎ E (6♂♂) Kastamonu: 20.05.201741° 52ˈ 48.9̎ N 33° 42ˈ 38.0̎ E (83 ♂♂, 13 ♀♀), 41° 58ˈ 25.00̎ N 33° 48ˈ 27.9̎ E (52 ♂♂, 28 ♀♀), 41° 55ˈ 31.9̎ N 34° 10ˈ 56.2̎ E (57 ♂♂, 54 ♀♀), 41° 41ˈ 34.8̎ N 33° 57ˈ 43.3̎ E (2 ♂♂, 1 ♀), 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (6 ♀♀); 15.06.2017 41° 52ˈ 49.0̎ N 33° 42ˈ 38.1̎ E (1 ♂); 16.06.2017 41° 34ˈ 34.1̎ N 33° 12ˈ 44.1̎ E (1 ♂), 19.05.2017 41° 38ˈ 35.8̎ N 33° 07ˈ 13.9̎ E (28 ♂♂, 15 ♀♀), 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (1 ♂,1 ♀) 41° 36ˈ 45.4̎ N 33° 07ˈ 02.9̎ E (6 ♂♂, 2 ♀♀). Distribution in Turkey: Antalya, Balıkesir, Bartın, Bursa, Eskişehir, Kastamonu, Kocaeli, Konya, Sakarya, Sinop (Altınayar, 1981; Demir, 2005; 2006; Önder et al., 2011; Tanyeri and Zeybekoğlu, 2020) Zoogeographic distribution: Albania, Austria, Belgium, Britain, Bulgaria, Croatia, Czech Republic, France, Germany, Greece, Hungary, Italy, Moldova, The Netherlands, Norway, Spain, Switzerland, Poland, Romania, Russia (Central Europe), Russia (Southern Europe), Slovakia, Slovenia, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021). Table 1. Measurements of body parts of Cercopis intermedia (N=number of individuals). Çizelge 1. Cercopis intermedia’nın vücut kısımlarının ölçüleri (N= birey sayısı). Table 2. Measurements of body parts of Cercopis vulnerata (N=number of individuals). Çizelge 2. Cercopis vulnerata’nın vücut kısımlarının ölçüleri (N=birey sayısı). Table 2. Measurements of body parts of Cercopis vulnerata (N=number of individuals). Çizelge 2. Cercopis vulnerata’nın vücut kısımlarının ölçüleri (N=birey sayısı). Male (N=30) Female (N=30) Body parts (mm) Min. Max. Ave. Min. Max. Ave. Material Body length 9.5 10.7 9.9 9 11 9.74 Head width 2 2.3 2.2 2.1 2.5 2.3 Vertex length 0.5 0.7 0.6 0.5 0.7 0.6 Pronotum length 1.4 1.9 1.7 1.6 2 1.7 Mesonotum length 1.2 1.7 1.5 1.4 1.7 1.5 Table 3 Measurements of body parts of Triechphorella geniculata (mm) (N=number of individuals) Table 3. Measurements of body parts of Triechphorella geniculata (mm) (N=number of individuals). Çizelge 3. Triechphorella geniculata’nın vücut kısımlarının ölçüleri (mm) (N=birey sayısı). Çizelge 3. Triechphorella geniculata nın vücut kısımlarının ölçüleri (mm) (N=birey sayısı). Male (N=31) Female (N=14) Body parts (mm) Min. Max. Ave. Min. Max. Ave. Body length 6 7 6.57 6.5 7.1 6.8 Head width 1.6 1.8 1.7 1.7 1.9 1.8 Vertex length 0.3 0.5 0.46 0.4 0.5 0.48 Pronotum length 1 1.3 1.2 1.2 1.4 1.3 Mesonotum length 1 1.2 1.06 0.9 1.3 1.1 Cercopis vulnerata Rossi, 1807 41° 36ˈ 45.4̎ N 33° 07ˈ 02.9̎ E (6 ♂♂, 2 ♀♀). 41° 36ˈ 45.4̎ N 33° 07ˈ 02.9̎ E (6 ♂♂, 2 ♀♀). Distribution in Turkey: Antalya, Balıkesir, Bartın, Distribution in Turkey: Antalya, Balıkesir, Bartın, Bursa, Eskişehir, Kastamonu, Kocaeli, Konya, Sakarya, Sinop (Altınayar, 1981; Demir, 2005; 2006; Önder et al., 2011; Tanyeri and Zeybekoğlu, 2020) Material Sinop: 18.06.2016, 41° 56' 995" N 34° 48' 238" E (32 ♀♀); 13.05.2017, 41° 52ˈ 31.3̎ N 34° 51ˈ 00.6̎ (15 ♂♂, 134 Araştırma Makalesi Research Article Africa, Poland, Portugal, Romania, Russia, Slovakia, Syria, Spain, Switzerland, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021) KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 Araştırma Makalesi Research Article Africa, Poland, Portugal, Romania, Russia, Slovakia, Syria, Spain, Switzerland, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021) Zoogeographic distribution: Albania, Algeria, Austria, Bulgaria, Czech Republic, France, Germany, Greece, Hungary, Iran, Israel, Italy, Lebanon, Morocco, North Figure 2. Cercopis intermedia Kirschbaum, 1868 (♂) (scale bars: 1 mm) Şekil 2. Cercopis intermedia Kirschbaum, 1868 (♂) (ölçek çubukları: 1 mm) Figure 2. Cercopis intermedia Kirschbaum, 1868 (♂) (scale bars: 1 mm) Şekil 2. Cercopis intermedia Kirschbaum, 1868 (♂) (ölçek çubukları: 1 mm) Figure 3. Genital structures of Cercopis intermedia a, aedeagus from lateral; b, stylus; C, genital plate; d, the tip of the female abdomen from ventral (scale bars: 0,5 mm) Şekil 3. Cercopis intermedia türünün genital yapıları a, aedegus yandan; b, stilus; C, genital plak; d, dişide abdomenin uç kısmı ventralden (ölçek çubukları: 0,5 mm) Figure 3. Genital structures of Cercopis intermedia a, aedeagus from lateral; b, stylus; C, genital plate; d, the tip of the female abdomen from ventral (scale bars: 0,5 mm) Şekil 3. Cercopis intermedia türünün genital yapıları a, aedegus yandan; b, stilus; C, genital plak; d, dişide abdomenin uç kısmı ventralden (ölçek çubukları: 0,5 mm) 135 KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 Araştırma Makalesi Research Article Table 1. Measurements of body parts of Cercopis intermedia (N=number of individuals). Çizelge 1. Cercopis intermedia’nın vücut kısımlarının ölçüleri (N= birey sayısı). Male (N=50) Female (N=50) Body parts (mm) Min. Max. Ave. Min. Max. Ave. Body length 8 9.55 8.7 8.25 9.50 8.8 Head width 2 2.25 2.15 2.1 2.5 2.2 Vertex length 0.5 0.75 0.6 0.6 0.75 0.7 Pronotum length 1.5 1.75 1.6 1.5 1.7 1.35 Mesonotum length 1.05 1.6 1.25 1.25 1.5 1.35 Table 2. Measurements of body parts of Cercopis vulnerata (N=number of individuals). Çizelge 2. Cercopis vulnerata’nın vücut kısımlarının ölçüleri (N=birey sayısı). Male (N=30) Female (N=30) Body parts (mm) Min. Max. Ave. Min. Max. Ave. Material Sinop: 13.05.2017 41° 53ˈ 05.9̎ N 34° 33ˈ 53.6̎ E (6♂♂) Kastamonu: 20.05.201741° 52ˈ 48.9̎ N 33° 42ˈ 38.0̎ E (83 ♂♂, 13 ♀♀), 41° 58ˈ 25.00̎ N 33° 48ˈ 27.9̎ E (52 ♂♂, 28 ♀♀), 41° 55ˈ 31.9̎ N 34° 10ˈ 56.2̎ E (57 ♂♂, 54 ♀♀), 41° 41ˈ 34.8̎ N 33° 57ˈ 43.3̎ E (2 ♂♂, 1 ♀), 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (6 ♀♀); 15.06.2017 41° 52ˈ 49.0̎ N 33° 42ˈ 38.1̎ E (1 ♂); 16.06.2017 41° 34ˈ 34.1̎ N 33° 12ˈ 44.1̎ E (1 ♂), 19.05.2017 41° 38ˈ 35.8̎ N 33° 07ˈ 13.9̎ E (28 ♂♂, 15 ♀♀), 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (1 ♂,1 ♀) Zoogeographic distribution: Albania, Austria, Belgium, Britain, Bulgaria, Croatia, Czech Republic, France, Germany, Greece, Hungary, Italy, Moldova, The Netherlands, Norway, Spain, Switzerland, Poland, Romania, Russia (Central Europe), Russia (Southern Europe), Slovakia, Slovenia, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021). Figure 4. Cercopis vulnerata Rossi, 1807 (♂) (scale bar: 1 mm) Şekil 4. Cercopis vulnerata Rossi, 1807 (♂) (ölçek çubuğu: 1 mm) Figure 4. Cercopis vulnerata Rossi, 1807 (♂) (scale bar: 1 mm) Şekil 4. Cercopis vulnerata Rossi, 1807 (♂) (ölçek çubuğu: 1 mm) 136 KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 Araştırma Makalesi Research Article Figure 6. Triechphorella geniculata (Horvath, 1881) (♂) (scale bar: 1 mm) Şekil 6. Triechphorella geniculata (Horvath, 1881) (♂) (ölçek çubuğu: 1 mm) Figure 6. Triechphorella geniculata (Horvath, 1881) (♂) (scale bar: 1 mm) Şekil 6. Triechphorella geniculata (Horvath, 1881) (♂) (ölçek çubuğu: 1 mm) Figure 5. Genital structures of Cercopis vulnerata a, aedeagus from lateral; b, stylus; c, genital plate; d, the tip of the female abdomen from ventral (scale bars: 0,5 mm) , Şekil 5. Cercopis vulnerata türünün genital yapıları a, aedegus yandan; b, stilus; c, genital plak; d, dişide abdomenin uç kısmı ventralden (ölçek çubukları: 0,5 mm) Figure 7. Genital structures of Triechphorella geniculata a, aedeagus from ventral; b, aedeagus from lateral; c, stylus; d, genital plate; e, the tip of the female abdomen (scale bars: a, b, c, d: 0,1 mm; d: 0,5 mm) Şekil 7. Triechphorella geniculata türünün genital yapıları a, aedegus ventralden; b, aedegus yandan; c, stilus; d, genital plak; e, dişide abdomenin uç kısmı ventralden (ölçek çubukları: a, b, c, d: 0,1 mm; d: 0,5 mm) Figure 7. Statement of Conflict of Interest Statement of Conflict of Interest There is no conflict of interest between the article authors. According to literature, Cercopis fauna of Turkey consists of 5 species. C. vulnerata distinguished from other Cercopis taxa by following character; in males, the anterior pair of processes on the apex of the aedeagus is two-thirds the length of posterior pair. In C. sanguinolenta and C. intermedia the anterior pair is half the length of the posterior pair. C. sanguinolenta differs from C. intermedia having totally black legs. C. intermedia has red knees (Holzinger et al., 2003). Material Distribution in Turkey: Adana, Amasya, Antalya, Balıkesir, Kahramanmaraş, Konya, Osmaniye, Samsun (Demir, 2005; 2006; Önder et al., 2011; Zeybekoğlu and Karavin, 2010) Zoogeographic distribution: Croatia, Greece, Near East, Turkey, Yugoslavia (Önder et al., 2011; Anonymous, 2021). Material Genital structures of Triechphorella geniculata a, aedeagus from ventral; b, aedeagus from lateral; c, stylus; d, genital plate; e, the tip of the female abdomen (scale bars: a, b, c, d: 0,1 mm; d: 0,5 mm) Şekil 7. Triechphorella geniculata türünün genital yapıları a, aedegus ventralden; b, aedegus yandan; c, stilus; d, genital plak; e, dişide abdomenin uç kısmı ventralden (ölçek çubukları: a, b, c, d: 0,1 mm; d: 0,5 mm) Figure 7. Genital structures of Triechphorella geniculata a, aedeagus from ventral; b, aedeagus from lateral; c, stylus; d, genital plate; e, the tip of the female abdomen (scale bars: a, b, c, d: 0,1 mm; d: 0,5 mm) Şekil 7. Triechphorella geniculata türünün genital yapıları a, aedegus ventralden; b, aedegus yandan; c, stilus; d, genital plak; e, dişide abdomenin uç kısmı ventralden (ölçek çubukları: a, b, c, d: 0,1 mm; d: 0,5 mm) 137 KSÜ Tarım ve Doğa Derg 25 (1): 133-139, 2022 KSU J. Agric Nat 25 (1): 133-139, 2022 Araştırma Makalesi Research Article Genus: Triecphorella Nast, 1933 Triecphorella geniculata (Horvath, 1881) (Figures 6-7) Material Kastamonu: 19.05.2017, 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (64 ♂♂, 45 ♀♀) DISCUSSION Members of the Triecphorella genus have a black-red pattern like Cercopis species. However, it is much smaller than Cercopis taxa in size. The only known taxon of this genus is T. geniculata. It is sampled from an area rich in herbaceous vegetation such as Graminae, Cyperidaceae at the base, at the edge of the river in Kastamonu (Ilıca). The specimens collected in a single locality are associated with the low population density. This may result from specialization to the host plant. However, there is no data on the ecology of this taxon. Cercopidae which is characterized by their bright color patterns consists of 7 species belonging to 3 genea in Turkey. 3 species, C. vulnerata, C. intermedia, and T. geniculata were determined from the study area. Because of the elytral color/pattern variation, the diagnosis of the Cercopidae species must be done according to the their genital structures. According to color/pattern, 4 variants belonging to C. intermedia were determined. Variation of C. vulnerata evaluated from the authors before (Tanyeri & Zeybekoğlu 2020). T. geniculata, has a transverse thin red band in the rear part of the fore wings and this feature did not show variation. When these data are evaluated, it is seen that the first results reported regarding the color/pattern variation displayed by C. intermedia. Contribution of the Authors as Summary Authors declare the contribution of the authors is equal. Acknowledgements This study is a part of the first author’s phd thesis approved by the Graduate School of Sciences of Ondokuz Mayıs University in August 2019 and supported by Ondokuz Mayıs University Research Fund (Project number: PYO.FEN.1904.16.013). C. vulnerata was collected on sunny slopes, pastures, dry or humid areas with rich herbaceous vegetation and around Kastamonu Hamidiye village over apple trees. Other taxa were collected from herbaceous vegetation. The first adult specimens were collected in the second week of May. It has been observed that adults belonging to the species of this genus exist in nature for a very short time. It has been determined that population densities have decreased considerably since the second week of June, but a few individuals could be collected. Contribution of the Authors as Summary Genus: Triecphorella Nast, 1933 Triecphorella geniculata (Horvath, 1881) (Figures 6-7) Material Kastamonu: 19.05.2017, 41° 39ˈ 33.2̎ N 33° 08ˈ 02.2̎ E (64 ♂♂, 45 ♀♀) there is different information about this taxon in the literature. C. septemmaculata originally described by Melichar, 1903, was accepted as the morph of C. sanguinolenta by Lindberg (1923). The intermittent red band behind the wings resembles 3 points. Nast (1933) accepted C. septemmaculata as the synonym of C. intermedia. Similarly, Lallemand (1949) evaluated this taxon as a morph. Dlabola (1965) evaluated C. septemmaculata as a separate taxon and gave the drawings of the lateral view of the aedeagus by stating its differences with C. intermedia. However, in the drawings, the differences between the two taxa are not fully understood. C. septemmaculata recorded from Kastamonu (Lodos and Kalkandelen, 1988) was not determined REFERENCES Anonymous 2021f. Fauna Europe (http//faunaeurope.org) (Access date: 10.01.2021). Altınayar G 1981. Orta Anadolu Bölgesi Tahıl Tarlalarındaki Böcek Faunasının Saptanması Üzerinde Çalışmalar. Türkiye Bitki Koruma Dergisi 21(2): 53- 88. Cryan JR and Svenson GJ 2010. Family-level Relationships of the Spittlebugs and Froghoppers (Hemiptera: Cicodomorpha: Cercopoidea). Systematic Entomology 35: 393-415. C. septemmaculata closely looks like C. sanguinolenta and C. intermedia in terms of genital structures. It is known with limited records from Turkey. 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